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https://openalex.org/W2108961704
https://europepmc.org/articles/pmc3723919?pdf=render
English
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Dosimetric accuracy and clinical quality of Acuros XB and AAA dose calculation algorithm for stereotactic and conventional lung volumetric modulated arc therapy plans
Radiation oncology
2,013
cc-by
7,610
RESEARCH Open Access Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 * Correspondence: kroon.p@bvi.nl 1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB Tilburg, the Netherlands Full list of author information is available at the end of the article © 2013 Kroon 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. Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Abstract Introduction: The main aim of the current study was to assess the dosimetric accuracy and clinical quality of volumetric modulated arc therapy (VMAT) plans for stereotactic (stage I) and conventional (stage III) lung cancer treatments planned with Eclipse version 10.0 Anisotropic Analytical Algorithm (AAA) and Acuros XB (AXB) algorithm. Methods: The dosimetric impact of using AAA instead of AXB, and grid size 2.5 mm instead of 1.0 mm for VMAT treatment plans was evaluated. The clinical plan quality of AXB VMAT was assessed using 45 stage I and 73 stage III patients, and was compared with published results, planned with VMAT and hybrid-VMAT techniques. Results: The dosimetric impact on near-minimum PTV dose (D98%) using AAA instead of AXB was large (underdose up to 12.3%) for stage I and very small (underdose up to 0.8%) for stage III lung treatments. There were no significant differences for dose volume histogram (DVH) values between grid sizes. The calculation time was significantly higher for AXB grid size 1.0 than 2.5 mm (p < 0.01). The clinical quality of the VMAT plans was at least comparable with clinical qualities given in literature of lung treatment plans with VMAT and hybrid-VMAT techniques. The average mean lung dose (MLD), lung V20Gy and V5Gy in this study were respectively 3.6 Gy, 4.1% and 15.7% for 45 stage I patients and 12.4 Gy, 19.3% and 46.6% for 73 stage III lung patients. The average contra-lateral lung dose V5Gy-cont was 35.6% for stage III patients. Conclusions: For stereotactic and conventional lung treatments, VMAT calculated with AXB grid size 2.5 mm resulted in accurate dose calculations. No hybrid technique was needed to obtain the dose constraints. AXB is recommended instead of AAA for avoiding serious overestimation of the minimum target doses compared to the actual delivered dose. Keywords: AAA, Acuros XB, Plan quality, Stage I lung cancer, Stage III lung cancer, VMAT Dose calculations All calculations were performed using the treatment planning system (TPS) Eclipse version 10 with beam algorithms AAA and AXB (Varian Medical Systems, Palo Alto, CA), which was installed on a standard clinical workstation (Dell T5500) with dual 2.40 GHz quad-core Intel processors (E5620), 24 GB RAM, and a 64 bit Windows 7 operating system, in a distributed calculation framework (DCF) network of 3 worksta- tions. The dose reporting mode dose-to-medium Dm was selected for AXB. Immediately after clinical introduction of the Varian Eclipse software, we also clinically introduced VMAT for lung SBRT stage I NSCLC as well as for lung stage III treatments. For this clinical introduction, we investigated the dosimetric accuracy and quality of stereotactic and conventional VMAT planning in Eclipse using AXB and AAA. Routinely, for all our patients, we perform pre- treatment verification measurements using an ionization chamber in the isocentre, combined with film measure- ments in the isocentre plane. Background could achieve at least comparable clinical plan qualities and skin dose levels than intensity modulated radio- therapy (IMRT) [10-12] and can successfully be used for stereotactic body radiotherapy (SBRT) for patients with stage I Non-Small-Cell Lung Cancer (NSCLC) [2,3,5]. Volumetric modulated arc therapy (VMAT) has been shown to be a powerful technique for irradiation of many treatment sites with obtaining higher dose conformity to the tumor while decreasing intra-fraction movements because of shorter delivery times [1-8]. Reliable and accu- rate dose delivery can be obtained using VMAT as shown by pre-treatment dosimetric plan validations [9]. VMAT In case of stage III large tumor lung cancers, it is diffi- cult to limit doses to organs at risks (OARs) such as heart and lung. High doses are preferred since local control in- creased significantly (p = 0.02) when patients are treated with higher doses than 64 Gy [13]. Rengan et al. [13] stated that the median survival time for patients treated to 64 Gy or higher was 20 months versus 15 months for * Correspondence: kroon.p@bvi.nl 1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB Tilburg, the Netherlands Full list of author information is available at the end of the article Page 2 of 8 Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 those treated to less than 64 Gy. Advanced planning tech- niques, IMRT and VMAT, have been shown to be able to increase the therapeutic dose with equal toxicity profiles compared to three-dimensional conformal radiotherapy (3DCFRT) [14]. Unfortunately, it is not always possible to deliver doses higher than 60 Gy to the planning target volume (PTV) using 3DCFRT, IMRT and VMAT because of dose limiting organs [14]. De Bree-Balk et al. [14] stated that possibly further improvements could be made by using hybrid techniques which combine two static fields with IMRT or VMAT, as also investigated by Verbakel et al. [15], who made a comparison between conventional static field plans, IMRT, hybrid-IMRT, VMAT and hybrid- VMAT; and by Chan et al. [16], who compared between 3DCFRT, VMAT and hybrid-VMAT. In both studies the VMAT plans consisted of at least 2 arcs and the hybrid plans of a combination of two static fields and IMRT or VMAT. They have concluded that hybrid techniques are superior in dosimetric outcomes for treating stage III lung tumours compared to the other techniques. Background The treat- ment planning for these studies has been performed using Varian Eclipse treatment planning version 8 or 10 with Anisotropic Analytical Algorithm (AAA). stage III lung tumors (average PTV 690 cm3), Fogliata et al. [26] have illustrated that AAA leads to a monitor unit underestimation of approximately 1-2% relative to AXB grid size 2.5 mm using a treatment planning comparison between 3DCFRT, IMRT and VMAT. Consequently, this suggests that AAA could overesti- mate the minimum target dose, which leads to lower target coverage than the prescribed dose, in case AXB represents the real dose distribution. Kan et al. [27] have illustrated that AXB was more accurate in predicting secondary build-up near and beyond air/tissue interfaces than AAA, using a comparison with measurements. Bush et al. [22] have shown that AXB was capable of modelling radiotherapy dose deposition in the low dens- ity regions. Dose distributions calculated by AXB were in good agreement with BEAMnrc/DOSXYZnrc Monte Carlo dose calculations. The purpose of this study was threefold. First, we wanted to justify the assumption that AXB represents bet- ter the real dose distribution than AAA by cross-checking the finding of Bush et al. [22] with measurements, for calculation grid sizes of 1.0 mm and 2.5 mm. Second, we investigated the dosimetric impact of using AAA instead of AXB, and grid size 2.5 mm instead of 1.0 mm for VMAT treatment plans for stage I and stage III lung tumors. Third, we investigated whether using AXB calcu- lations in Eclipse version 10.0, VMAT results in improved treatment plans compared to VMAT and hybrid-VMAT plans published in literature, again for stage I (lung stereo- tactic) and stage III (conventional lung) treatments. We have recently introduced the Varian Eclipse software version 10, with the AAA as well as the Acuros XB (AXB) algorithm for photon dose calculations in our institute. AXB solves the linear Boltzmann transport equation e.g., [17]. The dosimetric accuracy of AXB has already been in- vestigated in several studies [18-22]. Fogliata et al. [19,21] have concluded that AXB gives acceptable characteristics in homogeneous media for small and large fields (range 0.8×0.8 to 40.0×40.0 cm2) using comparisons of AXB with AAA and measurements. In heterogeneous situations, the AXB algorithm has been shown to provide a valid and ac- curate alternative to Monte Carlo calculations for field sizes ranging from 2.5×2.5 to 30.0×30.0 cm2 [18,20,22]. Treatment planning For the algorithm comparison, optimal VMAT plans were prepared using AAA algorithm grid size 2.5 mm consisting of two 180 degrees arcs using the VMAT optimization tool. Dose prescription was 54 Gy in 5 frac- tions to the 80% isodose, which covered at least 99% of the PTV, for stage I, and for stage III 66 Gy in 33 frac- tions, where at least 99% of the PTV received 90% of the prescribed dose. For stage III, an additional constraint of D≤1% = 72.6 Gy (110% of prescribed dose) was used and the dose to OARs, such as lung and heart, was opti- mized. The plans were recalculated using the same beam settings and monitor units as the AAA grid size 2.5 mm treatment plans for AAA grid size 1.0 mm, AXB grid size 2.5 mm and AXB grid size 1.0 mm. Therefore, it is now shown by simulations and measure- ments that calculations using AXB represent better the actual delivered dose distribution in case of narrow beams and heterogeneous situations than AAA. Of course, in most patients real lung density will be larger than the low density taken in this study (ρ = 0.03 g cm-3), and differences between AXB and AAA will be smaller as also stated by Bush et al. [22]. In the VMAT optimization process, a Clinical Protocol was used (and if necessary, optimized interactively) with Optimization Objectives for the PTV, heart, lungs and spinal cord (with a margin of 5 mm). Most dose volume histogram (DVH) objectives had a priority of 50, whereas the minimum and maximum PTV dose, defined as D100% and D0%, had a priority of 250 and the maximum spinal cord dose (10 Gy for stage I and 44 Gy for stage III) had a priority of 150 or 200. For stage III VMAT plans, a MLD objective of 12 Gy with a priority of 200 was applied; and a contra-lateral lung dose V5Gy-cont objective of 30% or 25% was used with a priority of respectively 450 or 500 for the 73 stage III patients. This study used the volume of both lungs minus internal target volume (ITV) for the cal- culation of MLD, and volume of both lungs minus PTV Justification of the assumption that AXB represents the real dose distribution The measurements and Eclipse dose calculations were performed for a field size of 1.0×1.0 cm2 and 4.0×4.0 cm2 using a 6 MV photon beam (Clinac 2100iX equipped with a Millenium 120-MLC, Varian Medical Systems, Palo Alto, CA) of 200 monitor units and a source to skin distance (SSD) of 100 cm. Justification of the assumption that AXB represents the real dose distribution The analyses in this study to cross-check the findings by Bush et al. [22] confirmed that AXB is much more ac- curate in heterogeneous situations than AAA (Figure 1). For single fields, there were no large differences between grid size 2.5 mm and grid size 1.0 mm. The relative doses were much higher for AAA than AXB in the mid- dle low density layer (ρ = 0.03 g cm-3) of the interface phantom. The difference in relative doses were even larger than 20% for a field size of 1.0×1.0 cm2 and larger than 10% for a field size of 4.0×4.0 cm2. Bush et al. [22] have also published large relative dose differences, e.g. larger than 30% for a very low density media (ρ = 0.001 g cm-3) and a field size of 4.0×4.0 cm2. Justification of the assumption that AXB represents the real dose distribution It was already shown by Gete et al. [23] that AAA cal- culations can slightly overestimate the minimum PTV dose relative to Monte Carlo calculations with BEAMnrc/ DOSXYZnrc for stage I lung tumors (PTV range between 19 to 62 cm3) with forward planning with multiple static non-coplanar conformal fields. It has also been shown by VMAT comparison studies that AXB leads to a slightly more accurate dose distribution than AAA [24,25]. For Before performing the main parts of our study, we vali- dated the findings of Bush et al. [22] that AXB repre- sents the actual dose delivery by performing percentage depth dose (PDD) measurements using EBT2 film (ISP, Wayne, NJ) in a simple heterogeneous interface phan- tom, which consisted of three layers: upper and lower of polystyrene with a density of ρ = 1.05 g cm-3 and thick- ness of 5 and 7 cm, respectively, and a middle layer of Page 3 of 8 Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 for V20Gy, and V5Gy. Most of the time, we applied a “Normal Tissue Objective” with a fall-off of 4 for stage I stereotactic treatments and of 1 for stage III conven- tional lung treatments. A help volume with a margin of 5 mm around the PTV was introduced with also re- latively high priorities when OARs were not spared suf- ficiently. It was almost never necessary to include constraints for the other OARs. foam, with a low-density ρ = 0.03 g cm-3 and a thickness of 8 cm. This very low density was chosen to investigate the accuracy of both algorithms in a very extreme situ- ation analogue to Bush et al. [22]. The measurements and Eclipse dose calculations were performed for a field size of 1.0×1.0 cm2 and 4.0×4.0 cm2 using a 6 MV photon beam (Clinac 2100iX equipped with a Millenium 120-MLC, Varian Medical Systems, Palo Alto, CA) of 200 monitor units and a source to skin distance (SSD) of 100 cm. foam, with a low-density ρ = 0.03 g cm-3 and a thickness of 8 cm. This very low density was chosen to investigate the accuracy of both algorithms in a very extreme situ- ation analogue to Bush et al. [22]. Patient selection Dose volume histograms were produced for all plans in order to analyze the doses to the PTV and OARs. The statistical differences were tested for the VMAT treat- ment plans between AAA grid size 2.5 mm, AAA grid size 1.0 mm, AXB grid size 2.5 mm and AXB grid size 1.0 mm. The statistical significance of the differences was tested with a paired two-tailed student t-test with significant level p < 0.05. Eight stage I and seven stage III lung patients were se- lected to investigate the dosimetric impact of using AAA instead of AXB, and grid size 2.5 mm instead of 1.0 mm, for dose calculations of VMAT treatment plans. The average PTV volume was 24.0 cm3 (range 5.1 to 56.9 cm3) for stage I and 418.9 cm 3 (range 140.3 to 762.6 cm3) for stage III tumors. OARs were delineated including heart and contra-lateral lung. The clinical quality of the VMAT plans was assessed using 45 stage I lung patients (average PTV 35.4 cm3; range 5.5 to 175.4 cm3 ) and 73 stage III lung patients (average PTV 344.4 cm3; range 25.1 to 1069.0 cm3). Dosimetric impact of algorithms and grid sizes on clinical lung VMAT plans Dose volume histograms were produced for all plans so that the relative differences between algorithms (AAA and AXB) and grid sizes (2.5 and 1.0 mm) could be analyzed. The dose calculation time by AAA grid size 1.0 mm in comparison to AAA grid size 2.5 mm, AXB grid size 2.5 mm and AXB grid size 1.0 was much larger. For ex- ample for stage I, the calculation times were 7, 10 and 3 times larger with AAA grid size 1.0 mm in comparison to Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 Page 4 of 8 Figure 1 Measured and calculated percentage depth dose curves in a slab-phantom. Predicted percentage depth dose curves by AAA and Acuros XB grid sizes 2.5 and 1.0 mm compared to measured data using a slab-phantom with (top) 1.0x1.0 cm2 and (bottom) 4.0x4.0 cm2 6 MV AP photon beams. Figure 1 Measured and calculated percentage depth dose curves in a slab-phantom. Predicted percentage depth dose curves by AAA and Acuros XB grid sizes 2.5 and 1.0 mm compared to measured data using a slab-phantom with (top) 1.0x1.0 cm2 and (bottom) 4.0x4.0 cm2 6 MV AP photon beams. AAA grid size 2.5 mm, AXB grid size 2.5 mm and AXB grid size 1.0 mm. We have decided to focus on the dose distribution differences between AAA grid size 2.5 mm and AXB grid size 2.5 mm; and on the dose distribution differences between AXB grid size 2.5 mm and AXB grid size 1.0 mm since AAA grid size 1.0 mm will probably never be used clinically and the differences with AAA grid size 2.5 mm were very small. AAA grid size 2.5 mm, AXB grid size 2.5 mm and AXB grid size 1.0 mm. We have decided to focus on the dose distribution differences between AAA grid size 2.5 mm and AXB grid size 2.5 mm; and on the dose distribution differences between AXB grid size 2.5 mm and AXB grid size 1.0 mm since AAA grid size 1.0 mm will probably never be used clinically and the differences with AAA grid size 2.5 mm were very small. a result, AAA grid size 2.5 mm treatment plans might be clinically approved with the real V20Gy being slightly too high. Dosimetric impact of algorithms and grid sizes on clinical lung VMAT plans The differences in lung doses calculated by AAA and AXB were patient dependent since they were dependent on field size, location of the target inside the lung and the density of the lung. Depending on the ac- tual combination of field size, target location and lung density, AAA can over- or underestimate the lung dose, as also stated by Bush et al. [22]. The relative dose differ- ences between both models for the other OARs were small. However, there was a large difference in calculation time between both algorithms. The dose calculation times required by AAA grid size 2.5 mm were about 2 times the calculation times required by AXB grid size 2.5 mm. A summary of the relative dose differences between algorithms and grid sizes is given in Table 1. For individual stage I lung patients differences occurred in dose distribu- tions between AXB and AAA. The maximum difference of near-minimum PTV dose (D98%) between AXB grid size 2.5 mm and AAA grid size 2.5 mm was −7.1 Gy corresponding to a relative difference of −12.3%, which indicated a serious underdosage of the delivered dose when this patient would have been planned with the AAA algorithm (Table 1, Figure 2). This finding is similar to re- sults published by Gete et al. [23]. They observed a 12.8% lower minimum PTV dose using Monte Carlo simulations than using AAA version 8.6 calculations for one patient plan. These relative differences can be explained by the significantly improved accuracy of AXB under the con- ditions of electronic disequilibrium compared to AAA. AAA only predicts little secondary build-up at regions beyond low-density media like lung [22,26,27]. In case of stage III patients, the difference in PTV doses were smaller than in case of stage I patients (Table 1, Figure 1) due to, e.g. larger fields and tumor sizes, resulting in smaller errors in the AAA algorithm. The differences in OAR doses were also small. There was again a significant (p < 0.01) difference in calculation time between AAA and AXB. The average final dose calculation time was respectively, 6 and 4 minutes, for AAA grid size 2.5 mm and AXB grid size 2.5 mm. Dosimetric impact of algorithms and grid sizes on clinical lung VMAT plans Table 1 Relative differences between algorithms and grid sizes for lung cancer patients Table 1 Relative differences between algorithms and grid sizes for lung cancer patients Relative difference p-value Relative Table 1 Relative differences between algorithms and grid sizes for lung cancer patients Relative difference p-value Relative d (AXB2.5 -AAA2.5)/ AAA2.5×100% (AXB1.0 - AXB2.5 *Significant relative difference tested by two-tailed student t-test. that the plan qualities were at least comparable. For example, McGrath et al. [3] have published an average MLD for stage I tumors of 4.6 Gy (with dose prescrip- tion: 99% of the PTVITV has to receive more than 43.2 Gy) with VMAT. In our study the average MLD was 3.6 Gy (with a higher dose description: 99% of the PTV has to receive more than 54.0 Gy). stage I patients with grid size 2.5 mm and 1.0 mm, re- spectively, and 4 and 27 minutes for stage III patients with grid size 2.5 mm and 1.0 mm. Therefore, we recommend using AXB grid size 2.5 mm for VMAT planning of lung tumors, since this results in accurate dose values with acceptable calculation times. For stage III patients, the clinical quality of treatment plans is a trade-off between high doses to gross tumour volume and limiting treatment related pneumonitis (TRP). Doses higher than 64 Gy are preferred [13] which could be obtained for all 73 clinical patients in this study. Simultaneously, lung doses should be minimized since these influenced the post-radiation acute TRP. Sev- eral studies describe analyses to determine indicators for predicting TRP [29-33]. Different predictors were indi- cated in these studies, e.g. V5Gy-cont by Song et al. [29], V5Gy by Wang et al. [30], MLD and V30Gy by Kim et al. [31], and V10Gy by Shi et al. [32] and Spych et al. [33]. Song et al. [29] have shown with an extensive multivari- ate analysis including V5Gy, V10Gy, V13Gy, V15Gy, V20Gy and MLD for total lung, ipsilateral lung and contra- Dosimetric impact of algorithms and grid sizes on clinical lung VMAT plans Only small significant differences were found between AXB grid size 2.5 mm and AXB grid size 1.0 mm for near-maximum PTV dose (D2%) (Table 1, Figure 2), while the calculation time will increase drastically when grid size 1.0 mm was used instead of grid size 2.5 mm. The average calculation time was 2 and 8 minutes for AXB showed a slightly but significant (p < 0.05) higher V20Gy (volume of both lungs minus PTV with a dose of 20 Gy or more) value than AAA for stage I patients. As Page 5 of 8 Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 Table 1 Relative differences between algorithms and grid sizes for lung cancer patients Relative difference p-value Relative difference p-value (AXB2.5 -AAA2.5)/ AAA2.5×100% (AXB1.0 - AXB2.5)/ AXB2.5×100% Average ± SDV (min-max) Average ± SDV (min-max) Stage I (N = 8) PTV D98% −3.2% ± 4.0% (−12.3% – 0.5%) 0.06 0.7% ± 1.3% (−0.8% – 3.1%) 0.19 D2% 0.2% ± 1.2% (−2.1% – 1.3%) 0.69 0.9% ± 0.4% (0.3% – 1.4%) <0.01* Dmean −0.6% ± 2.2% (−4.9% – 1.6%) 0.46 0.2% ± 0.5% (−0.6% – 1.2%) 0.45 Total lung V5Gy 1.3% ± 1.9% (−1.9% –3.2%) 0.12 0.1% ± 0.6% (−0.8% – 1.1%) 0.73 V20Gy 2.0% ± 2.3% (0.0% – 5.7%) 0.05* −0.7% ± 1.2% (−2.7% – 0.0%) 0.17 MLD 0.0% ± 0.5% (−0.7% – 0.7%) 0.60 0.1% ± 0.2% (0.0% – 0.4%) 0.08 Time Calculation −39.2% ± 13.5% (−50.3% – –8.2%) <0.01* 304.7% ± 48.7% (226.9% – 345.8%) <0.01* Stage III (N = 7) PTV D98% −0.3% ± 0.7% (−0.8% – 1.3%) 0.33 0.2% ± 0.8% (−1.4% – 0.9%) 0.57 D2% −0.6% ± 2.0% (−2.1% – 3.0%) 0.47 0.6% ± 0.4% (−0.1% – 1.0%) <0.01* Dmean −0.8% ± 0.7% (−1.6% – 0.3%) 0.02* 0.1% ± 0.3% (−0.5% – 0.4%) 0.40 Total lung V5Gy −1.8% ± 1.7% (−4.8% – 0.3%) 0.03* −0.4% ± 0.7% (−1.5% – 0.4%) 0.16 V20Gy 0.2% ± 0.7% (−0.5% – 1.5%) 0.69 0.0% ± 0.0% (0.0% –0.0%) 1.00 MLD −0.5% ± 0.6% (−1.3% – 0.0%) 0.09 0.2% ± 0.3% (0.0% – 0.6% ) 0.17 Time Calculation −31.4% ± 9.9% (−44.4% – –17.9%) <0.01* 556.9% ± 91.1% (412.3% – 646.0%) <0.01* *Significant relative difference tested by two-tailed student t-test. Clinical quality of VMAT The dose volume quantities of 45 stage I and 73 stage III lung patients planned with VMAT AXB grid size 2.5 mm were analyzed. The average MLD, total lung V20Gy and total lung V5Gy were 3.6 Gy, 4.1% and 15.7% for stage I and 12.4 Gy, 19.3% and 46.6% for stage III. The pre- scribed dose of 66 Gy could be delivered to all patients. The MLD was between 16.0 and 18.5 Gy for 15% of the stage III patients, for all other patients, the MLD was lower than 16 Gy. It was difficult to compare the obtained plan qualities with literature since different patients were used. How- ever, when we compared the dose volume quantities with published studies about VMAT techniques for stage I and stage III NSCLC patients [2,3,16,28], we concluded Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 Page 6 of 8 Figure 2 Dose volume histogram comparisons of PTV doses between algorithms and grid sizes. Comparison of dose volume histograms of the planning target volume (PTV) between AAA grid size 2.5 mm, Acuros XB grid size 2.5 mm and Acuros XB grid size 1.0 mm of (top) a stage I and (bottom) a stage III lung patient. The black vertical lines indicate the prescription: The dose that has to cover 99% of the PTV, respectively, 54.0 Gy for stage I and 59.4 Gy for stage III. Figure 2 Dose volume histogram comparisons of PTV doses between algorithms and grid sizes. Comparison of dose volume histograms of the planning target volume (PTV) between AAA grid size 2.5 mm, Acuros XB grid size 2.5 mm and Acuros XB grid size 1.0 mm of (top) a stage I and (bottom) a stage III lung patient. The black vertical lines indicate the prescription: The dose that has to cover 99% of the PTV, respectively, 54.0 Gy for stage I and 59.4 Gy for stage III. lateral lung that V5Gy-cont was the only remaining signifi- cant factor associated with TRP. They concluded that V5Gy-cont should be kept as low as possible and they sug- gested a cut-off value of 60% since incidences of grade ≥3 pneumonitis were 35% and 0%, respectively, for V5Gy-cont ≤ 60% and V5Gy-cont > 60% (p = 0.01). for only one patient. Kim et al. g 0Gy 95% 0 % 2 [15] Use the volume of both lungs minus PTV for V20Gy; PTV V95% > 97% and PTV V107% < 5%. 3 g 20Gy 5Gy 98% y 2% y 4 This study uses the volume of both lungs minus ITV for MLD, and volume of both lungs minus PTV for V20Gy and V5Gy. PTV V90% > 99% and PTV V110% ≤1%. y 3 [16] Use the volume of both lungs minus PTV for MLD, V20Gy and V5Gy; D98% > 57.0 Gy and D2% < 64.2 Gy. erage values and standard deviations are given. 4] Use the volume of both lungs minus CTV for MLD and V20Gy; PTV V95% > 99% and PTV V107% < 1%. Average values and standard deviations are given. 1 [ ] h l f b h l f d d Average values and standard deviations are given. 1 [14] Use the volume of both lungs minus CTV for MLD and V20Gy; PTV V95% > 99% and PTV V107% < 1%. 5 Median instead of average value is given. g 20Gy; 95% 107% e of both lungs minus PTV for V20Gy; PTV V95% > 97% and PTV V107% < 5%. Average values and standard deviations are given Conclusions We investigated the quality of VMAT treatment plans using Eclipse treatment planning system version 10.0 for stage I and III lung patients. All plans consisted of 2 partial arcs of 180 degrees. We showed that the AXB calculation algorithm was preferable to AAA since pos- sible PTV underdosage, as a result of inaccurate AAA calculations, can be avoided. In addition, the calculation time was much shorter for AXB. For clinical VMAT lung plans, the quality and accuracy of AXB grid size 2.5 mm was comparable with AXB grid size 1.0 mm. However, the calculation time increases drastically when grid size 1.0 mm was used. 7. Mancosu P, Castiglioni S, Reggiori G, Catalano M, Alongi F, Pellegrini C, Arcangeli S, Tozzi A, Lobefalo F, Fogliata A, Navarria P, Cozzi L, Scorsetti M: Stereotactic body radiation therapy for liver tumours using flattening filter free beam: dosimetric and technical considerations. Radiat Oncol 2012, 7:16. 7. Mancosu P, Castiglioni S, Reggiori G, Catalano M, Alongi F, Pellegrini C, Arcangeli S, Tozzi A, Lobefalo F, Fogliata A, Navarria P, Cozzi L, Scorsetti M: Stereotactic body radiation therapy for liver tumours using flattening filter free beam: dosimetric and technical considerations. Radiat Oncol 2012, 7:16. 8. Scorsetti M, Bignardi M, Alongi F, Fogliata A, Mancosu P, Navarria P, Castiglioni S, Pentimalli S, Tozzi A, Cozzi L: Stereotactic body radiation therapy for abdominal targets using volumetric intensity modulated arc therapy with RapidArc: Feasibility and clinical preliminary results. Acta Oncol 2011, 50:528–538. Feasibility and clinical preliminary results. Acta Oncol 2011, 50:528–538. 9. Fogliata A, Clivio A, Fenoglietto P, Hrbacek J, Kloeck S, Lattuada P, Mancosu P, Nicolini G, Parietti E, Urso G, Vanetti E, Cozzi L: Quality assurance of RapidArc in clinical practice using portal dosimetry. Br J Radiol 2011, 84:534–545. 10. Seppala J, Suilamo S, Kulmala J, Mali P, Minn H: A dosimetric phantom study of dose accuracy and build-up effects using IMRT and RapidArc in stereotactic irradiation of lung tumours. Radiat Oncol 2012, 7:79. 11. Bertelsen A, Hansen O, Brink C: Does VMAT for treatment of NSCLC patients increase the risk of pneumonitis compared to IMRT? – A planning study. Acta Oncol 2012, 51:752–758. The VMAT plans were compared to published treat- ment planning studies. The clinical VMAT AXB grid size 2.5 mm plans obtained in this study were at least compar- able to the published planning studies, e.g. Authors’ contributions PK designed the set-up of the study, performed the data collection and analyses, carried out the comparisons and drafted the manuscript. SH generated most of the treatment plans. ME supervised the project, helped to draft the manuscript, participated in the design of the study, data collection, treatment planning and analyses. All authors read and approved the final manuscript. 15. Verbakel WFAR, Van Reij E, Ladenius-Lischer I, Cuijpers JP, Slotman BJ, Senan S: Clinical application of a novel hybrid intensity-modulated radiotherapy technique for stage III lung cancer and dosimetric comparison with four other techniques. Int J Radiation Oncol Biol Phys 2012, 83(Suppl 2):297–303. Received: 8 February 2013 Accepted: 3 June 2013 Published: 24 June 2013 Received: 8 February 2013 Accepted: 3 June 2013 Published: 24 June 2013 Received: 8 February 2013 Accepted: 3 June 2013 Published: 24 June 2013 Received: 8 February 2013 Accepted: 3 June 2013 Published: 24 June 2013 study were at least comparable to the plans obtained with VMAT and hybrid-VMAT discussed in literature [14-16]. Historically, all our stage III plans were normalised as: 99% of the PTV is covered by 90% of the prescribed dose (66 Gy). Our normalisation is in close agreement with the recommended normalisation method by ICRU 83 [34], to the mean PTV dose. For our stage III patients, the mean PTV dose was 101.2% ± 1.4%. This im- plies that our normal tissue DVH values will even be slightly better when we use ICRU 83 for normalisation in the future. References V Zhang GG, Ku L, Dilling TJ, Stevens CW, Zhang RR, Li W, Feygelman V: Volumetric modulated arc planning for lung stereotactic body radiotherapy using conventional and unflattened photon beams: a dosimetric comparison with 3D technique. Radiat Oncol 2011, 6:152. 6. Wolff D, Stieler F, Welzel G, Lorenz F, Abo-Madyan Y, Mai S, Herskind C, Polednik M, Steil V, Wenz F, Lohr F: Volumetric modulated arc therapy (VMAT) vs. serial tomotherapy, step-and-shoot IMRT and 3D-conformal RT for treatment of prostate cancer. Radiother Oncol 2009, 93(Suppl 2):226–233. 7. Mancosu P, Castiglioni S, Reggiori G, Catalano M, Alongi F, Pellegrini C, Arcangeli S, Tozzi A, Lobefalo F, Fogliata A, Navarria P, Cozzi L, Scorsetti M: Stereotactic body radiation therapy for liver tumours using flattening filter free beam: dosimetric and technical considerations. Radiat Oncol 2012, 7:16. 5. Zhang GG, Ku L, Dilling TJ, Stevens CW, Zhang RR, Li W, Feygelman V: Volumetric modulated arc planning for lung stereotactic body radiotherapy using conventional and unflattened photon beams: a dosimetric comparison with 3D technique. Radiat Oncol 2011, 6:152. 5. Zhang GG, Ku L, Dilling TJ, Stevens CW, Zhang RR, Li W, Feygelman V: Volumetric modulated arc planning for lung stereotactic body radiotherapy using conventional and unflattened photon beams: a dosimetric comparison with 3D technique. Radiat Oncol 2011, 6:152. 6. Wolff D, Stieler F, Welzel G, Lorenz F, Abo-Madyan Y, Mai S, Herskind C, Polednik M, Steil V, Wenz F, Lohr F: Volumetric modulated arc therapy (VMAT) vs. serial tomotherapy, step-and-shoot IMRT and 3D-conformal RT for treatment of prostate cancer. Radiother Oncol 2009, 93(Suppl 2):226–233. 6. Wolff D, Stieler F, Welzel G, Lorenz F, Abo-Madyan Y, Mai S, Herskind C, Polednik M, Steil V, Wenz F, Lohr F: Volumetric modulated arc therapy (VMAT) vs. serial tomotherapy, step-and-shoot IMRT and 3D-conformal RT for treatment of prostate cancer. Radiother Oncol 2009, 93(Suppl 2):226–233. Acknowledgements 16. Chan OSH, Lee MCH, Hung AWM, Chang ATY, Yeung RMW, Lee AWM: The superiority of hybrid-volumetric arc therapy (VMAT) technique over double arcs VMAT and 3D-conformal technique in the treatment of locally advanced non-small cell lung cancer – a planning study. Radiother Oncol 2011, 101:298–302. Acknowledgements The authors thank E. Gerrits, P. Korthuis, A. Rog, L. Vugts, J. van Wershoven and M. van Wieren for their assistance during the measurements. The authors thank E. Gerrits, P. Korthuis, A. Rog, L. Vugts, J. van Wershoven and M. van Wieren for their assistance during the measurements. Competing interest Competing interest Competing interest The authors declare no conflict of interest. p g The authors declare no conflict of interest. 14. De Bree-Balk I, Van Hinsberg MGE, Van Veelen LR: High-dose radiotherapy in inoperable nonsmall cell lung cancer: comparison of volumetric arc therapy, dynamic IMRT and 3D conformal radiotherapy. Med Dosim 2012, 37(Suppl 4):353–357. Conclusions planned with hybrid-VMAT, therefore being a quick and easy alterna- tive for this technique. 12. Holt A, Van Vliet-Vroegindeweij C, Mans A, Belderbos JS, Damen EMF: Volumetric-modulated arc therapy for stereotactic body radiotherapy of lung tumors: a comparison with intensity-modulated radiotherapy techniques. Int J Radiation Oncol Biol. Phys. 2011, 81(Suppl 5):1560–1567. 13. Rengan R, Rosenzweig KE, Venkatraman E, Koutcher LA, Fox JL, Nayak R, Amols H, Yorke E, Jackson A, Ling C, Leibel S: Improved local control with higher doses of radiation in large-volume stage III non-small-cell lung cancer. Int J Radiation Oncol Biol Phys 2004, 60(Suppl 3):741–747. References V 1. Vanetti E, Clivio A, Nicolini G, Fogliata A, Ghosh-Laskar S, Agarwal JP, Upreti RR, Budrukkar A, Murthy V, Deshpande DD, Shrivastava SK, Dinshaw KA, Cozzi L: Volumetric modulated arc radiotherapy for carcinomas of the oro-pharynx, hypo-pharynx and larynx: a treatment planning comparison with fixed field IMRT. Radiother Oncol 2009, 92:111–117. 2. Verbakel WFAR, Senan S, Cuijpers JP, Slotman BJ, Lagerwaard FJ: Rapid delivery of stereotactic radiotherapy for peripheral lung tumors using volumetric intensity-modulated arcs. Radiother Oncol 2009, 93:122–124. 3. McGrath SD, Matuszak MM, Yan D, Kestin LL, Martinez AA, Grills IS: Volumetric modulated arc therapy for delivery of hypofractionated stereotactic lung radiotherapy: a dosimetric and treatment efficiency analysis. Radiother Oncol 2010, 95:153–157. 1. Vanetti E, Clivio A, Nicolini G, Fogliata A, Ghosh-Laskar S, Agarwal JP, Upreti RR, Budrukkar A, Murthy V, Deshpande DD, Shrivastava SK, Dinshaw KA, Cozzi L: Volumetric modulated arc radiotherapy for carcinomas of the oro-pharynx, hypo-pharynx and larynx: a treatment planning comparison with fixed field IMRT. Radiother Oncol 2009, 92:111–117. p 2. Verbakel WFAR, Senan S, Cuijpers JP, Slotman BJ, Lagerwaard FJ: Rapid delivery of stereotactic radiotherapy for peripheral lung tumors using volumetric intensity-modulated arcs. Radiother Oncol 2009, 93:122–124. 3. McGrath SD, Matuszak MM, Yan D, Kestin LL, Martinez AA, Grills IS: Volumetric modulated arc therapy for delivery of hypofractionated stereotactic lung radiotherapy: a dosimetric and treatment efficiency analysis. Radiother Oncol 2010, 95:153–157. This study illustrates that it is possible with two 180 degree arcs to obtain clinical plan qualities compared to VMAT and hybrid-VMAT plans described in literature. Generating acceptable plans using VMAT with two arcs of 180 degrees required only 1 hour for plan optimization and dose calculations. Consequently, our clinical VMAT plans show comparable clinical plan quality as hybrid VMAT techniques, therefore being a quick and easy alternative. y 4. Ong C, Verbakel WFAR, Cuijpers JP, Slotman BJ, Lagerwaard FJ, Senan S: Stereotactic radiotherapy for peripheral lung tumors: A comparison of volumetric modulated arc therapy with 3 other delivery techniques. Radiother Oncol 2010, 97:437–442. 4. Ong C, Verbakel WFAR, Cuijpers JP, Slotman BJ, Lagerwaard FJ, Senan S: Stereotactic radiotherapy for peripheral lung tumors: A comparison of volumetric modulated arc therapy with 3 other delivery techniques. Radiother Oncol 2010, 97:437–442. 5. Clinical quality of VMAT [31] have used a MLD cut- off value of 16 Gy and indicated that the actual incidence of lung toxicity of grade ≥2 was 8% for MLD ≤16 Gy and 54% for MLD > 16 Gy (p < 0.01). Wang et al. [30] have de- termined an incidence of grade ≥3 TRP at 1 year of 13% for MLD ≤16.5 Gy and of 36% for MLD >16.5 Gy (p = 0.02). We have obtained a MLD ≤16.5 Gy for 90.4% of the patients and the maximum MLD was 18.3 Gy. In this study, a V5Gy-cont planning objective of 30% or 25% was used with priority of 450 or 500 for the 73 stage III patients planned with VMAT. Adding this objective decreased significantly the MLD, V5Gy and V5Gy-cont (p < 0.01) from 12.7 Gy, 53.5% and 47.7% to 12.4 Gy, 46.6% and 35.6%, respectively. The V5Gy-cont was larger than 60% The lung dose values, MLD, V20Gy, V5Gy and V5Gy-cont were compared to lung dose values published in the literature using VMAT and hybrid-VMAT techniques. It is shown in Table 2 that the clinical VMAT plans of this Table 2 Comparison of stage III VMAT and hybrid-VMAT plans Study Method N Dprescibed [Gy] VPTV [cm3] MLD [Gy] V20Gy [%] V5Gy [%] V5Gy-cont [%] De Bree-Balk et al. [14]1 VMAT 20 66 8385 20.0 36.6 NA 69.6 Verbakel et al. [15]2 VMAT 14 66 779 NA 30.3 ± 5.7 NA 44.6 ± 9.0 H-VMAT 14 66 779 NA 30.1 ± 5.8 NA 36.2 ± 15.0 Chan et al. [16]3 VMAT 24 60 508 14.4 ± 2.9 25.4 ± 6.0 64.0 ± 15.4 NA H-VMAT 24 60 508 14.0 ± 2.9 23.3 ± 5.3 59.5 ± 16.7 NA This study4 VMAT 73 66 344 12.4 ± 3.5 19.3 ± 6.8 46.6 ± 10.6 35.6 ± 7.1 This study4 VPTV > 500 cm3 VMAT 13 66 678 14.5 ± 2.1 22.1 ± 6.4 52.1 ± 10.3 38.7 ± 10.0 Table 2 Comparison of stage III VMAT and hybrid-VMAT plans Study Method N D [Gy] V [ Table 2 Comparison of stage III VMAT and hybrid-VMAT plans Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 Page 7 of 8 Page 7 of 8 Author details 1D f 1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB Tilburg, the Netherlands. 2Department of Radiotherapy, Institute Verbeeten, Brugstraat 10, 5042 SB, Tilburg, the Netherlands. 17. Vassiliev ON, Wareing TA, McGhee J, Failla G, Salehpour MR, Mourtada F: Validation of a new grid-based Boltzmann equation solver for dose calculation in radiotherapy with photon beams. Phys Med Biol 2010, 55:581–598. 17. Vassiliev ON, Wareing TA, McGhee J, Failla G, Salehpour MR, Mourtada F: Validation of a new grid-based Boltzmann equation solver for dose calculation in radiotherapy with photon beams. Phys Med Biol 2010, 55:581–598. 1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB Tilburg, the Netherlands. 2Department of Radiotherapy, Institute Verbeeten, Brugstraat 10, 5042 SB, Tilburg, the Netherlands. Page 8 of 8 Page 8 of 8 Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 Kroon et al. Radiation Oncology 2013, 8:149 http://www.ro-journal.com/content/8/1/149 18. Han T, Mikell JK, Salehpour M, Mourtada F: Dosimetric comparison of Acuros XB deterministic radiation transport method with Monte Carlo and model-based convolution methods in heterogeneous media. Med Phys 2011, 38(Suppl 5):2651–2664. 19. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Accuracy of Acuros XB and AAA dose calculation for small fields with reference to RapidArc stereotactic treatments. Med Phys 2011, 38(Suppl 11):6228–6237. 20. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Dosimetric eva 20. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Dosimetric evaluation of Acuros XB advanced dose calculation algorithm in heterogeneous media. Radiat Oncol 2011, 6:82. 21. Fogliata A, Nicolini G, Clivio A, Vanetti E, Mancosu P, Cozzi L: Dosimetric validation of the Acuros XB advanced dose calculation algorithm: fundamental characterization in water. Phys Med Biol 2011, 56:1879–1904. 22. Bush K, Gagne IM, Zavgorodni S, Ansbacher W, Beckham W: Dosimetric validation of Acuros XB with Monte Carlo methods for photon dose calculations. Med Phys 2011, 38(Suppl 4):2208–2221. 23. Gete E, Teke T, Kwa W: Evaluation of the AAA treatment planning algorithm for SBRT lung treatment: comparison with Monte Carlo and homogeneous pencil beam dose calculations. J Med Imaging Radiation Sci 2012, 43:26–33. 24. Fogliata A, Scorsetti M, Navarria P, Catalano M, Clivio A, Cozzi L, Lobefalo F, Nicolini G, Palumbo V, Pellegrini C, Reggiori G, Roggio A, Vanetti E, Alongi F, Pentimalli S, Mancosu P: Dosimetric comparison between VMAT with different dose calculation algorithms and protons for soft-tissue sarcoma radiotherapy. Acta Oncol 2013, 52:545–552. 25. Author details 1D f Han T, Followill D, Mikell J, Repchak R, Molineu A, Howell R, Salehpour M, Mourtada F: Dosimetric impact of Acuros XB deterministic radiation transport algorithm for heterogeneuous dose calculation in lung cancer. Med Phys 2013, 40(Suppl 5):2651–2664. 26. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Critical appraisal of Acuros XB and Anisotropic Analytic Algorithm dose calculation in advanced non-small-cell lung cancer treatments. Int J Radiation Oncol Biol Phys 2012, 83(Suppl 5):1587–1595. 27. Kan MWK, Leung LHT, Yu PKN: Verification and dosimetric impact of Acuros XB algorithm on intensity modulated stereotactic radiotherapy for locally persistent nasopharyngeal carcinoma. Med Phys 2012, 39(Suppl 8):4705–4714. 28. Scorsetti M, Navarria P, Mancosu P, Alongi F, Castiglioni S, Cavina R, Cozzi L, Fogliata A, Pentimalli S, Tozzi A, Santoro A: Large volume unresectable locally advanced non-small cell lung cancer: acute toxicity and initial outcome results with rapid arc. Radiat Oncol 2010, 5:94. 29. Song CH, Pyo H, Moon SH, Kim TH, Kim DW, Cho KH: Treatment-related pneumonitis and acute esophagitis in non-small-cell lung cancer patients treated with chemotherapy and helical tomotherapy. Int J Radiation Oncol Biol Phys 2010, 78(Suppl 3):651–658. 30. Wang S, Liao Z, Wei X, Liu HH, Tucker SL, Hu C, Mohan R, Cox JD, Komaki R: Analysis of clinical and dosimetric factors associated with threatment- related pneumonitis (TRP) in patients with non-small-cell lung cancer (NSCLC) treated with concurrent chemotherapy and three-dimensional conformal radiotherapy (3D-CRT). Int J Radiation Oncol Biol Phys 2006, 66(Suppl 5):1399–1407. 31. Kim M, Lee J, Ha B, Lee R, Lee K, Suk Shu H: Factors predicting radiation pneumonitis in locally advanced non-small cell lung cancer. Radiat Oncol J 2011, 29(Suppl 3):181–190. 32. Shi A, Zhu G, Wu H, Yu R, Li F, Xu B: Analysis of clinical and dosimetric factors associated with severe acute radiation pneumonitis in patients with locally advanced non-small cell lung cancer treated with concurrent chemotherapy and intensity-modulated radiotherapy. Radiat Oncol 2010, 5:35. 33. Spych M, Gottwald L, Klonowicz M, Biegala M, Bibik R, Fijuth J: The analysis of prognostic factors affecting post-radiation acute reaction after conformal radiotherapy for non-small cell lung cancer. Arch Med Sci 2010, 6(Suppl 5):756–763. Author details 1D f Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 34. ICRU Report 83: Prescribing recording and reporting photon beam intensity modulated radiation therapy (IMRT). Oxford: Oxford University Press; 2010. 34. ICRU Report 83: Prescribing recording and reporting photon beam intensity modulated radiation therapy (IMRT). Oxford: Oxford University Press; 2010. 34. ICRU Report 83: Prescribing recording and reporting photon beam intensity modulated radiation therapy (IMRT). Oxford: Oxford University Press; 2010. • Convenient online submission doi:10.1186/1748-717X-8-149 Cite this article as: Kroon et al.: Dosimetric accuracy and clinical quality of Acuros XB and AAA dose calculation algorithm for stereotactic and conventional lung volumetric modulated arc therapy plans. Radiation Oncology 2013 8:149. doi:10.1186/1748-717X-8-149 Cite this article as: Kroon et al.: Dosimetric accuracy and clinical quality of Acuros XB and AAA dose calculation algorithm for stereotactic and conventional lung volumetric modulated arc therapy plans. Radiation Oncology 2013 8:149.
https://openalex.org/W4236569036
https://www.biodiversitylibrary.org/itempdf/50506
English
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American game-bird shooting, by George Bird Grinnell ... with colored plates of ruffed grouse and bobwhite, forty-eight full-page portraits of game birds and shooting scenes, and many cuts in text.
null
1,910
public-domain
158,504
CopyriGHT, 1910, BY FOREST AND STREAM PUBLISHING COMPANY \ he \ \ Vagal a as \. \ v { its A\ } nN W \ \ f ~ AMERICAN GAME-BIRD SHOOTING By George Bird Grinnell Author of PAWNEE HERO STORIES BLACKFOOT LODGE TALES #¥ THE STORY OF THE INDIAN # THE JACK SERIES # AMERICAN DUCK SHOOTING, etc., etc. With Colored Plates of Ruffed Grouse and Bobwhite, Forty-eight Full-page Portraits of Game Birds and Shooting Scenes, and many Cuts in Text NEW YORK Forest and Stream Publishing Company 269465 AMERICAN GAME-BIRD SHOOTING By George Bird Grinnell Author of PAWNEE HERO STORIES BLACKFOOT LODGE TALES #¥ THE STORY OF THE INDIAN # THE JACK SERIES # AMERICAN DUCK SHOOTING, etc., etc. With Colored Plates of Ruffed Grouse and Bobwhite, Forty-eight Full-page Portraits of Game Birds and Shooting Scenes, and many Cuts in Text NEW YORK Forest and Stream Publishing Company 269465 269465 PREFACE OWPULE AND MGQ@w PL ETE work on American upland shooting has as yet been written. Volumes have been published, each of which covers some section of what 1s really a large subject, but from the broader sportsman’s viewpoint no one of them is complete. Of these works, the most useful was written by the veteran naturalist and sportsman, D. G. Elliott, whose “Game Birds of North America’ deals chiefly with the natural history side of the grouse and quails, and touches only lightly on the methods by which they are pursued for sport. It seems time now that a book should be written, covering the whole subject of upland American game- bird shooting; that is, the shooting of those birds in which the pointing dog is the assistant to the gun— the turkeys, grouse and quail, and the American wood- cock and Wilson’s snipe, called also “English” and “jack” snipe, a bird of continental distribution. Some years ago I brought together a large amount O Some years ago I brought together a large amount vii vii PREFACE of material on duck shooting in North America, and the gratifying reception met by that volume encour- ages me to believe that there 1s place for a companion work, which shall appeal especially to men who tramp the uplands with gun and dog. They may perhaps welcome a book which shall deal with their favorite sport, and shall touch on it as practiced in different parts of the country. No two men use the terms shooting and hunting in the same way, and if the question could be submitted to a general conference of sportsmen, wide differences of opinion as to what constitutes a game bird would of course be found to exist. In certain sections of the country, and among certain classes of people, bluejays and woodpeckers are regarded as legitimate objects of the gunner’s pursuit; if they are less highly esteemed than quail and prairie chicken, it is only because they weigh less. For the purposes of this book I have considered as game birds only the species that are commonly hunted with dogs. This naturally throws out of the list many birds which offer good sport and are excellent for the table. Such are many of the plovers and beach birds, the Bartramian sandpiper, and all the rails. PREFACE In the preparation of the volume I have not hesi- tated to draw for information on all available sources, Vili Vili PREFACE and I have striven to bring together much material of interest to the sportsman which is now scattered through many volumes and periodicals, and is thus not easily accessible. The plan of the book does not differ essentially from that of “American Duck Shooting.” Its first part is devoted to descriptions of the birds and their hab- its, the second part to the various methods employed in taking the birds and the aids to shooting—dogs, guns and ammunition; while in the third is discussed the shooting of the future. I have received at several hands valuable assistance, which I desire here to acknowledge. Dr. A. K. Fisher, of the Biological Survey of Washington, a keen sports- man and experienced ornithologist, kindly read over the manuscript. Mr. B. Waters, distinguished as a crack shot, and especially learned in the hunting dog, is chiefly responsible for the chapters especially de- voted to dog and gun. To the kindness of Mrs. Ver- non Bailey and of the Houghton, Mifflin Co. I owe the excellent plates of the scaled quail and Mearns’ quail, taken from her “Handbook of Birds of the Western United States.” Messrs. Little, Brown & Company give me permission to reproduce several cuts from Baird, Brewer and Ridgway’s “North American Birds.” The Forest and Stream Publishing Company 1X PREFACE has allowed me to enrich the book with a wealth of illustrations not always to be had in such a volume. The colored plates of the ruffed grouse and Virginia quail, so admirably depicted by Mr. Louis Agassiz Fuertes, I use by the kind permission of the Biological Survey of the Department of Agriculture, who also let me have the excellent figures of the sage grouse and of Gambel’s quail. Dr. C. F. Hodge, of Clark Um- versity, Worcester, Mass, has furnished me with a number of beautiful photographs of his ruffed grouse and bobwhites. Sportsmen and ornithologists will find here much that is familiar, but, I hope, also something that ts new. Gro. BirD GRINNELL. Gro. BirD GRINNELL. New York, October, r9Io. New York, October, r9Io. CONTENTS PAGE |S LR CIEE) B72 Gi bs WISNER tT ta at Re eg NEO vii J LutFO) 33 YB I jf Coe BU has I AR zs RS Pa Xvii AMERICAN GAME BIRDS PAGE rE CG HEN 2) 1 2 eR Sd 0 2d DS a ee aN er I NVO OSC OC Aon isk oatca any iMrn Me dN se er RAM cute Len hte 6 ACMUERICANIN SNEP EG rele uate insen Usa eis eui sat Be aaa Maa lly 30 ICIS RNIN GC EO) WS PB URIS ese Wee iced ac am eile tei Wala acletenesg 40 AE AINE REC AING GUA Doe ot inltaee dagtekince wk ecrennls 47 OVATE VE OBWHIEN SPAR TRIDGE Ren cmceeenioatineie 49 INEASIKE DS OB IWATE Reema eietn ceeacstatoleval al spevaeelay aro ceieie 61 VIO WA TING © UATE erctetaattrerverercinoheoterere aleve ote alert tateve eis 76 SCALED MO WATE prsercperctenraieia svertalee custavercncieresicleotcten tel ore 81 CALIFORNIA OUATEY VALLEY (OUATIe Joc 'sa clon ote sere 85 GAMBERIS) ©OUADET pis celc alee ical cleseminblorale sl airetece QI INVASSENAG ORIN EBARINS 1) OAT EN weieealocialvelelitoielee sree 100 SEE ACN i RUC AIN TI GiIRI@ IS Bek ior salsieieclstelarte cee sishe 114 DUSKY GROUSE Se iieise a cottctee Se ole ds ale elele sel atcteslete II5 CANADA GROUSE, SPRUCE PARTRIDGE........-0e2005 125 I RANIKDENGS «GROUSE c 24 sssis poireieecersieleleler'e toe ele are! oles 135 RUPPED GROUSE; PARTRIDGEvs secs os scicisiieiccic clewlolois 139 X1 CONTENTS PAGE PAGE THE AMERICAN GROUSE—(Continued). WALLOW! PTARMIGAN! oo. 0c)c cissica ree ine eee ae 168 ROCK /PTARMIGANG ..0./5iiscrelcclontos iets Sarcoma eens er 187 WHITE-TAILED -PTARMIGAN.<cc cite neenneesehenaieee 193 PINNATED: ‘GROUSE. isis cos te ee ee ee ne 206 SHARP-TAILED -GROUSERHonasceeaeee rete ntene 251 SAGE ~ GROUSE su. dont a Soctig clement ea 272 UPLAND SHOOTING PAGE UPLAND SHOOTING 2.6 2. 2 ee Cee ee eee 303 WOODCOCK SHOOTING b.c.sccke aoe ceeaiee hee eee 305 SNIPE) ‘SHOOTING serwseisle cies Seat oa lee helene eee ee oe 319 QUAIL SHOOTING 4h.:cclenne: ciclis le eee eee 332 BOBWHITE sacs Soa on eases Secu ols Se ee Ee 332 VALLEY (QUATED 2a Se cetee sion tennis enein Goer: 350 GAMBEL’S ‘OUAIL wos e on deen sake cere le 352 MOUNTAIN (OUATL 2 Goce tebe ene Ge ee ae er 353 SCALED (OUATE. nine Se teks c Manin Oe Lee 354 MEARNS’ “OUAIU. (cic taro lncnten teed eee eee 357 PAGE UPLAND SHOOTING 2.6 2. 2 ee Cee ee eee 303 WOODCOCK SHOOTING b.c.sccke aoe ceeaiee hee eee 305 SNIPE) ‘SHOOTING serwseisle cies Seat oa lee helene eee ee oe 319 QUAIL SHOOTING 4h.:cclenne: ciclis le eee eee 332 BOBWHITE sacs Soa on eases Secu ols Se ee Ee 332 VALLEY (QUATED 2a Se cetee sion tennis enein Goer: 350 GAMBEL’S ‘OUAIL wos e on deen sake cere le 352 MOUNTAIN (OUATL 2 Goce tebe ene Ge ee ae er 353 SCALED (OUATE. nine Se teks c Manin Oe Lee 354 MEARNS’ “OUAIU. (cic taro lncnten teed eee eee 357 SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350 RUPFED }\GROUSE® aici feidic ceneere stein aes SR oe eee 359 DUSKY! GROUSE. se iic cine seven ines a hee He eee 384 ELARIWIGAN: SHOOTIN Giacccucessingnnone senor eee 302 WIELO Wi PPAR MIGAN(. cro o:5 <teicitea’s S alata seine oo ocelot 392 SHOOTING THE PRATRIE GROUSE.¢.ns teen 308 PINNATED (GROUSE. : vistscde cle oo. o6 eo aoeieea ec eee eee 309 SHARP-TATLED GROUSE.0 «ss 04 cans ea eloe ee ee eee 412 SAGE GROUSE, os.05.0.4 voen'se bc cei» ce tbiceec eel Eeeenes 416 SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350 RUPFED }\GROUSE® aici feidic ceneere stein aes SR oe eee 359 DUSKY! GROUSE. se iic cine seven ines a hee He eee 384 SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350 RUPFED }\GROUSE® aici feidic ceneere stein aes SR oe eee 359 DUSKY! GROUSE. se iic cine seven ines a hee He eee 384 SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350 RUPFED }\GROUSE® aici feidic ceneere stein aes SR oe eee 359 DUSKY! GROUSE. se iic cine seven ines a hee He eee 384 ELARIWIGAN: SHOOTIN Giacccucessingnnone senor eee 302 WIELO Wi PPAR MIGAN(. cro o:5 <teicitea’s S alata seine oo ocelot 392 SHOOTING THE PRATRIE GROUSE.¢.ns teen 308 PINNATED (GROUSE. : vistscde cle oo. o6 eo aoeieea ec eee eee 309 SHARP-TATLED GROUSE.0 «ss 04 cans ea eloe ee ee eee 412 SAGE GROUSE, os.05.0.4 voen'se bc cei» ce tbiceec eel Eeeenes 416 CONTENTS PAGE AON ATEST MOY ON TUM CUR RN A Sei ere en a 422 ED SLOP SOO PUNG sh twee een le 437 SPOMEENG aera age ey sect enrc te eee tiene MALIN), oe. 457 GONSPANDIBORDS. Bitches sak cect. sera ua) Roky 462 DISVe S ac ti SSRIS Sirsa ans Sun anna ies aha) pea 470 THE SHOOTING OF THE FUTURE PAGE PAGE RLOOKSDAGKWARD 2. Mh bike kit! Avtar MANES Ua SII TRANSPLANTING AMERICAN GAME BIRDS................... 527 DESTRUCTION AND PROTECTION.<..2/0)-2-0.00005,..00000 6. 536 ONCLUSION ne es tees Gein te cle ih eee De 555 Xili Xili PART TO FACE PAGE Rurrep Grouse, (Colored: Plate):..<..... 6-5. Frontispiece WIG ODCOCKHON WNIEST Ys ier:dioidssieici els ls 010) oleleusisliars evekelers slelevarclsrateterare Nest OF WOODCOCK......... 4 Nevahs\4 sajtettacelo oualaovtane arene retaliate eliaiske Nest oF WILSON’S SNIPE..... Bi, Mista ave Whotala ctalateleaisietetete thetote MouNTATN OMA asceescen sidtcsouetordcaveteral e toredetelele le ener ener hore ae SCALED) WATE eerie AOU GNOC TaC silavekaelecgialeynens CAVIRORNEAW@UAME Spine aeiel andisib.a seleuel sis) otTAtetensiohs avelonsvelaretele tetera NEST OF CALIFORNIA QUAIL....... svavsliermveysholelaievers clerclelettus orevere GAMBEL S@OUADR ieee acca slate Vel sterereness eet tosobeteke sie MassENA OR MEARNS’ QUAIL....... SHAS HS CULO MEMES STERRAVOUSKY, GROUSE AND) MOUNGesiaciiee cilia cleiclterecieiectel: DD WSK is GROUSEN. Gies ciors eaere ers sveonreeiae =e eresetoreve craieta mete e CANADA Grouse. (After Radubonts s Plate). Bet Ser beeders faveilors terre STRUTTING RUEFED) GROUSES {aspen en cae Wialelevmrere Sieeronsete Sodtee THE DRUMMING GROUSE..........-. Yale ih lotactsterebane Eis eiores NESTOR IRUREED. GROUSE a domeuleoceercten chic neas Sieseverteen DAY=DREAMING oie scnicelereres ARS acess Syole evaretetesats ab. etelleis wets RURRED) GROUSETON INIEST Oe aarmeiecietemteisieiinel sete stoveteealnereuays WTLLOWJE TAR MiIGAN «DN IWAUGAS KeNuatnetslstelelctelere e eferele ssehionmeraens WHITE-TAILED PTARMIGAN.. Veal Sravdce aliens aves sieve re isteieternee PINNATED GROUSE AND Ween Ree aia vis) arate Sa tg calle leuctaverere SHARP-TAILED GROUSE. (ined einen si Blate) Nevetrs WVGTAD oR OKIE Yi ARUN IG eraleteteteteretotele toisiare aie’ ototeloreterete lavereternetete 15 27 Xiv LIST OF ILLUSTRATIONS PAGE PART 1 TO FACE PAGE BOBWHITE IN Potato Fietp. (Colored Plate)............. 305 ViooncocK) CROUGHING:.:. 5.5. 01s. Thais Ua ein eens Serereralehelsteleey [307 SETTER POINTING SNIPE....... WE SieYanata chaleteters PAIOSIOC OR ane 325 ONPEROIND las cisjelee alos aratacelavegieashas eicic ale alerts aeerericisioe ey Gat SHO OMMN GUNES SEA TNIE Sh cpl sale terete valsis seve term: oraterels eis raleteicreveln oars 345 SETTER POINTING, WITH BIRD IN MOUTH........... Naletelewter GOL RUFFED GROUSE Cock, REARED IN DOMESTICATION........ 377 DUSiveE GROUSE RON! DHE IVLOUNDATNS suericisiecinisiecis civic celelsicierl, S07, WVitireTe OVW WAR MOKA Sa tet ctceclel teevetel ene ia fie Mislenatarstes sewies she gOS PRAIRIE CHICKEN SHOOTING IN KANSAS........ Rede ae etrelisy OS POINTERS HAST ONY GHICKENGEayelcicisin ss icicee ticle caletere ot oretere ae ALT WILD TURKEYS IN THE PINES..... clare oleracea se setae AS SELTER STANDING A RUPFED (GROUSE...) -2 ues ee oe Ayes Bate pAzaL DELIVERING YEE) tESURD). fel cte lo pias’ s cle loicieislotioe 6. etejole FR taal ela 487 SHOOTING IN Mississippi. (Painting by Edmund Osthaus). 503 PART, IIL TO FACE PAGE BonwaHrre Carck,/TBREE WEEKS ‘ORDs)i« wos syigce scioeresiee « S11 EUROPEAN OWADI soit cise tieieleas is 4 ele ays ae oreitvtetatrake Wiel sts AAA ace Salis asRi Dusky (GROUSE-PHEASANT:.. <4 iecesc.elsinee's ss cies eens 519 EUROPEAN PARTRIDGE... ace stole ele Siatuvavincatal Clabes cetera petals 521 DoOURE ET EVANDEDY OF) OUATE We sjesmele acre Hae vel be evotetel ey bas 537 WINTER QUARTERS OF Dr. HopGE’s QUAIL........ susthalstete 539 RUFFED GROUSE CHIcKs Just HATCHED....... Sr eae sea calcite 541 BEEDING BOBWHITE) HICKS... ...05+0.+-<- Bieta etepeteheaheienve teks 545 BOBWHITE REARED BY Dr. C. F. Hopce..... syotevertiektehentetens 547 RUFFED GROUSE FEEDING FROM HAND.).. csicciccccssescness SSE XV XV LINE (‘CUTS IN TEx PAGE RANGE OF THE WOODCOCK: | (Map): .cuscciee aniline cee ig BILe, Foor, TAIL. AND WING OF BOBWHITEs. + -2o2ceeccoee en ho BIEL AND) FOOT. OF: MOUNTAIN: ©UATI pce ced cree sericiice hike enya BIELPAND) HOOT OF SCALED OUATE Ann eae Wave oe este MO? Bic AND FooT OF CALIFORNIA QUAIL... .105-> cemeuniinsc GOO Bier, Foor AND’ WING OF MEARNS’ QUAIL. secs cemeene cee 07, BiLit, Foot, Tart AnD W1NG oF Dusky GROUSE.......... 116 WAIL OF, CANADA, (GROUSE. jee ceeelececetiat ees eee eee cO ‘(AIC OF FRANKLIN'S, GROUSE! cheese ccc dee ee eee ee Briuxi, Foot, Tain anD WING OF RUFFED GROUSE.......--. 140 BILL, Foot, Tart AND WING OF WHITE-TAILED PTARMIGAN. 104 Bit, Foot, TaAiL AND WING OF PINNATED GROUSE..... 568 Alay) BiLt, Foot, TArL AND WING OF SHARP-TAILED GROUSE.... 252 Briiy, Foot, Par AND WING OF SAGE GROUSE..-2e6s)eeee) 273 Biti, Foot, TAIL AND WING OF WILD TURKEY............ 283 XVi INTRODUCTION The astonishingly rapid increase of population in the United States has resulted in an equally startling decrease in America’s larger fauna, especially in those mammals, birds and fishes which are useful for food. The story of the extermination of large animals over vast areas is familiar to all, and men not yet beyond middle life have themselves seen the extermination of food birds over much of the country east of the Mis- sissipp1 and north of the Ohio River. Even young men can remember when the prairie chickens abounded in Iowa, Minnesota and Nebraska, in regions where now there are but few. As the game became more scarce the importance of preserving it began gradually to be appreciated. Yet in a thickly settled region it is very difficult to effi- ciently protect the game. Obviously, the best way to accomplish this is to interest the general public in it, to point out the economic value of the game birds, and to secure for the authorities, whose work it is to enforce the laws, the backing of public opinion in be- half of those laws. XVii INTRODUCTION The number of people who are trying to accom- plish something in this good work in America is greater now than ever before, and the promise of good results is brighter than it has been. On the other hand, in many sections the stock of native birds is pitifully reduced, so that while once the only per- sons who used the shotgun were the field shooters, to-day the number of men who shoot afield is small by comparison with those who use the shotgun at clay targets thrown into the air from a trap. The good sportsman—good in the sense of being devoted to the ethics of sport, and in the sense of being successful in his pursuit of game—wishes to know all he can about the life ways of the beautiful birds that he seeks for, and in getting together the material for this book I have endeavored to keep this fact con- stantly in mind. XVili Limicole. The great order Limicole stands between the galli- naceous birds on the one hand, and the herons, cranes, rails, and their allies on the other. This order, known as shore birds, or the plover-snipe group, is a large and cosmopolitan order. The species are usually of small size and the legs are long, as is usually—but not always —the bill. In certain of its characters the skeleton resembles those of the gulls and auks. The birds live in open places on the ground and usually near water or in moist situations—though to this rule there are marked exceptions. The young leave the nest and run about as soon as hatched; in other words, as the natur- alists say, they are pracocial. The eggs are few in number, averaging about four. The food is chiefly insects, worms, small shellfish or other soft animals which are found in sand or mud. The wings as a rule are long, flat and pointed, but are sometimes rounded. The tail is usually short. The head is almost always I I I AMERICAN GAME BIRD SHOOTING 2 covered with feathers and in some cases there is a dis- tinct crest. The bill varies greatly, from short in some species to long in others. In one species it turns up, in another is bent to one side, often it is curved downward, and sometimes it is oddly expanded at the end, or it may be compressed to a knife shape. Usually it is covered with a soft skin, is often much swollen and in some cases is a very sensitive organ of touch. The nostrils open above ina groove. They are never feathered. The sexes are usually alike, though they differ in the phalaropes, the jacana and the European ruff. The breeding dress is often quite different from that of the autumn, and some species are extremely showy in their spring plumage. Within this order are found two great families that are peculiarly familiar to American gunners. These are the Charadriude, or plover family, with a dozen or fifteen species, and the Scolopacide, or snipe family, which contains more than forty species, and in which are found the only two here to be considered. Besides these two great families, there are other smaller ones, which include the oyster catchers and turnstones, the avocet and stilt, and the phalaropes. Limicole. Not very distantly related to this group is the bustard Not very distantly related to this group is the bustard family of the Old World, some of which reach a size near that of the American wild turkey. This bustard family, according to Coues, perfectly connects the shore birds with the Alectorides, or cranes, rails and their allies. SNIPE-LIKE BIRDS 3 The only birds that we have to consider in this con- nection belong to the snipe family, which may be sep- arated into several groups. All snipe-like birds have long bills usually covered with a sensitive skin, which is soft throughout and furnishes to the bird a useful organ of touch. While the bill is long, it is never wide —though in one or two cases expanded at the tip; the nostrils are short, narrow slits. The toes are usually four, though in two or three cases there are but three. Usually they are separated, but in a few species they are palmated or semi-palmated. The neck and legs are usu- ally long and the legs seldom feathered down to the tarsal joint. Most of the birds belonging to this family are of small size, but occasionally, as in the curlews, the birds are as large as a small domestic fowl. Snipe and sandpipers are exceedingly gregarious, Snipe and sandpipers are exceedingly gregarious, traveling in flocks whose numbers can hardly be counted. This makes them especially subject to danger from gunners, and the birds being gentle and unsus- picious may often return to the decoys over and over again after being shot at. Over-shooting and lack of enforced protection has almost put an end to the shore- bird shooting on a great part of the Atlantic coast. build in or near marshy Snipe-like birds usually build in or near marshy places or by water, and as a rule lay four eggs. The voice is a shrill, but often sweet, whistle, readily imi- tated and used to lure the birds to decoys. Ornitholo- gists state that there are about ninety well-marked species of these birds, which are divided into five groups, the first containing the woodcock and snipe, the AMERICAN GAME BIRD SHOOTING 4 second the godwits, the third the sandpipers, the fourth the tattlers, and the fifth the curlews. Limicole. In the woodcock and true snipe the eye is placed high In the woodcock and true snipe the eye is placed high up on the head, so that the ear is below it, and, as Dr. Coues says, if the brain be examined it will be found curiously tilted over so that its anatomical base looks forward. The bill is straight and is much longer than the head. It is deeply grooved almost to its very end, where it is conspicuously swollen and soft. The soft, sensitive covering of the bony bill is abundantly supplied with nerves, and the bill is an instrument of touch by which the bird feels in the soft earth where it feeds for the food which it desires. Not only is the bill used as a direct probe, but it is capable of being somewhat bent in one way or another. Any one who has ever compared the bill of the dried skin of a woodcock or a snipe with that of a fresh specimen has probably noticed that the dried bill is much shrunken and is also pitted, showing where the more or less thin skin which covers the bill has shrunken into the pits in its bony substance. Woodcock and the snipe differ from many of their al- Woodcock and the snipe differ from many of their al- lies in not being gregarious. They do not gather in great flocks and so cannot be destroyed in immense numbers. On the other hand, they are very simple, gentle birds, and the woodcock living in cover is easily shot, though the snipe is better able to care for himself. The godwits are large birds with long, grooved bills The godwits are large birds with long, grooved bills slightly turned up instead of down. They frequent wet meadows and marshes as well as bays and estuaries, and SNIPE-LIKE BIRDS iE are as truly bay and shore birds as are the sandpipers. There are not many species. The sandpipers are a large group, and among them are some species with peculiar bills. The bill is soft and sensitive, though less so than in the two previous groups. All the sandpipers are extremely gregarious, and while they vary somewhat in size, as a rule they are small birds. The tattlers, of. Limicole. which the winter and the summer yel- lowleg and willet are familiar examples, are larger than the sandpipers and longer legged. In these birds the bill is less sensitive than in the sandpipers. They are not less gregarious than the previous group, and are noted for their restlessness and for the noise they make, whence their common name. It is a large group. T The curlews are distinguished by a down-turned sen- sitive bill of great length, and in most cases by their large size. These also are noisy birds and remind one somewhat of the godwits, which nearly equal some of the curlews in size. Both godwits and curlews are found all over the world. The flesh of all the The flesh of all these birds is usually very delicious, though of course varying in excellence with the foods of the species. Some of these which feed along the shore and largely on shellfish are not particularly well flavored, while the woodcock and snipe are among the most delicate of our game birds. Philohela minor Philohela minor The American woodcock is a small bird, weighing only from five to nine ounces. He is eleven or twelve inches in length, and of this the bill occupies from two and one-half to three inches. The plumage below is rich russet-brown, paling, on the upper breast, sides of the neck and forehead, to ashen-gray. The crown is black, with two or three crosslines of tawny, and the back is curiously mottled with tawny, ash-gray and black, the latter predominating. The tail feathers are black, barred with tawny, their tips smoky-gray on the upper side and snow-white beneath. The legs and feet are pale flesh-color, the bill dark horn-color at the tip, becoming paler at the base, and the large, soft, humid eyes are brown. The range of the woodcock is from Canada south to the limits of the United States and west to beyond the Mississippi River, but the high, dry plains of the trans-Missouri region limit the extension of his range westward, for he is a bird that loves moisture and cool, dark thickets. Huis range is shown on the accompany- ing little map taken from Dr. Fisher’s article, in the Year Book of the Department of Agriculture for Igo1. 6 6 WOODCOCK 7 The English woodcock, so called—by which is meant the woodcock of Europe—is twice the bulk of the American bird, and though in a general way sim- ilar in color, the pattern is so different that the two could never be confused, even though they were of the same size. This European bird has occasionally been RANGE OF THE WOODCOCK RANGE OF THE WOODCOCK taken in America. Any sportsman who may chance to kill a woodcock twice the size of the ordinary bird, paler in color and distinctly cross-barred, should by all means have it at once examined by a naturalist; and if unable to reach a naturalist, he should have the skin preserved, and should report the occurrence. Many of the woodcock pursue their winter journe Many of the woodcock pursue their winter journeys as far south as the Gulf of Mexico, while others winter much farther to the north. Philohela minor In fact, it is not altogether AMERICAN GAME BIRD SHOOTING 8 unexampled for them to winter as far north as Con- necticut, provided they can have a certain amount of shelter and the weather is mild enough, so that their feeding grounds are not sealed up by the frost. On the other hand, the birds which do not go farthest to the southward may occasionally suffer by unexpected and severe freezes, such as took place in February, 1899, to be spoken of further on. Wherever it goes, the woodcock starts early for its summer home and is often found breeding in New Jersey in the month of March. The woodcock is thus one of the early spring mi- grants, and, soon after his arrival, which is usually in March or early April, according to the weather, he makes his presence known, to those who understand where and when to listen for him, by the curious night song with which he wooes his mate. This song has been interestingly described by Walter H. Rich in the Journal of the Maine Ornithological Society for June, 1910. He says: “As we walked along in the dusk, . . . my com- panion called my attention to a bird note which was new to him. I listened a moment, and then said, ‘The first night hawk, I think.’ An instant later I doubted, and at once my doubt was confirmed when there sounded again the nasal p-a-a-nck! p-a-a-nck! of a woodcock. Soon we heard him whistle somewhere on wing, and presently he appeared flying about, high up under a particularly bright star, where he sang and trilled and twittered. I do not remember having ever WOODCOCK 9 before heard a woodcock make so much, so varied and so good music. It was a veritable song, and many a so-called songbird does not possess such a repertoire as did he. “The night was clear and cloudless, with no moon, but bright with starlight, so that there was no difficulty in following every development. 1 think he went through his performance at least a dozen times, with intervals of rest between of not more than a minute. On each occasion he came to earth near by us, always within fifty yards, and nearly always within thirty feet, seeming to have some curiosity as to our presence and intentions, but showing very little fear. Philohela minor In one in- stance he had almost settled upon the ground, within three feet of me, before noticing my motionless figure; either that, or, as may be possible, he was examining his disturbers at close range, for, flying first close to my companion, and making a half circle about his head, he approached me, poised an instant within hand reach, and then came to earth not over ten feet away. “When I approached him as he rested upon the ground he made a peculiar guttural note, which I can- not describe better than to say that it was like the faint, muffled b-o-o-mp of a bullfrog. I stood quietly wait- ing until he should resume, which he presently did, jerking out, with much bobbing of head and twitching of tail, a series of nasal, rasping quancks! After a little he whistled away again, circled, twittered, sang, and dropped to earth. He must have made eight or AMERICAN GAME BIRD SHOOTING Io ten flights before I left him, and from all indications was ready to make as many more. “A week later a party of four of us made an excur- “A week later a party of four of us made an excur- sion into the cover to listen to my twilight musician, and, though the wind was high and conditions seemed somewhat unfavorable, we were treated to a very fine exhibition of his powers. “This evening there were two birds present, and at “This evening there were two birds present, and at times we had both in the air together. Once there came a sudden whistling of wings, and we saw one bird chase the other out of this territory. Pursuer and pur- sued were very plainly visible as they darted and twisted over the tops of the pines on the western bor- der of the cover. Presently one returned and resumed the serenade. I wondered whether this performance was a part of the courtship, or whether my little friend was driving an interloper away from his lady’s case- ment. If the latter was the case, no serious harm seems to have resulted from the combat, if such it was, since we heard what was probably the intruder performing a little distance away a short time after. “One of my companions carefully timed several of “One of my companions carefully timed several of the flights. ~%*Some years ago I contributed to the Century Magazine arti- cles on the woodcock and the snipe, from which, by the cour- tesy of the Century Company, I am permitted to make extracts for the present chapters on those birds, Philohela minor They ranged from forty-five to fifty-five seconds in duration, divided approximately as follows: first, a period of whistling, circling flight while the bird mounted to his proper height—anywhere between sixty and one hundred yards, as nearly as I could esti- mate it in the uncertain light—lasting about thirty-five seconds; second, another period of short but somewhat WOODCOCK DE varying extent, when he piped a few preparatory notes at little intervals before starting into his full tide of song; third, the song itself, filling perhaps fifteen sec- onds; and finally, the dart to earth, with wings half closed, made in silence. The intervals between flights ranged between thirty and sixty seconds. “The song notes were as clear and liquid as a bobo- “The song notes were as clear and liquid as a bobo- link’s, and fully as musical. During the production of these the whistle of the wings could be plainly heard as a sort of accompaniment to the vocalism.” I think no such good description of the woodcock's song has before been given. Many observers mention only a single note, somewhat like the ordinary call of the night hawk, which he continues for some time, and then suddenly pitches downward from his height, and drops into cover. Here the female is waiting for him, and about her he struts, with head thrown back, wings trailing and tail spread, a parody on the turkey cock of the farmyard.* The nest is a rude structure of dead leaves and grass, and is usually placed under a fence, or by a log in some thick swamp, or perhaps on a tussock or bit of high ground in an alder run. The eggs are generally four in number, and are of a dull cream color, marked with large spots of bright or dull brown. In No. 2 of Vol IX, of the Bulletin of the Buffalo Society of Natural Sciences, are found “Notes on AMERICAN GAME BIRD SHOOTING 12 the American Woodcock,” by Ottomar Reinecke, of Buffalo, IN. Y: Mr. Reinecke—as have many other people familiar with the bird—points out that it is one of the earliest of the birds to nest. Sometimes this early nesting is followed by unhappy consequences, as in cases which took place in the spring of 1904. The author says: “Tn our locality the 13th of April is usually the time to look for full sets of woodcock’s eggs. Philohela minor That year was no exception to the rule, although at the time re- ferred to we had been visited by a heavy snowfall, which covered the ground to the depth of eight inches. The snow had been partially melted by the sun, but was frozen hard during the ensuing night. Two days later another snow storm occurred. The next morn- ing found us on the ground, which is an ideal locality for the nesting of this bird. The ground was then covered with about five inches of crusted snow. In company with several friends we immediately began our search for the woodcock. We soon found the tracks of a pair where they had been feeding or try- ing to find food around partly frozen water holes. “We flushed the birds and began looking the snow- covered ground over carefully for the nest. We found none in that vicinity, but following the tracks of a single bird, which evidently were made the previous day, we came to the spot at least a quarter of a mile distant where the bird had started on its walk to the water holes. On examining the place nothing but a WOODCOCK 13 slight depression was found, appearing somewhat dirtier than the otherwise clean snow. “We were beginning to think that the bird had taken “We were beginning to think that the bird had taken this place in a small clump of bushes as a refuge from the snow storms, when it was suggested that perhaps after all the nest might be under the hard frozen snow. This was no sooner suggested than we were down on our knees, taking turns at melting the snow with our breath. After a few minutes we were rewarded by the sight of one egg, and, continuing our efforts, we found a fine set of four eggs, the first lying directly in the middle and on top of the three other eggs and being separated from them by a layer of at least an inch of snow. From this I take it that when the first snow storm came, the bird had laid only three eggs and found difficulty in keeping on them for any great length of time. Philohela minor The woodcock lives on worms from soft, marshy ground, and, consequently, she could not remain a long time on the eggs, or, possibly, the large amount of falling snow made the bird con- tinually shift her position until, by this constant mov- ing, the eggs were covered with snow; then the next day the other egg was deposited as before mentioned. After this the second snowfall occurred, which the bird could not withstand, and so finally deserted nest and eggs. We found several more nests in the same way on that day, and in each case the eggs were found as described.” Mr. Reinecke records the finding in April, 1901, of Mr. Reinecke records the finding in April, 1901, of five eggs in a woodcock’s nest on which the bird was AMERICAN GAME BIRD SHOOTING 14 sketched and from which she was afterward fright- ened. The number is very unusual, if not previously unexampled. As soon as the young emerge from the egg, they leave the nest and follow the mother. Thencefor- ward their development is rapid, and young birds have been found well able to fly by April 10. Two broods are usually reared in the Middle States. A curious habit of the woodcock, which, though well attested, is as yet but little understood, is its practice of carry- ing its young from place to place, apparently to avoid danger. Exactly how the mother bird does this is not certainly known, but the weight of evidence seems to show that she holds it clasped between her thighs, as a rider does his horse, and does not carry it in her weak and slender claws. She will sometimes thus transport her young for a hundred yards or more, and if pursued will even make a second flight with it. By the last of July, in favorable seasons, the young of the second hatching are quite fit to look out for themselves, and early in August the woodcock disap- pear—that is to say, can no longer be found by those who search for them. They retire to the dry hillsides among the heavy undergrowth, and remain there until the moult is complete. From such places—often among thick growths of hazel or witch-hazel—they may some- times be flushed by the ornithologist who is searching for early migrants. Philohela minor In September they collect once more in their accustomed haunts, and then are fat, in good plumage, and fit for the gun. As soon as the young emerge from the egg, they leave the nest and follow the mother. Thencefor- ward their development is rapid, and young birds have been found well able to fly by April 10. Two broods are usually reared in the Middle States. A curious habit of the woodcock, which, though well attested, is as yet but little understood, is its practice of carry- ing its young from place to place, apparently to avoid danger. Exactly how the mother bird does this is not certainly known, but the weight of evidence seems to show that she holds it clasped between her thighs, as a rider does his horse, and does not carry it in her weak and slender claws. She will sometimes thus transport her young for a hundred yards or more, and if pursued will even make a second flight with it. By the last of July, in favorable seasons, the young By the last of July, in favorable seasons, the young of the second hatching are quite fit to look out for themselves, and early in August the woodcock disap- pear—that is to say, can no longer be found by those who search for them. They retire to the dry hillsides among the heavy undergrowth, and remain there until the moult is complete. From such places—often among thick growths of hazel or witch-hazel—they may some- times be flushed by the ornithologist who is searching for early migrants. In September they collect once more in their accustomed haunts, and then are fat, in good plumage, and fit for the gun. *JS9U UO YIOIPOOM *J UO YIOIPOOM WOODCOCK Ti Twenty years ago there was much discussion as to the manner in which the startled woodcock produces the whistling sound usually heard as it springs from the ground. The ranks of sportsmen were divided into two factions, one of which held that the whistle was vocal, while the other was as firmly convinced that it was produced by the wings. Oddly enough, able ornithologists, who were also sportsmen, were divided on the question—and are probably still divided, for the matter has never been satisfactorily settled. Philohela minor Such distinguished men as William Brewster, of Cambridge, and the late Gur- don Trumbull, of Hartford, whose “Names and Por- traits of Birds Which Interest Gunners” will always be remembered, took opposite sides on this question and argued at length about it. The ever-increasing scarcity of the woodcock and consequent inability to observe it put an end to the discussion. Formerly it was legal all over the country to kill this Formerly it was legal all over the country to kill this species during the month of July, at which time many of the young were barely able to fly, and when, after a late spring, some of the mother birds were even still brooding the eggs of their second hatching. This practice was most pernicious and is no longer per- mitted in most States. The coming together in September of the birds which have been mysteriously hidden away, no one knows where, is often loosely spoken of as “‘the first flight’”—in other words, is regarded as the beginning of the southward migration. It is, however, nothing AMERICAN GAME BIRD SHOOTING 16 more than a collecting in favorite food localities of the home-bred birds—those which have spent the summer, or been reared, in the neighborhood. The first true migratory movement of the wood- The first true migratory movement of the wood- cock usually follows a sharp frost early in October. The birds are not gregarious, and for the most part move singly; though two, three, and even four have been seen flying together, and sometimes six or eight may be started in succession from a single small piece of cover. The migration is performed during the night, though in dull, cloudy weather there is some movement in the daytime. Their flight is low over the fields. This low flight is swift, and the birds are often killed by flying against telegraph wires, and some- times dash themselves against buildings. In New York and New Jersey the woodcock may almost be considered as resident, for in mild winters a few birds are to be found late in December and early in February. The bird does not seem especially to dread the cold, but the freezing up of the ground cuts off the supply of food, and so obliges it to move southward. Philohela minor Often, however, in the coldest weather, an old fat bird may be found about some warm spring hole, where the ground never freezes; and here, if undisturbed, it may remain all through the winter. The principal food of woodcock is the earth-worm, though they also devour many insects which are to be found in the damp situations which they affect, and have been seen to catch butterflies. The “angle- worm,” however, is the main reliance of this species, WOODCOCK. 17 and the number of these which a single bird will devour in a given time is astonishing. Audubon says that a woodcock will devour in a single night more than its own weight in worms, and some experiments on this point, made on a captive bird, entirely confirm the observations of the great naturalist. This speci- men was apparently a male, and weighed, at the time of its capture, five ounces. His cage was two feet long and one deep, and had been fitted up for him by covering the bottom with long, dry moss, except in one end,- where there was a box of wet earth, eight inches square and three deep. The bird was fed alto- gether on earth-worms, and these were buried, a few at a time, in the mud. From the first this woodcock manifested very little fear of man; and it was but a short time before he so well understood what the open- ing of his cage door meant, that at the approach of his owner he would run to his ‘‘feeding-ground” in anticipation of the meal. So eager was he that it was necessary to push him away to the other end of the cage while the worms were being buried. As soon as he was permitted he would run to the mud and “bore” for the worms. This was a very inter- esting proceeding. He would push the point of his bill into the earth at an angle of about sixty degrees, and by two or three deliberate thrusts bury it to the base. While doing this the left foot was slightly ad- vanced and the body somewhat inclined forward. When the bill was wholly buried, he stood for a few seconds perfectly still, as if listening. Philohela minor Perhaps he AMERICAN GAME BIRD SHOOTING 18 was doing so, but it seems more probable that he was waiting to see if he could perceive any movement in the earth near his bill. If none was felt, he would withdraw his probe and thrust it in again a little farther on. If, however, he detected any movement, the beak was hastily withdrawn, rapidly plunged in again in a slightly different direction, and the unfor- tunate worm was brought to the surface and devoured with evident satisfaction. When the supply of worms was exhausted the bird carefully cleansed the mud from his bill by means of his feet and, after giving himself a shake or two, retired to the farthest corner of his cage, buried his long beak among the feathers of his back and settled himself for a quiet after-dinner nap. Sometimes he would thrust his bill down among the moss once or twice, and then walking to his water dish would wash it by slowly moving his head from side to side. After he had been confined for over a month, the worms fed to the bird during twenty-four consecutive hours were counted and weighed, and he was found to have eaten two hundred worms, weigh- ing five and one-quarter ounces. At the end of this time he was still eager for food. Somewhat later he had increased one ounce in weight, and he now ate in twenty-four hours no less than eight ounces of worms. For nearly twenty years now the woodcock has been growing more and more scarce, and it has been generally accepted as true that it is on the way to ex- tinction. Not many years ago so distinguished a WOODCOCK 19 naturalist as Dr. A. K. Fisher published in one of the Year Books of the Department of Agriculture an ac- count of Two Vanishing Birds, one of which was the woodcock. Until within a few years summer shoot- ing of woodcock has been permitted in a number of States, a practice which resulted in the practical de- struction of almost all the birds breeding and reared in certain territories. So much shooting at a time when the birds were but recently from the nest and many of them able to fly only short distances, was extremely destructive. Philohela minor The birds were so gentle and at that season fly so easily that summer shooting was really a sport for children rather than for men. If kept up long enough, this would have resulted in If kept up long enough, this would have resulted in the extermination of the bird over considerable areas, or in such a reduction in its numbers that some natural change, which under ordinary circumstances might be wholly harmless, might almost wipe the bird out of existence. For many years far-seeing persons have recognized this danger and have been laboring faith- fully to have the practice of summer shooting stopped. It is only within a comparatively short time that their efforts have been successful. Besides this danger, to which the woodcock were Besides this danger, to which the woodcock were formerly exposed over much of the country, was an- other which destroyed them by wholesale, although such destruction came only at very long intervals. This was the occurrence of periods of severe cold in their southern wintering grounds. Though such cold periods do not come often, yet orange growers in AMERICAN GAME BIRD SHOOTING 20 Florida know to their cost that they do occur at in- tervals. Frosts which merely chill the ground may prevent the woodcock from feeding and so may force them to congregate at a few open spots or warm spring holes, where if found they can be readily killed, but severe colds long continued are exceedingly destructive. In February, 1899, such a cold wave swept over In February, 1899, such a cold wave swept over parts of the South, and its influence on bird life in general was so interesting that I need not apologize for quoting the gist of the observations made by Mr. Arthur T. Wayne, of Mt. Pleasant, S. C. I give this account* of the great cold wave of February 13 and 14, 1899, chiefly in his own words, omitting only the technical names of the various species: “The cold wave which struck the coast of South Carolina was the severest recorded for two hundred years. On Monday, February 13, the thermometer registered 14° above zero, with the ground covered with snow from four to five inches deep on a level, while drifts were two feet deep. This is a remarkable occurrence for the coast region and to be seen scarcely in a lifetime. *Auk, Vol. XVI, 1800, p. 197. Philohela minor On Tuesday, at 6:55 a.m., the ther- mometer registered 6° above zero. This exceedingly cold weather came upon us very suddenly. It was sleeting all day Sunday, February 12, but toward mid- night grew suddenly colder, and when morning dawned the whole country was covered with snow. The destruction of bird life caused by this cold wave WOODCOCK 2I can scarcely be conceived. To say that fox sparrows and snow birds were frozen to death by the millions is not an exaggerated statement, but a conservative one. There was a tremendous migration of fox spar- rows on Monday, February 13, following the coast line of the mainland, They apparently came from the northeast in a southwesterly direction. Thou- sands tarried in my yard all day long and swarmed on the piazza, fowl-yard and other places that would afford protection. They would scratch away the snow in order to find a bare place, singing—that is, the stronger birds—the whole time, while their compan- ions were freezing by the hundreds. While they were numbed by the intense cold, boat-tailed grackles and red-winged blackbirds would peck them at the base of the skull, killing them and eating them. The stronger fox sparrows would also eat their dead com- panions. It was a most pathetic sight. I caught quan- tities of fox sparrows, grass finches, snow birds and chipping sparrows and put them into a large cage, which I brought into the house and placed before a large fire, with the hope of saving them from destruc- tion ; but, despite this, they all died. Very few of these birds were emaciated, and the great majority were fat. “The “The woodcock arrived in countless thousands. Prior to their arrival I had seen but two birds the entire winter. They were everywhere and were com- pletely bewildered. Tens of thousands were killed by would-be sportsmen, and thousands were frozen to AMERICAN GAME BIRD SHOOTING 22 death. The great majority were so emaciated that they were practically feathers, and, of course, were unable to withstand the cold. One man killed two hundred pairs in a few hours. I shot a dozen birds. Late Tuesday afternoon I easily caught several birds on the snow and put them into a thawed spot on the edge of a swift-running stream in order that they would not perish, but upon going to the place next morning I found one frozen. Philohela minor These were fearfully emaciated and could scarcely fly. Two birds were killed in Charleston, in Broad Street. It will be many years before this fine bird can establish itself under the most favorable conditions.” Mr. Wayne gives a list of sixteen species which he found frozen to death, among them such hardy birds as the meadow lark and hermit thrush. He goes on to say: “Bluebirds and pine warblers were decimated. “Bluebirds and pine warblers were decimated. Mocking birds, cardinals, Florida towhees, Carolina wrens and all woodpeckers escaped.” It must take any species many years to recover from a wholesale sweeping off of its individuals, such as took place on this occasion, and if such a destruction of the woodcock took place all along a section of its winter home, as did in South Carolina, it is not strange that this species should have been regarded by naturalists as a vanishing bird. For many years in Louisiana, and possibly in other For many years in Louisiana, and possibly in other portions of the Southern States where the conditions WOODCOCK 23 are favorable, the woodcock are killed during their winter sojourn by firelighting. Audubon, in his “Ornithological Biographies,” says that in Louisiana the negroes commonly killed wood- cock at night by firelighting and striking the birds with a pole or long stick. A resident of Louisiana described in Forest and Stream one method of this destruction, by which enor- mous numbers must be killed. He says: “This bird is migratory in this country and does not breed here at all, yet our State has protected him. If the States of his nativity were to protect him entirely for a few years, in conjunction with the protection afforded by the Southern States, no doubt he would soon become abundant. “The birds are night feeders. Their favorite feed- “The birds are night feeders. Their favorite feed- ing grounds are old sedge fields burned off clean, and pasture lands that have been pastured closely, but they can also be found in the cotton, corn and cane fields. They must be hunted on ground that is fairly clean of weeds and grass, else they cannot be seen. “They are hunted with a torch. Philohela minor The primitive way was a pine torch in an old-fashioned fire basket with a long handle, extra fuel being carried in a sack, but the torch has been superseded by the kerosene lamp. A large lamp is placed in a tin frame with a glass front. The lamp has a strong reflector behind it and should illuminate the ground for fifty feet. It is carried by a man who does no shooting, suspended by a strap around his shoulders and about waist high. Walking AMERICAN GAME BIRD SHOOTING 24 through the fields, as the light is veered around so as to cover the ground within its range, the woodcock can be seen squatting in his feeding place. “The darker the night the better; a drizzly night “The darker the night the better; a drizzly night is the best of all. On starlight nights it is not easy to get close enough to kill them with a long cane, which the darkeys frequently use, or even to shoot them with squib loads, but on dark, drizzly nights one can almost catch them with the hands before they become accus- tomed to the light, which temporarily dazzles them. The birds are usually found in pairs—unless one has been killed—and squatting from three to six feet apart, and not infrequently, if the night be very dark, the hunter can kill both before flight. “A muzzleloader is preferred on account of the “A muzzleloader is preferred on account of the cheapness of the ammunition. The birds are rarely ever shot at more than thirty feet and frequently under ten feet. For this reason squib loads are used. An or- dinary charge of black powder is divided into two loads, a wad run down on it, and the charge of No. 8 shot is about what a man can hold between his thumb and fore finger, say fifteen or twenty pellets; more would tear the birds. “The woodcock rarely spends over sixty to seventy “The woodcock rarely spends over sixty to seventy days in Louisiana, but during this period many thou- sands were killed before the enactment of prohibitory laws, not only by the colored man, but by the whites as well, in the manner mentioned. Philohela minor No doubt your sportsmen critics will denounce the method, and, under any circumstances other than those which actually exist WOODCOCK: 25 in Louisiana, their denunciation would be just, but I opine if these same critics could be transported to Louisiana in the woodcock season they would be sur- prised to find in how short a time they would become lovers of the night pothunt. “T know from personal experience. I was born a “T know from personal experience. I was born a sportsman and I can recall, some thirty-five years ago, when I scorned to shoot a woodcock on the ground, but then I was new in the State. It did not take me long to get broken in to the method. The great deli- cacy of the bird and the almost impossibility of getting him by daylight hunting begets the habit of night pot- hunting, and, like other bad habits, it grows apace. “This section of the State is rather out of the wood- “This section of the State is rather out of the wood- cock country, and I have not hunted them for twenty- five years. They are here every year, but not plentiful enough to warrant night hunting, but well do I re- member a noted hunt of about thirty-five years ago. I was a visitor to Louisiana then. One dark, drizzly night my brother-in-law was lamp-carrier for me, and I killed seventy-two from 9 until I a.m. with a muzzle- loading gun. No, it was seventy-one that I killed with the gun, but when my ammunition became exhausted on the way home we found a bird on the side of the path. I drew the ramrod and killed it with a blow on the head, making an even six dozen. “T cannot refrain from telling of my last woodcock hunt. It was in January, 1885, just twenty-five years ago. On a starlight night three of us started out for a hunt, one gunner on each side of the light. The “T cannot refrain from telling of my last woodcock hunt. It was in January, 1885, just twenty-five years ago. On a starlight night three of us started out for a hunt, one gunner on each side of the light. The AMERICAN GAME BIRD SHOOTING 26 birds were plentiful, but were so wild that we could not get shots at half of those we found. Philohela minor The two of us killed fourteen. “The next night was bright also, and I suggested a new scheme. I, being a crack wing shot, was to load my gun with the ordinary bird load, while my com- panion was to use the squib; he to shoot them on the ground and I to shoot those on the wing when flushed. “T instructed the light-carrier that when a bird “T instructed the light-carrier that when a bird rose for flight he should keep the rays of light on him as he flew. This was easily done by tilting the lamp with the hands. The results of the hunt were twenty-nine; my companion killed fourteen and I killed fifteen, all flying. I do not now recall that I missed a single shot. Several times my companion, shooting at too long range for his squib load, missed, and I killed the bird in the air. But it was not re- markable shooting at all, for when the light was thrown on him he seemed practically to stop in the air. I could almost have killed them with a rifle.” The vast alluvial region of southern Louisiana has many large areas peculiarly favorable to woodcock life. Years ago, before the pothunter had effected so much destruction, the woodcock congregated in those regions in vast numbers in November and December. The heavy tropical rains, of whose volume and persistency the northern resident has little conception, softened vast areas of land, cultivated and uncultivated, and thus fitted them for the feeding grounds of the wood- “yYI0IP00 M JO JSON “yYI0IP00 M JO JSON WOODCOCK 27 cock. At the same season snipe abounded in enormous numbers, and for the same reason. Another casual, but purely local, agent of destruc- tion to the woodcock is the forest fires, which burn so frequently in many of the Eastern States and which run through groves or swamps where the woodcock have their nests. In such States as New York, New Jersey, Pennsylvania, Connecticut and Massachusetts, small brush or forest fires started by careless railroad engineers or thoughtless boys, occur in autumn and spring, and these may travel over considerable stretches of country, destroying great numbers of young seedling trees, burning through the leaves, dry underbrush and dead branches of swamps, and destroying the nests of quail, woodcock and ruffed grouse, and sometimes even injuring the birds themselves. Philohela minor These fires, while doing harm here and there, are not of regular occur- rence and work but little injury compared with those prairie fires which in old times used to sweep over the fertile States of the West, destroying the nests of the prairie chickens, leaving the country bare of food for them and often causing the farmer the loss of some of his haystacks, or even of some of his buildings. Yet the harm done by these eastern fires is a serious matter in a region where game birds are few. An area so burnt over is not likely to be occupied by woodcock for several years. The birds will not breed there, nor, in most cases, will they resort to such a burnt area for food or rest. The years 1908 and 1909 seem to have shown dur- The years 1908 and 1909 seem to have shown dur- AMERICAN GAME BIRD SHOOTING 28 ing the fall migration a greater number of woodcock than have been seen for many years. We have heard of bags being made of fifteen or twenty birds to a gun, and gunners have told us of starting thirty or forty woodcock in a single day. One gunner of great experience and knowledge of the habits of the birds and the particular sections to which they delight to resort, has told us that in the autumn of 1909 he started in a single day by actual count not less than fifty birds. The number that he and a companion killed was some- what less than one-third this number. They kept within the law of the State in which they were shoot- ing, but the gunner confessed that the temptation to overrun the limit was strong. The observations which have been carried on as to the present abundance or scarcity of woodcock are not sufficient to justify us in concluding that they are really increasing, but two or three more favorable seasons should make us hope that before long the birds will be back with us again in something like their old-time abundance. It is certain that of late years in parts of New Eng- land birds have bred where they have not been seen for many years before; and under present conditions, when woodcock are killed only in the autumn, they are usually fat, strong-winged birds, very different from the little cheepers of mid-July. Philohela minor This improved condition of things is no doubt due This improved condition of things is no doubt due to the adoption of proper game laws in the British Provinces, New England and the States north of Mary- WOODCOCK 29 land. In this large area, which includes a considerable portion of the summer range of the woodcock, shoot- ing lasts for from one to two and a half months, begin- ning from September 15 to November 1 in different States, and ending November 15 to January 1. The abolition of the barbarous practice of spring and sum- mer shooting and the shortening of the open season has resulted in this encouraging increase in the number of the birds. Gallinago delicata. The snipe is a small bird, not much more than one- half the weight of a woodcock. Its bill, which is dark brown and considerably swollen at the end, is more than twice as long as the head. The lower portion of the tibia is naked; in other words, the feathers do not come down to the tarsal joint, which they reach in most other birds, and perhaps in all land birds. The eyes are brown, and the feet are bluish or green- ish gray. The upper parts are generally black or blackish brown, divided lengthwise by streaks of brown and whitish. The chin is whitish and the neck reddish brown spotted with darker. The scapulars are streaked with reddish brown, as are also the wing coverts and secondaries. The tail feathers are blackish, with a broad band of brownish red near the end, and the tips white. The bird’s length is 10% inches, its extent of wings from 16 to 17, and the bill is over 2% inches long, and sometimes longer. As in the wood- cock, so in the snipe, the bill is a sensitive organ of touch. Allied to the woodcock, but wholly different from it in appearance, habits and home, is the American snipe, often wrongly called “English” snipe. Birds 30 30 30 AMERICAN SNIPE 31 of this genus are to be found all over the world, and almost everywhere their habits are more or less alike. During their migrations they frequent fresh meadows or wet ground, where the worms and insects which constitute their food are to be found. They are swift of wing, make long journeys, and, for the most part, breed well to the North. It is not, however, the cold which causes them to migrate, but rather the scarcity of food which precedes the advent of cold weather. Many of the snipe along the Atlantic coast migrate to a locality only just below the frost line. Often they may be seen at any time in the winter on the fresh meadows of Virginia and North Carolina, and if cold weather should come and a freeze take ‘place, they move on forty or fifty miles southward, to return again as soon as milder weather has softened their feeding grounds. The snipe goes southward to the Gulf of Mexico and beyond. Gallinago delicata. As worked out by the American Ornith- ologists’ Union, the range of the snipe is essentially as follows: North America and South America. Breeds from northwestern Alaska, northern Mackenzie, cen- tral Keewatin and northern Ungava, south to northern California, southern Colorado, northern Iowa, northern Hlinois, Pennsylvania and New Jersey. Winters from northern California, New Mexico, Arkansas and North Carolina, through Central America and West Indies to Columbia and southern Brazil; remains in winter casually and locally north to Washington and Mon- tana, Nebraska, Illinois and Nova Scotia. Accidental AMERICAN GAME BIRD SHOOTING 32 in Hawaii, Bermuda and Great Britain. Cold affects them little. My friend, Colonel W. D. Pickett, ob- served snipe wintering in Wyoming, where the mercury often went down to from twenty to thirty degrees below zero. Here, in warm springs that were never frozen, the birds remained and seemed to prosper all through the winter. In the same way mallard ducks winter in open water holes in the interior of Alaska. Usually the snipe reaches the middle and southern New England States early in the month of April, though this is a matter which depends largely on the weather. Here they loiter for some time, the greater part of them moving on farther to the North, where they breed. Nests have been found in New York State, and in the summer of 1908 I saw in August on the banks of the Housatonic River, in Connecticut, snipe which I believe had been hatched there. -Most of the snipe, however, go as far north as Canada; and New Brunswick, Nova Scotia and suita- ble localities just north of the boundary line between the United States and the British possessions are favorite breeding grounds. The nest is usually a hollow in the open marsh, where The nest is usually a hollow in the open marsh, where the moss or grass has been pushed aside and bent down to make a concavity which will hold the four eges. These, like those of many of this group, are sharply pointed—pyriform—and always lie in the nest with the four small ends together. They are grayish green or olive in color, thickly dotted, blotched and spotted with dark brown, the spots growing larger at s Snipe. ’ Nest of Wilson s Snipe. ’ Nest of Wilson AMERICAN SNIPE 33 the large end. Gallinago delicata. They measure more than an inch and a half in length by a little over an inch in breadth. When hatched, the young leave the nest at once. They are tiny little things covered with yellow and brown down. At this age the bill is short, and the young are unable to probe for food. Audubon says that at first they seem to feed on minute insects found on the surface of the mud, or amid grass and moss. It is possible that they do so, but probably no one knows very clearly just how they are nourished for the first few days of their lives; but as they grow older and the bill increases in length and strength, they begin to feed as do the old ones, and probe the mire. On their return flight the snipe make their appear- On their return flight the snipe make their appear- ance quite early and are often found on good ground in small numbers in late August or early September. If the weather has been very dry, so that the area soft enough to admit of their food being procured is contracted, snipe may often be found in considerable numbers on small wet places; but as the autumn flight takes place gradually and slowly, the birds moving on for short distances at a time, they are usually not found in any such numbers as sometimes occur in spring at the height of the migration, when some cold wave checks the onward advance. It is commonly believed that snipe spend all their time in wet and marshy places, and very likely they do so, when undisturbed. Once, however, many years ago, I visited a snipe ground near Vincennes, Ind., where the birds were astonishingly abundant. We AMERICAN GAME BIRD SHOOTING 34. killed many, and finally the snipe became wild and flew away, not as if going to great distances, but as if deserting this especial place. In the effort to learn where the birds had gone I passed over a little rise of ground on to a small, cultivated plateau of the prairie running out between two arms of the marsh, and to my great astonishment started a large number of snipe from the dusty ground between the rows of potatoes. Gallinago delicata. A little later, entering a piece of timber, where the ground was dry under foot, several snipe sprang up in front of me and flew away. It was apparent that these birds, being much harried on the marsh, had taken refuge in this place, waiting until the cause of the disturbance should disappear. I have never seen snipe so abundant anywhere as they were in this place. The following morning, returning to the marsh, very few were found, and those few extremely wild. The food of the snipe consists chiefly of earth-worms and various insects which are found in the soft mire of the marshes which they frequent.. Similar in its food to the woodcock, its flesh is quite as toothsome, and, indeed, it would be difficult by the taste alone to tell which bird one was eating. The snipe is commonly called jack snipe, or English The snipe is commonly called jack snipe, or English snipe, both misnomers, which should not be used by sportsmen. It should properly be called snipe, common snipe, or American snipe. In the South, according to Audubon, the Creoles call it cache-cache, no doubt from its cry, while Mr. Nuttall gives “alewife bird,” “from its arrival with the shoals of that fish.” Years AMERICAN SNIPE 35 ago I called attention to a parallel name in Connecticut and elsewhere in New England: “As the bird arrives about the same time as the shad and is found on the meadows along the rivers where the nets are hauled, the fishermen, when drawing their nets at night, often start it from its moist resting places, and hear its sharp cry as it flies away through the darkness. They do not know the cause of the sound, and from the asso- ciation they have dubbed its author the shad spirit.” In the same way, Krider, in his sporting anecdotes, In the same way, Krider, in his sporting anecdotes, speaks of the snipe as called shad-birds by many of the fishermen along the Delaware. Until the time of Wilson, the American snipe was Until the time of Wilson, the American snipe was regarded as identical with the English snipe, and it is, of course, from this similarity of appearance that one of its common names is derived. Gallinago delicata. Until within a comparatively short time, the snipe, Until within a comparatively short time, the snipe, like many of our wild fowl, has been almost without protection. To be sure, in the Northern States it was not practicable to kill it in winter, but whenever to be found it was fair game for the gunners of all the North- ern States. On the other hand, it has always been famous for the twisting flight, which renders it very difficult for inexperienced gunners to kill; yet at times, when the birds are fat, tame and unsuspicious, this difficulty is greatly reduced, and great numbers may be killed. The coloring of the snipe is markedly protective in The coloring of the snipe is markedly protective in character. White below, barred on sides with black, with a breast more or less streaked, and upper parts AMERICAN GAME BIRD SHOOTING 26 black or gray, longitudinally striped with white, whitish and cream color and tawny, it is extremely difficult to see on the ground. Once, in Montana, riding near a little mud flat, dotted with tussocks of yellow grass, I saw at a little distance a snipe feeding in the mud. For some reason he had not noticed me until I got quite close to him. Then he stopped, looked for a moment, and took two deliberate steps which brought him between me and a yellow tussock, the yellow of which was, of course, constantly interrupted by shad- ows of darker—the spaces between the blades of grass. Without taking my eye off him I looked at the tussock, and after I had adjusted my field glasses, could make out a snipe standing there in plain sight, but invisible because of his background. After a mo- ment or two the bird seemed to think that it had been needlessly alarmed, and moved out again against a dark background, where it was plainly seen; but when I started my horse forward, it again became alarmed and retreated to its position of shelter, which again was in front of, and not behind, the tussock. It seemed to understand that this background would ab- solutely conceal it. This is, of course, only one of the common devices of wild animals to escape the obser- vation of their enemies. Gallinago delicata. The snipe is not likely to be taken for any other one of our game birds, though the dowitcher, or brown- back, one of its nearest relatives, resembles it rather closely. Mr. Trumbull, in his admirable book, ““Names and Portraits of Birds which Interest Gunners,” gives AMERICAN SNIPE 27 the derivation of the old word, dowitch, as being originally “the Dutch or German snipe (Duitsch, Deutscher), probably employed to distinguish the red- breasted snipe from the common ‘English’ snipe.” In spring and, to a less extent, in fall, the snipe often practice a curious performance, called by some sports- men booming and by others drumming. The birds, of which there are likely to be a considerable number, rise high in the air, circle more or less about overhead, often uttering a twittering song, and then may sudden- ly drop from a great height toward the ground, caus- ing a humming or drumming sound, which is believed to result from the air passing rapidly between the quill feathers. An interesting account of this action and, in- deed, of several habits of the snipe, was written by Mr. Austen to Forest and Stream, years ago, and commented on by Dr. Nicols, of Cookstown, both these gentlemen residing in Canada. We quote Dr. Nicols’ remarks : “Tn your issue of April 23, Mr. H. Austen, of Hali- fax, N. S., claims that the snipe does its booming (I take it he means drumming) with his voice, and not, as supposed, with his wings, and that this noise is only made at dusk or long after dark. If Mr. Austen will go out to any good snipe ground when these birds are in, especially in the earty part of May, in the spring, or September or October, in the fall of the year, on a dull, overcast day, he will find numbers of snipe in the air circling and soaring about and drumming to their heart’s content, when he will be satisfied—or I am AMERICAN GAME BIRD SHOOTING 38 much mistaken—that the noise a snipe makes when drumming or booming is made with his wings. “In the same letter Mr. Gallinago delicata. Austen writes: ‘All the sound that I have ever heard in the daytime has been their scaipe, except that on August 11, 1885, when shooting on the marsh, I heard about and around me who, who, who, only not in very loud tones, and for quite a while was puzzled, until I found running about me at my feet’ three tiny young snipe, which must have been a very late, or, possibly second, brood.’ If Mr. Austen will go out where:these snipe breed, during the mating season—that is, with us, in the month of May —he will find that snipe sing, twitter and call. During this season, snipe call one another pete, pete, pete. The cock bird springs into the air, flying twenty or thirty yards before lighting again, with his tail and head up, singing, and twittering much like a bobolink. One could hardly think that these tame, foolish birds were the wild, swift-flying, hard-to-hit birds of the previous month. “The snipe and woodcock both drum with their wings. Woodcock drum about dusk, letting themselves down from an elevated position plump on to the ground, with wings set edgewise. Snipe generally drum on dark and dull days, letting themselves down from a high position with wings set edgewise, fifty or a hun- dred feet, immediately soaring up again to circle around as before. This they repeat for hours together. Years ago, when snipe were plentiful in the Holland marshes, a few miles from here, I have seen upon a dull day AMERICAN SNIPE 39 fifty or sixty snipe in the air at one time, circling and drumming; in fact, every snipe on the marsh appeared to be up in the air. I need hardly say that on such occasions the bag was small. In those good old times I remember in the month of October making a bag of 105 snipe in one day; the day was a bright, warm, still day.” Galline. Among the gallinaceous birds, also called Galline— cock family—and formerly Rasores, scratchers—are to be found by far the most numerous and economically the most important of the upland game birds. The group includes the turkeys, guinea fowl, pheasants, grouse, partridges, quail and some other groups, wild and domesticated, which are especially used for food. On the one hand it is allied to the pigeons, and on the other to the cranes, and through them to the limicoline birds, which include the snipe, woodcock, plover and so on. The great systematist, Huxley, divided the galli- naceous birds into two groups, one of which he called Alecteropodes, or fowl-footed, in which the hind toe is small, and elevated above the others—as, for example, in the domestic hen, the turkey and the grouse—while to the other division he gave the name Peristeropodes, or pigeon-footed, in which the hind toe is well devel- oped and long, and all four toes are in the same plane and rest equally upon a flat surface in walking—as the pigeons, currassows and others. The English naturalists consider all the gallinaceous The English naturalists consider all the gallinaceous game birds which we know here in northern North 40 40 40 GALLINACEOUS BIRDS 4I America, as well as a number of others, to belong to the pheasant family (Phasianide) which in that view includes the turkeys, the pheasants, the partridges, the grouse and some others. Until recently American natu- ralists were disposed to regard the pheasant family as limited to the true pheasants, the turkeys, domestic fowl and guinea fowl; and to place the grouse and the par- tridges and quail in a family by themselves (Tetraont- de), the grouse family. For the partridges of the Old World the sub-family Perdicine was established, and another sub-family (Odontophorine) for the quails of the New World. By American ornithologists these two groups have recently been given family instead of sub-family rank, and are called Perdicide and Odon- tophoride. The turkeys have also been given family rank, and are now called Meleagride. Although the various birds belonging to this group Although the various birds belonging to this group may differ widely in many respects, all have certain characteristics in common. In all the body is heavy and round, owing to the great development of the pec- toral muscles—what we call the breast in a bird—the head is small and the neck rather long. Galline. The bill is short and stout, much arched, and overhangs the lower mandible. The digestive system has certain peculiarities corre- lated with their habits of life. There is a large crop capable of great distention; a strong gizzard—except in the sage grouse—lined with a tough, almost horny, coating, peculiarly adapted to grinding the hard sub- AMERICAN GAME BIRD SHOOTING 42 stances, as grain, nuts, and seeds, on which the birds very largely feed. The wings are generally short and rounded, and the flight, while often extremely swift, is more or less heavy and labored and seldom greatly protracted. In some species certain feathers of the wing are enormously developed. The tail varies extremely. In some species it is very long and pointed, in others extremely short; again nar- row but long, or less long and very wide. In the pea- cock, one of the large and showy pheasants, the coverts of the tail greatly exceed the quills in length. In the domestic fowl the tail develops a number of oddities. In the blackcock the quills on the outer sides are curi- ously bent outward, whence one of its names, Lyrurus —lyre-tailed. In the grouse and the partridges, the metatarsus, that portion of the “leg” which is without the body, and which in most birds is not covered with feathers but scales, is short, whereas in such birds as the turkey or the pheasant it is relatively much longer. This so-called leg is really a part of the bird’s foot, and corresponds to that part of the foot in man which lies between the ankle and the toes, and in the horse to that portion which lies between the hock and the pastern—the can- non bone. In the pheasants, quails and partridges the feet are naked, but in the grouse they are always more or less protected by a covering of hairlike feathers, not un- like those found on the feet of certain hawks and owls. GALLINACEOUS BIRDS 43 Sometimes these feathers descend only as far as the toes, but in the ptarmigan (Lagopus—harefoot) the whole foot is thickly covered with dense hairlike feath- ers, which serve to protect it from cold and no doubt to some extent facilitate progression over soft snow. Galline. In the grouse, too, the toes are pectinated—provided with a horny comblike structure—on either side, and these are the only members of the group that are so pro- vided. In some species, if not in all, these pectinations are deciduous. Spurs with a bony core and a horny sheath, thus resembling the horn of a cow—such as we see in the turkey and the male of the domestic fowl— are common to all the male pheasants. In some species there are two or more spurs on each foot and in some, spurs are present in the female. They are not found in the grouse or the partridges. In the grouse family a little tuft of projecting feath- ers runs forward on each side of the bill and covers the nostrils. In the partridges the nostril is naked, but is protected by a scale which overhangs it. In the gallinaceous birds the plumage is as varied In the gallinaceous birds the plumage is as varied as it is possible to conceive, ranging from the most bril- liant metallic sheens of all hues in some of the pheas- ants, to the plainest, dullest clay color in some par- tridges and grouse, as the sage grouse, or the white- tailed ptarmigan in summer plumage. Many of the American quails are singularly beautiful birds, but less by the brilliancy of their colors than by the fine con- trasts or harmonies which their plumage exhibits. Of all these birds in America, however, only the gorgeous AMERICAN GAME BIRD. SHOOTING 44 ocellated turkey of Yucatan and Guatemala can com- pare in brilliancy with many of the pheasants of Asia. Grouse inhabiting arctic or alpine countries—called ptarmigan—are notable for their seasonal changes of plumage. They turn white in winter and are said to be in a continuous state of moult at all seasons, except for a brief period when they have assumed their sum- mer dress, and again in winter after the completing of the winter plumage. An exception to this rule is the Scotch grouse, which does not turn white in winter, presumably having lost that protective adaptation, be- cause such a change of coloring is not required in the region which it inhabits. Galline. The birds of this group are all formed for life upon The birds of this group are all formed for life upon the ground, in this respect contrasting markedly with that other division of gallinaceous birds, the so-called pigeon-footed group, which shows a tendency toward life in trees. This, of course, does not mean that fowl- footed birds necessarily avoid trees, for as a matter of fact many of them commonly roost in trees, and all re- sort to them for refuge and often for feeding purposes. In many of the pheasants and in all the grouse there In many of the pheasants and in all the grouse there are patches of naked skin on the body, which are usu- ally particularly obvious during the breeding season. The comb and the wattles of the domestic hen offer ex- amples of such naked patches. The turkey, of course, has the head entirely naked, covered with roughnesses and caruncles, with a notable process on the forehead. The grouse of North America have narrow, naked patches over the eyes, which during the breeding season GALLINACEOUS BIRDS 45 become congested and large, and by popular writers are sometimes called combs. Besides, several American grouse, such as the sage hen, the pinnated grouse and the dusky grouse, have on the sides of the neck naked patches of skin, which in the breeding season are capa- ble of being inflated, and when so inflated completely change the appearance of the bird. In the prairie chicken, these, when inflated, have been compared to small oranges ; in the sage grouse they are much larger ; while in the dusky grouse they appear smaller, and can only just be seen through the white feathers which sur- round them. The sharptail grouse has a naked place in the same position on the neck, and so has the ruffed grouse. All the grouse have peculiar and noisy methods of ushering in the breeding season, of which the drumming of the ruffed grouse, the booming of the prairie chicken, the hooting of the blue grouse and the dancing of the sharptail are familiar examples. These various actions and sounds are not, however, exclusively confined to the breeding season. Grouse are generally supposed to be polygamous, but Grouse are generally supposed to be polygamous, but not all species are so. Galline. The quails, or American par- tridges, are monogamous, the male and female remain- ing together during the season of incubation and hatch- ing. In fact, in case of accident to the female, the male sometimes hatches out and rears the brood. The Ameri- can quails possess shrill and rather pleasing voices, while the calls of the grouse are often rough, hoarse and disagreeable. AMERICAN GAME BIRD SHOOTING 46 In the north and in temperate regions America is well provided with grouse, and in the south with quails. The American quails reach their greatest development toward the tropics. Odontophoride. Of true partridges belonging to that family of galli- naceous birds known as Perdicide there are none in America, but their place is taken by another family, known as Odontophoride, which has a wide distribu- tion, with a number of species in the United States, and a still greater expansion to the southward. While the grouse are all large birds, some of them While the grouse are all large birds, some of them approaching the wild turkey in size, the American par- tridges, or quails as they are commonly called, are all small. The grouse have the lower legs and feet more or less covered with hairlike feathers, and the nostrils also covered with points of feathers reaching out on to the bill, known as antiz. The grouse have over the eye a naked strip of skin which in the breeding season becomes to some extent enlarged and congested, so that it is sometimes loosely spoken of as a comb, though it is not a comb like that of the common hen. The Ameri- can partridges on the other hand have the feet and nostrils naked, lack the bare skin above the eye and usually have short tails. The character which distin- guishes them from the partridges of the Old World (Perdicide) is found in the cutting edge of the mandi- ble, which is toothed or notched. Sometimes this char- 47 47 AMERICAN GAME BIRD SHOOTING 48 acter is so faintly marked as hardly to be noticed, and when this is the case it is not easy to point out any dis- tinctive character separating them from the Old World group. The division, however, is a convenient one, and the New World birds are sufficiently alike to be grouped together. Though most numerous in tropical America, a num- ber of beautiful species are found within the United States. Such are the plumed quail of the California mountains, the helmeted birds of the California low- lands and of the interior, the scaled quail of the south- west and the Massena, or Mearns, quail. In the United States and Mexico there are ten genera In the United States and Mexico there are ten genera with forty species and sub-species belonging to the Odontophoride. No members of the group range north of the United States, except that the valley quail of California has been introduced on Vancouver Island, B. Odontophoride. C., and that formerly the Virginia quail was found in small numbers in southwestern Ontario. QUAIL, BOBWHITE, PARTRIDGE. Colinus virgmianus, Colinus virginianus floridanus, Colinus virginianus texanus. Colinus virgmianus, Colinus virginianus floridanus, Colinus virginianus texanus. Of all these forms, by far the best known is the bob- white—the partridge of the Southern States, and quail of the north. It has been divided by naturalists into several sub-species or geographical races inhabiting southern New England and the Middle States, the Southern States, Florida and Cuba, Texas and southern Arizona; and in Mexico are found many other differ- ent forms of the genus. All these, except those of the extreme southwest—Mexico—differ from each other chiefly in size and in the varying intensity of the blacks and browns which mark their plumage. Of those of the United States the northern form, known as the quail or Virginia partridge, is the largest; the southern races, called the Florida bobwhite, the smallest and blackest ; while the Texas bobwhite is midway between the two in size, and in color is paler and grayer than either. . The general color of the typical bobwhite is reddish The general color of the typical bobwhite is reddish brown, paler below and streaked, spotted or crossed with black markings. The lower breast and belly be- come whitish, and all the upper parts are crossed with 49 49 AMERICAN GAME BIRD SHOOTING 50 fine lines of blackish or dusky. In the male, the throat and a broad line over the eye are white, the rest of the head black or blackish streaked with brown. The sides of the neck are spotted with triangular dots of black and white, the back is reddish brown mottled with gray and more or less streaked with tawny. The female is paler than the male, and has the throat patch and the eet OE EER BOB WHITE QUAIL BOB WHITE QUAIL stripe over the eye buffy. Sometimes the young males have this buff throat patch and superciliary stripe. The tail is bluish gray, the under tail coverts white streaked with reddish brown. The feathers of the flanks and sides are reddish brown, striped with whitish. Length about ten inches and wing about four and a half. Ex- tent of wings fifteen to sixteen inches. Weight from about six and a half to eight ounces. stripe over the eye buffy. Sometimes the young males have this buff throat patch and superciliary stripe. QUAIL, BOBWHITE, PARTRIDGE. The tail is bluish gray, the under tail coverts white streaked with reddish brown. The feathers of the flanks and sides are reddish brown, striped with whitish. Length about ten inches and wing about four and a half. Ex- tent of wings fifteen to sixteen inches. Weight from about six and a half to eight ounces. QUAIL, BOBWHITE, PARTRIDGE SI The Florida bobwhite (C. virginianus floridanus) averages somewhat smaller and considerably darker, the black markings being wider and occupying relatively more space on the feathers, The Texan bobwh The Texan bobwhite (C. virginianus texanus) is slightly smaller than the typical bobwhite, but larger than that of Florida. Mr. Ridgway says an olive gray- ish tint prevails over the whole back without conspicu- ous black spots, and the general surface is usually dis- tinctly barred with lighter; the black markings of the lower parts are usually broad and nearly transverse, as in C. virginianus floridanus. Bobwhite is the most widely distributed of our galli- naceous birds except perhaps the turkey and the ruffed grouse. In one form or another it is found from Mas- sachusets south to Florida and the Gulf of Mexico, and from southern Ontario, Canada, west and south through eastern Minnesota, Southern Dakota, Nebraska, Kan- sas, Oklahoma and Texas, into Mexico. Besides, it has been introduced at various points west of its natu- ral range and apparently has become thoroughly estab- lished in Utah and Idaho. Jt is reported also to be quite abundant in portions of California and in the Willamette Valley, Oregon, as well as on certain islands of Puget Sound, Washington. Though introduced and doing well at various points—Denver, Fort Collins, etc. —in Colorado, some naturalists believe that it has of itself advanced west with the farmers into eastern Colorado. 52 AMERICAN GAME BIRD SHOOTING Being a bird that lives on the ground, bobwhite can- not survive in a country of deep snows. Along the northern border of its range it has often been almost ex- terminated in certain regions, because a heavy snowfall during the night has covered up the roosting birds, and then a change in the weather melting the surface of the snow has been followed by another change to freezing, forming a hard crust through which the quail cannot break, resulting in starvation and death. QUAIL, BOBWHITE, PARTRIDGE. There are many records of bevies being found after the snow of some severe storm has melted, close huddled together precisely as they roosted. Long continued periods of severe weather, when the ground is covered with snow and ice, sometimes make food exceedingly hard to ob- tain and at the same time render the birds more visible to their many enemies. At the present time the quail may be said to be almost At the present time the quail may be said to be almost extinct in Massachusetts, Connecticut and much of New York State, and in many sections they seem to be con- stantly growing fewer in numbers. This is due largely to over-shooting, but partly also to the destruction of the covers which once afforded them so much more pro- tection than at present. The reduction in their numbers is a misfortune not merely to the sportsman, but to the whole country, for investigations have shown the quail to be a valuable ally to the farmer, destroying multi- tudes of harmful insects, and also devouring vast quan- tities of noxious weed seeds. The value of the services which it performs is now coming to be understood by the public at large, and it is reported that in certain QUAIL, BOBWHITE, PARTRIDGE 53 sections of the country—as in parts of Oklahoma—far- mers refuse to permit gunners to venture on their prem- ises in pursuit of bobwhite. The quail does not migrate and therefore is always exposed to the dangers of winter, and where they have once been exterminated efforts to replace them by other quail imported from the south have seldom been success- ful. The most hopeful indication for the protection of the species is in the growing comprehension of its eco- nomic value. Quail are extremely prolific, and under ordinary conditions might survive the attacks of all natural enemies, but when the climate sweeps off all the birds in a particular region the progress of re-establish- ment is slow. In old times in the south the practice of netting quail In old times in the south the practice of netting quail was very generally indulged in. Powder and shot were costly, while the net could be used over and over again. QUAIL, BOBWHITE, PARTRIDGE. This method of taking them, at first practiced only as a means of procuring food, was carried on later for the purpose of securing living birds to sell to persons who desired to restock their covers with quail. Presumably it has been in vogue up to within a short time, 1f not now practiced in parts of Oklahoma and in Texas. A chief danger to the quail of the south is the non- A chief danger to the quail of the south is the non- enforcement of the game laws, and the market shooting by negroes, many of whom gun persistently almost the whole year round and are excellent shots. The ordinary bobwhite is the northernmost and best The ordinary bobwhite is the northernmost and best known species of this genus. Yet the smaller, darker quail of the south and of Florida and the grayer form The ordinary bobwhite is the northernmost and best known species of this genus. Yet the smaller, darker quail of the south and of Florida and the grayer form AMERICAN GAME BIRD SHOOTING 54. of Texas are at once recognized by northern gunners as essentially the same bird that they know at home in the north. The uneducated birds of the southwest, how- ever, do not afford the same sport furnished by birds frequently pursued, but show the disposition to run be- fore the dog exhibited by the other quail of the dry country—Gambel’s, the scaled and the valley quails. It is said of bobwhite that years ago it was scarcely It is said of bobwhite that years ago it was scarcely found west of the Missouri River, but that it has fol- lowed the settlements north in Minnesota and west in Nebraska. This may be true, but it is quite as likely that it has always existed in this region, but was not observed there until the country became more or less thickly settled. Wherever found, the quail is resident and breeds. Al- though occasionally large flocks occur, consisting of twenty-five or thirty birds, it never packs, as do many of the grouse of the open, and where such large flocks are found it is probable that they consist of the first and second broods of the same parents, or of the birds hatched by two hen quail that have occupied the same nest. QUAIL, BOBWHITE, PARTRIDGE. , It must be said, however, that there are at least two records where packs of quail have been seen by good sportsmen of great experience. One of these was Edmund Orgill, who made the following interesting report: “A curious experience occurred a short time ago to a friend of mine who went on a hunt to north Missis- sippi, where he had been earlier in the season and found QUAIL, BOBWHITE, PARTRIDGE 55 birds plentiful. He was accompanied by his brother, and they had a brace of good dogs. They hunted for hours without the sign of a bird, and could not account for the apparent scarcity. At last their dogs made game, but before they could establish a point the birds commenced getting up all around and in front of them. They were confident that there were from 70 to 100 birds, and instead of their taking the usual quail flight, they rose clear up in the air like a flock of pigeons, and went away for half a mile. . They followed the line, which was in an open country, and found a few scat- tered birds in an old cotton field, fully the distance named from where the birds took their flight. They gave up finding more, and started again; and after a prolonged tramp they had a duplicate of their first ex- perience, finding a second flock or drove—not a bevy, for they say there were nearly a hundred. Like the former they made an immense flight. “Now, I know that it is common for the grouse in Scotland to pack, occasionally the English partridges do, and our prairie chickens; but I never saw or heard of quail doing so before.” On this report S. T. Hammond, referring to the On this report S. T. Hammond, referring to the year 1852, commented as follows: “In Iowa thirty-eight years ago, when crossing “In Iowa thirty-eight years ago, when crossing through a piece of woods from one prairie to another, we came upon a clearing of two or three acres of wheat stubble, and a large drove of quail got up, at least 200 or 300, but they scattered in every direction. To this day we can see that clearing and how the quail filled AMERICAN GAME BIRD SHOOTING 56 the air. QUAIL, BOBWHITE, PARTRIDGE. This is the only thing that has come under our personal observation that looked like the Missis- sippi case.”’ The quail is a bird of the open, building its nest in the fields, in little patches of brush, or in fence corners, but after the broods are grown, commonly taking refuge when alarmed in woods or tangled brushy patches. Usually the winter home of the bevy is in some swamp or woodland adjacent to fields where grain or weed seeds or other food is accessible, and a few years ago their wanderings were limited to a comparatively small area. The birds went to their feeding grounds in the morning, walking from them to some place where they sat in the sun and dusted for two or three hours; toward night they fed again; and then often walked in among high grass, where they roosted. Of late years, since they have been so con- stantly pursued, their habits appear to have undergone some change, and they are believed often to roost in the woods or swamps, but in old times it was common to find these roosting places out among the stubbles or in the open fields. Years ago, a gunner familiar with a certain section of country, who had traveled over it enough to learn where the different bevies of quail lived, could almost certainly start each one of them and have a couple of shots before they took refuge in swamp or woods. In those days, however, a man who had such knowledge—if he went shooting frequently—usually contented himself with three or four birds a day. If his QUAIL, BOBWHITE, PARTRIDGE 57 outings came but seldom, he was likely to demand more and to follow up the bevies. The broods of quail which have kept together all The broods of quail which have kept together all through the winter usually break up as warm weather approaches, and soon after this comes the mating. This takes place earlier in the south, and in New England it is often the first of June before the birds are mated and the clear whistle of the male is heard. The nest is a depression in the ground, lined to some extent with bits of grass and weed stems and occasionally with a feather or two dropped from the mother’s breast. Rarely the parents build over the nest a dome-shaped roof. QUAIL, BOBWHITE, PARTRIDGE. Cap- tain Bendire quotes Judge John M. Clark of Saybrook, Conn., who saw a male bobwhite engaged in the work of constructing a domed nest. Judge Clark says: “Tn May, 1887, while on a hill back of my house one morning I heard a quail whistle, but the note, which was continually repeated, had a smothered sound. Tracking the notes to their source I found a male bob- white building a nest in a little patch of dewberry vines. He was busy carrying in the grasses and weaving a roof as well as whistling at his work. The dome was very expertly fashioned and fitted into its place without changing the surroundings, so that I believe I would never have observed it had he kept quiet.” Captain Bendire also says that Mr. G. E. Beyer of Captain Bendire also says that Mr. G. E. Beyer of New Orleans, La., found a nest constructed of pine nee- dles, arched over and the entrance probably a foot or more from the nest proper. Such constructions are very unusual. The only concealment commonly found is that Captain Bendire also says that Mr. G. E. Beyer of New Orleans, La., found a nest constructed of pine nee- dles, arched over and the entrance probably a foot or more from the nest proper. Such constructions are very unusual. The only concealment commonly found is that AMERICAN GAME BIRD SHOOTING 58 offered by the weeds and grass that grow about the nest. Captain Bendire says that in the south, cotton rows are a favorite nesting place. It rarely happens that for some reason or other a second brood of quail may be very late. Such a case was mentioned in Forest and Stream in February, 1879, where a quail’s nest is stated to have been found in the month of January. From twelve to eighteen white eggs are laid. Some- From twelve to eighteen white eggs are laid. Some- times many more are found in a nest. In such cases it is probable that two hens have shared a single nest. Captain Bendire tells of a nest of nine eggs taken in Texas, all of which were more or less spotted and streaked with reddish brown and lilac markings, es- pecially about the larger end. QUAIL, BOBWHITE, PARTRIDGE. While the hen is sitting on her eggs, the male from some elevated perch not far off whistles through much of the day the cheerful notes which give him his name and which are also interpreted as more wet. When one is near the bird, a third note is heard preceding the two most striking ones, and much lower pitched, mak- ing the whole call seem more like ah bobwhite. It is generally supposed that this call is peculiar to the male, but the female also utters it, though not with the same fullness and vigor as the male. On one occasion while driving slowly along a narrow grassy lane I heard a quail whistling at some distance ahead of the horse. We drew nearer and nearer, yet the sound seemed hardly strong enough for that of a full-grown male, and presently I distinctly saw whistling, a female quail, QUAIL, BOBWHITE, PARTRIDGE 59 which did not leave her perch on a fence post until the horse had passed her and I was within four or five feet of her. Then she dropped down into the grass close to the fence. The rallying cry uttered by members of a scattered bevy is a call of three notes, but entirely different from the mating cry bobwhite. I believe the male bobwhite usually takes part in the I believe the male bobwhite usually takes part in the work of incubation, and all writers are agreed that if an accident happens to the female, the male incubates the eggs and rears the young. When the eggs hatch, the little ones, then scarcely larger than bumble-bees, at once follow the parents, who look after them with every manifestation of affection. Sometimes it happens that the farmer while driving his mowing machine through the tall grass, may see a male and a female quail rise in front of him and flying but a few feet drop down again. He knows then that somewhere in the grass near his team are the tiny young whose lives are now in jeopardy, and often he will turn his horses about and go the other way, in order to give the parent birds an opportunity to lead their young away from the dan- ger spot. QUAIL, BOBWHITE, PARTRIDGE. Sometimes the little ones may be seen, a dozen of them, hurrying after their parents across the newly shorn grass, half hidden by its short stems. Oft- en the parents strive to lure man or dog away from the tender young by feigning injury, and on a signal from the mother the young lie close hidden until the danger is passed, 60 AMERICAN GAME BIRD SHOOTING During their earliest youth the young quail feed al- most exclusively on soft insect food, but as they grow larger they eat more and more seeds, and when autumn comes, fruit of one kind and another and grain. In New Jersey barrens, where grain fields are few and far between, I have taken quail that seemed to have fed exclusively on the acorns of the scrub oak. Most carnivorous reptiles, birds and mammals are enemies to quail, and yet against the mammals and rep- tiles they are fairly well able to protect themselves. Occasionally a snake devours the eggs in a nest or may destroy a few of the very small young, but the depreda- tions they commit are slight. Hawks are the enemies from which the quail have most to fear, and of these the goshawk, Cooper’s hawk and sharp-shinned hawk are the most destructive. JI have more than once seen a marsh hawk stoop at a quail, but I never saw this hawk catch one. I have also seen a marsh hawk stoop at a crow, and even at a great sage grouse that would weigh many times what the hawk weighed. The grouse seemed alarmed and ran away, but the crow merely threatened the hawk with its beak and seemed not at all disturbed. Colinus ridgwayi. The masked bobwhite is very little larger than the Virginia bobwhite. It has the whole under side of the head black and the white stripe over the eye is very narrow or sometimes disappears entirely. The neck and chest below the black throat is uniformly cinnamon or reddish, like the other lower parts. The female is almost exactly like the female of the Texas bob- white, but usually has a strong band of cinnamon color across the upper part of the chest. There are no special d There are no special differences in habit among the various forms of bobwhite quail, except those which de- pend on their surroundings—the character of the coun- try which they inhabit. The masked bobwhite, or Ridg- way’s quail, was described by Mr. Brewster from speci- mens sent on from Mexico by F. Stevens. Previ- ous to that, however, Herbert Brown of Tucson, Arizona, had sent on specimens which were erroneously identified as Grayson’s bobwhite, a Mexican species not known to occur in the United States. In a paper entitled “Arizona In a paper entitled “Arizona Quail Notes,” published in Forest and Stream in 1885, Mr. Brown writes about this species in the following words: 61 In a paper entitled “Arizona Quail Notes,” published in Forest and Stream in 1885, Mr. Brown writes about this species in the following words: 61 61 61 62 AMERICAN GAME BIRD SHOOTING “The masked bobwhite is found in the country lying between the Barboquivari Range in Arizona and the Gulf coast in Sonora, more especially between Barbo- quivari and the Plomoso, where this species is quite abundant. ‘They are also found on the Sonoita Creek, about 60 miles north of the Sonora line. From the Sonoita Valley they range in a westerly direction fully 100 miles, and through a strip of country not less than 30 miles in width within Arizona Territory. Very probably they may go beyond this, both to the east and west. The habits of the masked bobwhite, so far as we know them, appear to resemble very closely those of the common quail, only slightly modified by the con- ditions of their environment. They utter the character- istic call of bobwhite with bold, full notes, and perch on rocks or bushes while calling. Colinus ridgwayi. They do not appear to be a mountain bird, but live on the mesas (table- lands), in the valleys, and possibly in the foothills. “The masked bobwhite was, three years since, abun- dant in the neighborhood of Bolle’s Well, a stage sta- tion on the Quijotoa Road, near the northern end of the Barboquivari Range, 29 miles southwest of Tuc- son and about 40 miles north of the Mexican boundary line. As the station was then comparatively new, the grass thereabouts was high, and these quail could be had for the taking; but now that the stock has eaten away the grass, the birds have not for a year or more been seen about the place. On the road from Bolle’s Well, west to the Coyote Range (about 25 miles), these quail were frequently to be met with, but the teamsters BOBWHITE IN MEXICO 63 and travelers have killed or frightened them off. One of the former assured me that he had killed as many as five at one shot. Ten miles south of Bolle’s, in the Altar Valley, we came across a small covey, perhaps a dozen inall. The bright, deep chestnut breast plumage of the males looked red in the sun, and gave the birds a most magnificent appearance. We secured but one, a male, the rest secreting themselves in the tall sacaton grass, which at this point was between 4 and 5 feet high, and as we had no dog we did not follow them. Our next place to find them was on the mesa, southeast of the peak, where we camped to hunt for them, but they were scarce here, and we managed to secure but few. “In addition to their bobwhite they have a second call of hoo-we, articulated and as clean-cut as their bobwhite. This call of hoo-we they used when scattered, and more especially when separated toward nightfall. At this hour I noted that although they oc- casionally called bobwhite, they never repeated the first syllable, as in the day time they now and then at- tempted to do. In body they are plumpness itself; in this respect, considering size, they overmatch the Ari- zona quail (Callipepla gambeli), with which I com- pared them. In actual size of body, however, the latter is the larger. Colinus ridgwayi. Of three stomachs of this species exam- ined, one contained a species of mustard seed, a few chaparral berries, and some six or eight beetles and other insects, ranging in length from a half inch down AMERICAN GAME BIRD SHOOTING 64 to the size of a pinhead. The second was similarly provided, but contained, in lieu of mustard seed, a grasshopper fully an inch in length. These two were taken on the mesa. The third, from a bird taken in the valley, contained about twenty medium-sized red ants, several crescent-shaped seeds, and a large num- ber of small, fleshy green leaves.” Mr. Brown pointed out, years ago, that the intro- duction of live-stock into southern Arizona bade fair to exterminate the masked bobwhite in that territory, by the destruction of its nests and eggs by horses and cattle, as well as by the eating of the cover among which it lives. His prediction has been verified, and as recently as June, 1909, he wrote, saying: “Colinus ridgwayt is a dead bird so far as Arizona is, as yet, con- cerned, but it is again getting a good foothold in So- nora, about 75 miles south of the line. I am almost afraid to say anything about it, however, as I fear I might send skin hunters into the country.” Other writers have noted in our common bobwhite the same tendency to disappear in sections where gen- eral farming has given place to stock-raising. The bobwhite family attains its greatest development in Mexico. Though differing greatly in color, the pat- tern of that color is somewhat similar in all the differ- ent forms. Several are black, or black mottled with white, on the breast and tail, where our bird is pale in color, just freckled with black. All have the same whistle with which we are so familiar in our own bird, 65 BOBWHITE IN MEXICO and generally their habits are much alike. They range from the sea-level to an altitude of 7,500 feet. They have not the shyness or swiftness of wing of They have not the shyness or swiftness of wing of the much hunted form of the East and South, and are gentle, unsuspicious little birds, ready to run along on the ground before the traveler or to dodge out of the trail to one side. Colinus ridgwayi. Of the dozen forms described from Mexico, perhaps only two, Ridgway’s bobwhite and the Texas bobwhite, cross the boundary into United States territory. No one is so familiar with this group in Mexico as Mr. FE. W. Nelson, the distinguished naturalist, whose work in various branches of science, in Alaska, Mexico and Central America, is so well known. In the Auk, for April, 1898, he published a charming account of this group, which is accompanied by an excellent plate of Godman’s bobwhite, a species described by Mr. Nelson, from the lowlands of southern Vera Cruz. Mr. Nelson calls his sketch “With Bobwhite in Mexico,” and we copy it here: “While traveling in Mexico a few seasons ago, I “While traveling in Mexico a few seasons ago, I arrived at a small town near the southern end of the tableland in the State of Puebla. The first business in hand was to secure suitable quarters for myself and assistant. Having accomplished this, I was ready at an early hour the following morning for a tramp into the surrounding country. It chanced to be market day, and passing the outskirts of the town I met a straggling procession of Indians, in picturesque cos- tumes, some driving heavily loaded donkeys, others car- AMERICAN GAME BIRD SHOOTING 66 rying on their own backs crates of fruits, vegetables, hand-made pottery and other simple wares. All were pushing forward, eager to take part in the keenly rel- ished pleasures of petty chaffering, which would enable them to return home with a few decimos knotted in the ends of their sashes. Some of the men saluted me with a polite ‘Buenos dias, sefior,’ but I noted that their conversation was carried on in the Aztec tongue, as spoken by their fathers centuries ago. “Once free of the last houses, a convenient opening “Once free of the last houses, a convenient opening in the fence was soon found, and I crossed into a great field, which reached for miles down the broad, open valley. Areas covered with wheat and corn stubble indicated the character of the last crops, while farther away broad belts of brilliant green sugar-cane were in vivid contrast to the dry browns and yellows of the general surface. The sun was shining brightly, and the fresh, balmy air seemed full of life-giving power. Colinus ridgwayi. The musical notes of meadow-larks were heard at in- tervals, and on one side of the valley flocks of red- winged blackbirds were swirling back and forth over some small marshy spots grown up with tules. Through the valley bottom flowed a little stream of clear, spark- ling water, which, before reaching the distant shore of the Pacific, runs a wild course through the mountain gorges of Guerrero. Behind me arose the mysterious pyramid of Cholula, crowned by a white-walled chapel, which now occupies the place of ancient sacrifice. Over to my right stood the gigantic form of the Smoking Mountain—hoary old Popocatepetl—with the gleaming BOBWHITE IN MEXICO 67 robe of the White Lady—Iztaccihuatl—shining over his shoulder. In front a sweeping plain descended for many miles, through a district of great sugar estates, to the far horizon, where it was walled in by the blue front of distant mountains. “Turning to one side, I approached some scrubby bushes which appeared to offer shelter for birds or other game. Suddenly the familiar accents of my mother tongue fell on my ear. I listened with bated breath. Again arose in clear, round tones, the calls so familiar in my boyhood days, bobwhite, bobwhite. With eager steps I hastened forward to a small group of acacias, and there, quietly perched on top of a bush, was an old friend, the author of the notes. It is diffi- cult to describe the mingled pleasure and exultation caused by this unexpected meeting. It proved to be the Puebla bobwhite (Colinus graysoni nigripectus, Nel- son), and during the following days a number of oth- ers were seen, and it became evident that my friend of the first morning was one of a colony located in the neighborhood. “Afterward, during my Mexican travels, I learned that the bobwhites are widely spread in that country, and although many of them have changed the color of their dress more or less, yet their customs and tricks of speech remain much the same as in their northern home. “Ata later date during this same season, while work- ing down the eastern slope of the Cordillera in Vera Cruz, near the city of Orizaba, we found others of the AMERICAN GAME BIRD SHOOTING 68 family, known as the black-breasted bobwhite (Colinus pectoralis, Gould). Colinus ridgwayi. They were living in brush-grown and weedy old fields—sometimes straying about the coffee plantations—and were on friendly terms with most of their tropical neighbors. Fortunately, in these parts guns and dogs are few, and mostly harmless, so that Bob’s days were generally peaceful and contented. But even here life was not without its cares, for the spotted tiger-cats and woolly-haired opossums, with sad lack of consideration, were given to nocturnal raids that filled them with terror and sometimes lessened their numbers. “From Orizaba our wanderings led far away over plains and mountains to the city of Tehuantepec, on the hot lowlands bordering the Pacific coast. There we found our friends again, but known as the Coyolcos bobwhite (Colinus coyolcos, Miull.). They were com- mon, and although their garb had changed consider- ably, yet their voices and mode of life remained true to the family traditions. Indeed, so fixed are old hab- its among them that even long association with the suave and politic Mexican has failed to cure Bob of one custom that I often deplored during my youthful days, when, gun in hand, I sought to make his acquaintance. I refer to that abruptness of manner which is shown in such a disconcerting way when one comes upon him in his favorite haunts. “Near Tehuantepec their home is on the partly wood- “Near Tehuantepec their home is on the partly wood- ed and partly grassy plains. Old fields and grassy prai- ries, that extend irregularly amid the scrubby forests BOBWHITE IN MEXICO 69 of that district, are their favorite haunts. Here the mesquites, mimosas, acacias, cassias, Brazil-wood, eb- ony, mahogany, Spanish cedar, and other tropical trees and bushes, give the landscape quite a different aspect from that which Bob is accustomed to see in his north- ern home. Old cornfields and weedy indigo planta- tions are popular resorts, and furnish an abundance of food. Brush fences of thorny scrub are built about these fields, and serve as fine places of shelter in times of dan- ger. The quails do not penetrate heavily wooded bot- toms along streams, where the moisture causes a vigor- ous tropical forest growth, unless some farmer hews out a clearing for his cornfields. In these forest belts the motmots, trogons, red-and-yellow macaws, several spe- cies of parrots and other tropical birds, abound, and a little farther south troops of spider monkeys are en- countered. Colinus ridgwayi. In many places it is but a few steps from the dense shade of the bottoms, where the harsh screams of the macaws dominate all other woodland notes, to the borders of grassy prairies where our friends pass their sedate lives, associated with meadow-larks and sparrows. Throughout this region, where deer, pec- caries, tree pheasants and other game is plentiful, small- er birds are considered unworthy of powder and shot, all of which conduces greatly to peace of mind among the bobwhites. “While traveling down the coast from Tehuantepec into Chiapas, we found them numerous most of the way, and they were a constant source of interest and pleasure. Their cheerful notes were frequently heard AMERICAN GAME BIRD SHOOTING 70 from the scrubby bushes near the trail, and the neat, trimly-built little fellows carried on their small affairs with little regard for our presence. While riding at the head of the pack train, I frequently found them scratching in the sandy trails, dusting themselves, or searching for food. At such times it was amusing to note the pretty air of doubt and hesitation with which they awaited my approach before finally moving rather deliberately a few yards to one side when I came too near. Now and then the male could be heard uttering little querulous notes, as if in subdued protest at being disturbed. After entering Chiapas the coast was left behind, and we passed into the interior through a se- ries of beautiful open valleys ornamented with scat- tered bushes and belts of trees. It was during the rainy season, and the vegetation was growing luxuri- antly ; everywhere were myriads of flowers, and the in- numerable plumelike heads of tall grasses nodded grace- fully in the passing breezes. In these valleys the bob- whites were very common. It generally rained during the night, but the clouds broke away at dawn, leaving a brilliantly clear sky. We were up and on our way at sunrise, amid the invigorating freshness of early morn- ing, when every leaf and twig bore a pendant water- drop that sent out quivering rays of light with the first touch of the sun. On every hand were new flowers and strange birds. Colinus ridgwayi. Now and then the Central American mockingbird, in full-throated ecstasy, poured out its rich song, and over it all, at short intervals, the clear call of bobwhite arose from a bush or low tree. At BOBWHITE IN MEXICO 71 an altitude of about 3,000 feet we passed out of their range, and did not find them again until we reached the valley of Comitan, on the Guatemalan border, where their notes were heard. A few miles farther on, just after entering Guatemala, a single female, which proved to be quite different from those taken in Mexico, was brought me by an Indian. This specimen served as the type of the Guatemala bobwhite (Colinus insignis, Nelson). Beyond this nothing was learned of them in these remote parts. “From Comitan Valley we made a long circuit over the Guatemalan highlands and reached the Pacific coast again, on the border of Chiapas. There, on some grassy prairies in the midst of the forested coast plain, a few miles back from the sea, we found many bobwhites of a previously unknown branch of the family (Colinus salvini, Nelson). In this vicinity an attempt was made, many years ago, to establish a large colony of Americans. They came with great flourish of trum- pets and large expectations, but the climate did its silent work so effectually that two or three stranded relics were all that remained. Over the desolate sun- scorched flats near by the same cheery call of the quail sounded in the ears of the Mexican oxdrivers and muleteers as they carried their cargoes of coffee and cacao to the coast, that I heard from many a field and thicket over thousands of miles of varied country to the north. Among these sturdy little Americans there appeared no sign of degeneration, and it was pleasanter to meet them than some of my countrymen AMERICAN GAME BIRD SHOOTING 72 of a larger growth. So many failures at colonizing people from the north in these hot southern lands had come to my notice that I had become skeptical of its successful accomplishment in any instance; yet here in the tropics were the bobwhites, essentially a group of the temperate regions, living as cheerfully as pos- sible, and upsetting my preconceived ideas. Colinus ridgwayi. “After passing some time in this district we hired “After passing some time in this district we hired an oxcart one evening, and were trundled across the plains to the coast during the cool hours of the night. There, on the sandy shore, we waited ten days for a steamer, which finally carried us back to Tehuantepec. From this place a railroad crosses the Isthmus to the port of Coatzacoalcos, on the Gulf of Mexico, and we took advantage of it to reach the eastern coast. Coat- zacoalcos is a curious little town, destined to play an important part in the development of southern Mexico and western Guatemala. It is one of the few places in Mexico where small frame houses are the prevail- ing style, and reminds one more of some small mining camp in the Far West than of a seaport on the Gulf of Mexico. Here, where yellow fever, malaria and other ills stalk about, according to the season, we heard of howling monkeys, jaguars, tapirs, and other tropical creatures with which we still desired to be- come more familiar. For this purpose we ascended the Coatzacoalcos River about twenty miles, to the town of Minatitlan, a place once noted for its enor- mous trade in Spanish cedar and dyewoods. We re- mained here some days, in the midst of the coast low- BOBWHITE IN MEXICO ie; lands, where the tropical forest is interrupted by grassy prairies of considerable extent. In visiting these prai- ries we were surprised and delighted to find another of the bobwhites that had not been previously known even to those most familiar with the ramifications of this good old stock (Colinus godmani, Nelson. Aft- erward we found them a few miles out of Coatzacoal- cos, and they were also seen a little farther north, in the open country about the shores of beautiful Lake Catemaco. This latter point is probably near their limit in gat direction. “The me ananen of the “The me ananen of the Mexican bobwhites is curi- ous, and shows that the family has been long in the land. They range over parts of the cool tableland, and extend down to the tropical lowlands of both coasts, but are unaccountably absent from many apparently suitable places. Colinus ridgwayi. “Many “Many changes have taken place in their garb owing to the influences and requirements peculiar to such va- ried situations, but the general style is retained, so that their relationship cannot be mistaken. “A representative of “A representative of this group lives in Yucatan, which, it is said by some, belongs to the family proper; but if this is so, there must be a bar sinister on its escutcheon to account for some of its peculiarities. “At present eleven branches of bobwhites “At present eleven branches of bobwhites are known to live in various parts of Mexico, and our work has enabled us to introduce four of them to the friends of the family. Wherever they were encountered over this great area it was interesting to observe how closely AMERICAN GAME BIRD SHOOTING 74 they continue to resemble one another in notes and habits. From the border of Canada to Guatemala they hold true to a general style of speech and manners that always betrays their connection, with the possible ex- ception of the Yucatan branch, of which I am unable to give any definite information. “For the charming qualities and pretty ways of these “For the charming qualities and pretty ways of these little friends of the field, I trust their days may be many and their numbers never grow less. “As it is quite possible that some of our mutual “As it is quite possible that some of our mutual friends may have the opportunity to call upon these Mexican connections of ‘our Bob,’ I have taken some trouble to secure their names and addresses, which are given below. The directory is complete, I believe, up to date. “Tt. Colinus ridgwayi (Brewster). Ridgway’s bob- white. Sonora; ranging south from the Arizona bor- der. (Between 1,000 and 2,500 feet above sea-level. ) “2. Colinus virginianus texanus (Lawr.). Texas bobwhite. Northeastern Mexico; Nuevo Leon and Tamaulipas. (From near sea-level up to 2,500 feet.) “3. Colinus graysoni (Lawr.). Grayson’s bobwhite. Southern part of tableland; from San Luis Potosi and northern Jalisco to Valley of Mexico (3,000 to 7,500 reet)': “Tt. Colinus ridgwayi (Brewster). Ridgway’s bob- white. Sonora; ranging south from the Arizona bor- der. (Between 1,000 and 2,500 feet above sea-level. ) “2. Colinus virginianus texanus (Lawr.). Texas “2. Colinus virginianus texanus (Lawr.). Texas bobwhite. Northeastern Mexico; Nuevo Leon and Tamaulipas. (From near sea-level up to 2,500 feet.) “3. Very different in appearance from the bobwhite group are the striking quails of the western and south- western portions of the United States. Of these birds there are almost a dozen forms, some of them plumed, others helmeted, others with full, soft crests, but all very unlike the bobwhites. These are among the most beautiful of North American birds, and in many of the regions which they inhabit are extremely abundant. Colinus ridgwayi. Colinus graysoni (Lawr.). Grayson’s bobwhite. Southern part of tableland; from San Luis Potosi and northern Jalisco to Valley of Mexico (3,000 to 7,500 reet)': “4. Colinus graysoni nigripectus (Nelson). Puebla bobwhite. Tableland of southern Puebla (3,000 to 6,000 feet). “cs. Colinus pectoralis (Gould). Black-breasted bobwhite. Eastern base of Cordillera in Vera Cruz; BOBWHITE IN MEXICO 75 from Jalapa to Isthmus of Tehuantepec (500 to 5,000 feet). “6. Colinus godmani (Nelson). Godman’s bob- white. Lowlands of southern Vera Cruz; probably also ranging into Tabasco. (From sea-level to 1,500 feet. ) “7 Colinus coydlcos (Mull.). Coyolcos bobwhite. Pacific coast of Oaxaca and Chiapas; from city of Tehuantepec to Tonala. (From sea-level to 3,000 feet. ) “8. Colinus atriceps (Ogilvie-Grant). Black-headed “8. Colinus atriceps (Ogilvie-Grant). Black-headed bobwhite. Putla, western Oaxaca. (About 4,000 feet.) “9. Colinus salvini (Nelson). Salvin’s bobwhite. “9. Colinus salvini (Nelson). Salvin’s bobwhite. Coast plains of southern Chiapas, near Guatemalan bor- der. (Sea-level to 500 feet.) “to. Colinus insignis (Nelson). Guatemala bob- white. Valley of Comitan in Chiapas, into adjacent border of western Guatemala (3,000 to 6,000 feet). “T1. Colinus nigrogularis (Gould). Yucatan bob- white. Yucatan. (Sea-level to 500 feet.”’) “T1. Colinus nigrogularis (Gould). Yucatan bob- white. Yucatan. (Sea-level to 500 feet.”’) Very different in appearance from the bobwhite group are the striking quails of the western and south- western portions of the United States. Of these birds there are almost a dozen forms, some of them plumed, others helmeted, others with full, soft crests, but all very unlike the bobwhites. These are among the most beautiful of North American birds, and in many of the regions which they inhabit are extremely abundant. MOUNTAIN QUAIL. Oreortyx pictus. Oreortyx pictus plumiferus. Oreortyx pictus confinis. This is one of the largest of our quails. Its upper parts are brown or olive, or sometimes even bluish, the inner webs of the tertiary feathers being buff, so that when the wings are closed a distinct stripe is seen on each side of the rump. The breast and head are in part lead color; the long, backward directed crest of narrow feathers growing from the top of the head is black; the throat is chestnut, and from this a black stripe runs up to the eye. The chin, front of cheeks, lower portion of the lores, a line bordering the throat patch from the posterior angle of the eye and forehead, are whitish. The effect of this is to surround the bill with a border of whitish. The flanks are deep chestnut barred with black and white. The thighs are reddish and the under tail coverts black. In color, the female is very similar to the male, but usually has the crest plumes smaller. The length is from 11 to 12 inches, and the wing from 514 to 514 inches. This is the typical mountain quail, ranging in the California mountains from Santa Barbara, Cal., north to Washington. In Oregon its range seems to extend 76 76 MOUNTAIN QUAIL 77 out to the eastward, where it has been found near Mt. Hood. It was first found north of the Columbia River a good many years ago, and later was introduced near Seattle, at Vancouver Barracks, and also on Whidbey Island, in Puget Sound, where the birds have done well. The plumed partridge (Oreortyx pictus plumiferus) is a paler race, grayish olive above, but often with the neck more or less lead-colored, like the breast, the edges = ons: OS a asp eo Sta DP Gee Ss Cog (sees See. aatses So YS R MOUNTAIN QUAIL MOUNTAIN QUAIL MOUNTAIN QUAIL of the tertiary feathers whitish, and the forehead white. It is found on both sides of the Sierra Nevada, from eastern Oregon southward, and through California south to northern Lower California. The San Pedro quail (Oreortyx pictus confinis) is a still paler race, with a thicker bill, first found in the San Pedro Mountains of Lower California. According to Mr. Anthony, it ranges high up on both sides of the mountains, to the highest peaks, estimated at about 12,500 feet, and is not common below 2,500 feet. AMERICAN GAME BIRD SHOOTING 78 The mountain quail is perhaps the most beautiful American quail, though in such an attractive group it is hard to say that one is handsomer than another. The range of the species is limited to the mountains of the Pacific coast, from Washington south through Oregon and California, as stated. Captain Bendire quotes Prof. O. B. Johnson, of the Captain Bendire quotes Prof. O. B. Johnson, of the University of Washington, to the effect that its north- ern range has been extended by artificial means. He says: “Twenty years ago this species was found but little north of the Willamette Valley, Oregon, but they grad- ually worked down the south side of the Columbia River, toward Astoria, and in 1872 I was informed that some of these birds, shot at Kalama, Washington, were the first seen north of the Columbia. A crate of trapped birds sent to the Seattle market were, some time afterward, purchased by the Young Naturalists’ Society and set free. These have since multiplied nice- ly, and others have been sent to Whidbey Island, forty miles north of Seattle, where I understand they are also doing well. A covey wintered in a barn lot with the hens just at the outskirts of Seattle this winter.” Quite a number were also liberated near Vancouver Quite a number were also liberated near Vancouver Barracks, and did well. It is a moisture-loving species, and delights in a country where the rainfall is heavy. The paler race of this species is found in the drier re- gions of the Sierra and some of the desert ranges. This is quite an abundant species, found high up on the mountains in summer, and also low down toward Mountain Quail. Mountain Quail. MOUNTAIN QUAIL 79 the desert in winter. MOUNTAIN QUAIL It is said that in spring many fol- low up the snows as they melt, spending the summer high up on the mountains, and then in the autumn re- turn down the slopes, below the point where snow lies. The birds have a call not very unlike that of the bob- white, a whistle of three syllables, and the attitude as- sumed by the male while calling is not unlike that of the common quail, the wings being drooped and the point of the bill raised. The food consists of insects, seeds, berries, and the buds and tops of tender plants. In the early spring they feed so much on the tops of the wild garlic—one of the first green things to appear —that the flesh sometimes tastes of the plant. This quail is a prolific This quail is a prolific breeder, the eggs usually vary- ing in number from 9 to 15, though sometimes they are much more numerous, a nest containing 19 eggs being reported, as also a brood of 20 young birds. The time of nesting varies from early April to late May. The eggs are white. In northe In northern Lower California Mr. Anthony found that the San Pedro partridge does not differ greatly in habit from the other forms. The race is confined tc southern California and northern Lower California. The mountain quail is a bird only casu The mountain quail is a bird only casually pursued by the gunner. Often they are flushed and shot, and in some localities they are regularly pursued with dogs and shot over points. In such places they are reported to lie close, and to offer good opportunity for sport. The situations in which they are often found, rough mountain slopes, overgrown with manzanita, chemisa! The mountain quail is a bird only casually pursued by the gunner. Often they are flushed and shot, and in some localities they are regularly pursued with dogs and shot over points. In such places they are reported to lie close, and to offer good opportunity for sport. The situations in which they are often found, rough mountain slopes, overgrown with manzanita, chemisa! AMERICAN GAME BIRD SHOOTING 80 and other chaparral, will long protect them, and make the work of shooting them very difficult for the dog, and hardly less so for the gunner. MOUNTAIN QUAIL When the birds take wing they are swift fliers, and soon out of sight. It has been suggested that these birds would do well It has been suggested that these birds would do well in various localities in the southern Alleghany Moun- tains, where they might take the place of the native species, which seem to be growing scarcer. I do not know, however, that any serious effort has been made to acclimate them in such localities. Callipepla squamata. Callipepla squamata castanogastris. Callipepla squamata. Callipepla squamata castanogastris. In size, not far from the Virginia quail; with a short, thick crest, white at the tips. The rest of the head is brownish or grayish, growing paler on the throat. The back part of the neck, back and breast are bluish gray. A black border to each feather warrants the name “scaled.”” The wings are pale brown, and the flanks streaked with white. Other lower parts are buffy or yellowish, the belly sometimes with a patch of chestnut brown. Sexes alike. Length, g to 10 inches. Eggs white or buff dotted with brown. Inhabits northwest- ern Mexico, and the borders of the United States, from western Texas to southern Arizona. The chestnut-bellied scaled partridge has the wings The chestnut-bellied scaled partridge has the wings tipped with brown, tail bluish gray, the lower parts behind deeper buff, sometimes yellowish, and a large patch of rusty chestnut on the belly in the male and sometimes in the female. This bird is found in east- ern Mexico and in the lower Rio Grande Valley of Texas. In northern Mexico, along the southwestern border of the United States, from western Texas through southern New Mexico and Arizona, the scaled partridge is abundant. 81 82 AMERICAN GAME BIRD SHOOTING The birds seem to shun the timber, and to be most abundant on the high mesas, preferring the dry and barren country. Mr. Herbert Brown, however, speaks of having seen them immediately about water as well as far away from it. Captain Bendire, however, intimates that his experience leads him to think that the bird scarcely requires water. Mr. E. W. Nelson tells us that, while he has often found them far from water, they nevertheless make regular visits to the watering SCALED QUAIL SCALED QUAIL places. All observers agree that they are exceedingly shy and hard to approach, and that they are swift run- ners, dodging in and out among the bushes with the greatest ease, and soon out of sight. Even if flushed they fly but a short distance, when they alight and run again. Like many other gallinaceous birds, they greatly enjoy taking dust and sand baths, and at such times they act much like young chickens. In the eve- ning they retire to roost, to ridges and knolls, and the birds call to each other until the bevy has come to- Scaled Partridge. Scaled Partridge. SCALED PARTRIDGE; BLUE QUAIL 83 gether again. Sometimes the birds collect in consid- erable numbers, 60 or 80 having been seen together. The breeding season begins in March, and the young birds are usually hatched by the first of June, or earlier. There is some reason to think that two broods are reared. The number of eggs ranges from 9 to 16, and is commonly about 11 or 12. The eggs are sometimes dead white, or again pale buff in color, and are dotted with very small reddish brown spots scattered over the entire egg. The scaled partridge is not a bird pursued for sport. The scaled partridge is not a bird pursued for sport. It is true that many of them are killed by gunners, but merely for the food they afford, the birds being potted on the ground at every opportunity. Of all the quail, this is the most difficult to make lie to dog or man. In Arizona the army officers used to hunt them on horseback, following up the birds and shooting them whenever and however they could. The scaled quail, which is also sometimes called The scaled quail, which is also sometimes called white topknot quail, or cottonhead, does not differ in habits from its relative, the chestnut-bellied scaled partridge. Concerning the scaled partridge in southern Arizona, Mr. Herbert Brown, of Tucson, writes me: “The blue quail are less common than Gambel’s quail, “The blue quail are less common than Gambel’s quail, and do not, as a rule, live on the desert proper, but in- cline rather to the higher and rougher foothills. I saw a dog tried on only one, but am inclined to believe in their sprinting qualities. I have shot them in the foot- hills of the Tucson Mountains, west of here, and as far AMERICAN GAME BIRD SHOOTING 84. east as the Graham Mountains, where 1 found them quite plentiful. They are quick, active birds, and as pretty as pictures. 1 take it for granted that they are found much further east, but the eastern slope of the Grahams is as far as my personal acquaintance goes.” California Quail. California Quail. CALIFORNIA QUAIL; VALLEY QUAIL. CALIFORNIA QUAIL; VALLEY QUAIL. Lophorty« californica. Lophortyx« californica vallicola. Two forms of this species are found on the Pacific coast : a northern race, inhabiting the coast of Califor- nia north of Monterey, and which has been introduced in British Columbia, Washington and Oregon, and a paler race found in the interior valleys and foothills of California, south through Lower California to Cape St. Lucas. In the California quail the flanks are streaked with white on a brown or grayish ground. The feathers of the belly, in the male, are edged with black, and have a central patch of chestnut. The upper parts generally are smoky brown, the inner edges of the tertiary feath- ers buffy, the throat black, and the forehead whitish. The female has no black or white on the head, is plain grayish or brownish, lacks the chestnut belly patch, and has the scale-like markings of the under part less dis- tinct. The crest is much shorter than in the male. The length is 9% or Io inches, and the wing about 4%. This is the bird found in the coast valleys of Oregon and Washington and California. The valley quail (L. californica vallicola) is paler, The valley quail (L. californica vallicola) is paler, being grayish brown, the inner edges of the tertiary 85 85 86 AMERICAN GAME BIRD SHOOTING feathers whitish, and the flanks grayish brown. It is a bird of the interior districts of California and Oregon, south to Cape St. Lucas. The valley quail is an abundant species on the coasts of California, Oregon and Washington, and has been introduced on Vancouver Island and on a number of the islands of Puget Sound. This, according to Dr. Suckley, was done by Governor Charles H. Mason and Mr. Goldsborough, as.early as the year 1857. They {(iijpRwwMBbSes. daira, cy \, soe = »s SSO LT CALIFORNIA QUAIL CALIFORNIA QUAIL have long been common on Whidbey Island and on Vancouver Island, where they make, according to my experience, far better shooting than they do in the dry country of California. Of the two races, the California quail belongs espe- Of the two races, the California quail belongs espe- cially to the coast, and the paler grayer form is found in the drier interior, from western and southern Ore- gon south, through western Nevada and interior Cali- fornia, to, Cape St. CALIFORNIA QUAIL; VALLEY QUAIL. Lucas: owen 'Caltiornta:’) Whis species is still abundant, and many broods are found on some California ranches. In some places they CALIFORNIA QUAIL; VALLEY QUAIL 87 are not popular, because believed to eat the grapes. Others more justly believe that the number of noxious insects devoured by the valley quail far exceeds in importance the small amount of fruit which they eat. In many places the too long open season and unrea- In many places the too long open season and unrea- sonable bag limit have sadly depleted their numbers, and unless complete protection or restocking is re- sorted to this beautiful bird will become locally rare. The habits of this species differ very much in differ- The habits of this species differ very much in differ- ent localities. They are tough, hardy birds, and some- times surprise the ornithologists by turning up in places where they would not at all be expected. On the other hand, they sometimes suffer severely from cold and snow. Mr. A. C. Lowell, writing from Ft. Bidwell, in northeastern California, reported to Captain Bendire: “These birds are unable to stand the severe cold of this region, especially when accompanied by a heavy fall of snow. In the winter of 1887-88 about two feet of snow fell, followed by three very severe nights in which the thermometer reached 28° below zero. This killed most of the birds. In the following fall I heard of but three or four coveys of quail within a radius of sixty miles, where thousands had been the year before. They ranged from the northern end of Warner Valley south to Reno, Nevada, and were especially numerous in Buffalo Canyon and along the western shore of Pyramid Lake. They were very common up to the summits of the Warner Mountains, which attain here an altitude of about 6,000 feet. Though the canyons Mr. A. C. Lowell, writing from Ft. Bidwell, in northeastern California, reported to Captain Bendire: “These birds are unable to stand the severe cold of this region, especially when accompanied by a heavy fall of snow. In the winter of 1887-88 about two feet of snow fell, followed by three very severe nights in which the thermometer reached 28° below zero. This killed most of the birds. CALIFORNIA QUAIL; VALLEY QUAIL. In the following fall I heard of but three or four coveys of quail within a radius of sixty miles, where thousands had been the year before. They ranged from the northern end of Warner Valley south to Reno, Nevada, and were especially numerous in Buffalo Canyon and along the western shore of Pyramid Lake. They were very common up to the summits of the Warner Mountains, which attain here an altitude of about 6,000 feet. Though the canyons 88 AMERICAN GAME BIRD SHOOTING and water courses along those slopes were their favorite resorts, I have never seen nor heard of a covey of these quail down in the cultivated fields of the valleys. Here, at least, they prefer to live exclusively on the brushcovered hillsides.” On the other hand, we have known of cases in Cali- fornia where a brood of quail came regularly every evening to drink from the fountain immediately in front of a ranch house. In certain parts of southern California the quail has found the orange trees safe roosting places, and in the evening comes down from the brush-covered hills to the orchards. In Lower California, Mr. A. W. Anthony found the valley partridge very common in the mountains up to an altitude of about 9,000 feet. He adds: “Both in southern and Lower California I was told by the Indians and native Mexicans that during very dry seasons the valley quail do not nest, but remain in flocks during the entire summer. This statement I was able to verify by personal observation during the summer of 1887. ‘These birds were seen by me in large flocks throughout the spring and sum- mer months, and only two or three broods of young were noticed. Birds taken during April, May and June showed but little development of the ovaries. Should the winter rains, however, be sufficient to in- sure an abundance of seeds and grasses, the coveys begin to break up early in March, and from every hill in the land the loud challenge of the male is heard. The call notes of this sub-species are quite varied, fre- . Nest of California Quail . Nest of California Quail CALIFORNIA QUAIL; VALLEY QUAIL 89 quently the same bird changing his call six or seven times within half an hour.” Mr. CALIFORNIA QUAIL; VALLEY QUAIL. William Proud wrote Captain Bendire from Butte County, California, concerning this species, as follows: “Hundreds of these birds roost every night in the shrubbery around my house. Some of them are very tame, feeding among the chickens and coming on the verandah. They appear to know that they are pro- tected. They mostly roost in thick brush, and on the ground when the brush is not at hand. In early sea- sons they begin to pair in the last week of February, but the time varies somewhat according to the season. During this period there is considerable fighting among the males for the favor of the coveted female. This is kept up until they are suitably mated and the nest- “ing season arrives. This usually begins here about the last week in March, when the pairs scatter among the shrubbery along the banks and creeks and in adja- cent ravines, along hedgerows and brush fences, and on the borders of cultivated fields. The earliest nest I ever found was on March 15, and on April 15 I met young birds probably a couple of days old. I consider 14 eggs to be about the average number laid by these birds, and have found as many as 24 in a nest. The large sets I attribute to other hens laying in the nest, probably young birds which have failed to make preparation for their own eggs. On May 21 my dog pointed a valley partridge on her nest, which contained 22 eggs, and every one hatched. AMERICAN GAME BIRD SHOOTING go “During incubation the male is very attentive and watchful, usually taking an elevated position near the nest, where, with crest erect and tail spread, he bids defiance to all intruders, uttering an oft repeated whew-whew-whew. When the breeding hen leaves the nest to feed, should he be absent from the post of duty, her cry of tobacco-tobacco, very plainly given, brings him wp at.once. ... “As soon as the young are hatched they immediately leave the nest, keeping under cover as much as possi- ble. Should the brood be disturbed, the old birds will run and flutter along the ground to draw the attention of the dog, or whatever may have frightened them, to themselves and away from the young. In about ten days these can fly a short distance. CALIFORNIA QUAIL; VALLEY QUAIL. The valley par- tridge feeds on insects and the young and tender leaves of clover and green peas; later, on grain and various small seeds; in the fall they eat wild grapes, and are also very partial to the seeds of the amaranth, alsc those of Mentzclia levicaulis. Here only one brood is raised in a season, and incubation, as nearly as I can ascertain, lasts about twenty-eight days.” The eggs of this species range from 12 to 16, nests The eggs of this species range from 12 to 16, nests occasionally, as already stated, containing many more. The eggs are cream-white in color, spotted and blotched with different shades of dark and light brown and drab scattered over the whole egg. Lophortyx gambeli. Lophortyx gambeli. This is a richly-colored bird, and has the flanks bright chestnut streaked with white. There are no scalelike markings on the belly, and the central patch is black, the forehead dark, and the back of the head red- dish. The upper parts are much as in L. californica. The female resembles that of L. californica vallicola, but has the flanks chestnut and the belly without any trace of scalelike markings. It is, perhaps, a little larger than the California partridge. Its range is north- western Mexico and the neighboring portions of the United States, from southern California and Arizona to western Texas, and north as far as southern Utah. Besides this, other species of this genus are found Besides this, other species of this genus are found in Mexico, and in one or two of these the sexes are markedly unlike. Gambel’s quail is a dweller in the desert country of the south, and ranges from eastern southern Califor- nia through Arizona, and much of New Mexico, into western Texas. It is also found in southern Nevada and portions of Utah, and south into western Mexico. With a general similarity to the valley quail, it has With a general similarity to the valley quail, it has gI gI AMERICAN GAME BIRD SHOOTING 9Q2 habits not unlike that species, except so far as these habits are modified by its different surroundings. Like many gallinaceous birds, Gambel’s quail is very social in habit, and at the proper season they get to- gether in great flocks, and when alarmed and driven to wing may get up all about one, only to disappear almost at once among the thick cover or in the distance. Dr. Coues, in his article on this species published in Dr. Coues, in his article on this species published in “The Birds of the Northwest,” designates the valleys of the Gila and Colorado as its centers of abundance. This article is well worth quoting in part, as painting charming pictures of a region little known to most sportsmen, but one of extreme interest. He says: “An interesting fact in the distribution He says: “An interesting fact in the distribution of this species is the effect of the Colorado desert in shutting it off from the fertile portions of California. This dreary, sterile waste offers a barrier to its west- ward extension that is only exceptionally overcome. Lophortyx gambeli. Although the birds enter the desert a little way, they rarely reach far enough to mix with the representative species of California (L. californicus). The strip of country that mostly assists in their occasional passage westward is along the Mojave River, a stream rising in the San Bernardino Mountains, and flowing eastward toward the Colorado, from which it is shut off by a range of hills, and consequently sinks in the desert at Soda Lake. Among other birds, the two kinds of plumed quail—Gambel’s and the California—meet along this comparatively fertile thoroughfare upon neu- tral ground, as Drs. Heermann and Cooper, as well as GAMBEL’S QUAIL 93 myself, have witnessed. Much further south Colonel McCall found birds at Alamo Mucho, forty-four miles west of the Colorado; but still the desert is in effect the barrier I have represented, and the two quails, speaking generally, do not meet. One wonders the less at this who has any good idea of the Colorado desert, such as may be gained, for example, from the following passage from Colonel McCall’s article, which remains associated in my mind with the plumed quails, with all the freshness of first impressions. Speaking of the Alamo, where he shot a pair, “Here is in truth a desert!’ exclaims the colonel. ‘Figure to yourself, if you can, a portion of this fair earth where for some hundreds of miles the whole crust seems to have been reduced to ashes by the action of internal fires; behold a vast plain of desolation, surrounded, and at intervals intersected, by abrupt mountain ranges which are little better than gigantic heaps of scoria. Imagine this scen- ery to be actually glowing under the direct rays of the midsummer sun, and you may have some idea of the prospect that meets the eye of the traveler who looks out upon the desert from the well of the Alamo. You may perceive in his rear a few stunted cottonwood trees, scattered along the edge of a channel in which appar- ently water once was, but now is not; while around him, here and there, is a light-leaved mesquite that stretches forth its slender arms and appears to invite him to a shade that is but a mockery. Lophortyx gambeli. Here it was that I first heard the plaintive voice of this bird as he strove to cheer his mate while occupied in the tedious task AMERICAN GAME BIRD SHOOTING 94 of indubation.’ And singularly enough, the bird is al- most equally hemmed in by desert to the eastward as well; for according to the same writer’s accounts, ‘A sandy desert between the Pecos and Devil’s River is the barrier beyond which the species under considera- tion has not extended its range.’ “Such a glimpse of the haunts of the plumed quail “Such a glimpse of the haunts of the plumed quail makes one wonder how it ever became a game bird at all; how sportsmen could be able to make game of it without being themselves rather made game of. But great as are the natural disadvantages of the surround- ings, the bird’s attractions are still greater, and partly so from this very fact ; but Arizona is a large place, and one need not always endure desert in his quail-shoot- ing. There is a much brighter side to the picture. We have just seen only the darkest possible. Perhaps no territory rivals Arizona in variety of climate, physical geography and natural productions. Between rugged mountains that lift snow-capped peaks among the clouds, lie hidden pleasant green valleys whose fresh verdure contrasts with surrounding desolation. Vast primeval forests stretch for leagues, or are only inter- rupted by oak and cedar openings. Bright fringes of cottonwoods and willows mark the devious courses of streams, where walnut and cherry are scattered, and grape vines cling to them and roses bloom beneath leafy boughs. Here is plenty, at least, if not peace. Noth- ing mars the pleasures of the chase but the chances of being chased. Were it not for Indians, we should have here the acme of quail-shooting. GAMBEL’S QUAIL 95 “Gambel’s quail may be looked for in every kind of cover. Where they abound it is almost impossible to miss them, and coveys may often be seen on exposed sand heaps, along open roads, or in the cleared patches around settlers’ cabins. If they have any aversion, it is for thick, high pine woods without any undergrowth; there they only casually stray. Lophortyx gambeli. They are particularly fond of the low, tangled brush along creeks, the dense groves of young willows that grow in similar places, and the close-set chaparral of hillocks or mountain ravines. “T have often found them also among huge granitic boulders and masses of lava, where there was little or no vegetation except some straggling leaves, and have flushed them from the dryer knolls in the midst of a reedy swamp. Along the Gila and Colorado they live in such brakes as I have described in speaking of Abert’s finch, and they frequent the groves of mes- quite and mimosa that form so conspicuous a feature of the scenery in those places. These scrubby trees form dense interlacing copses, only to be penetrated with the utmost difficulty ; but beneath their spreading, scrawny branches are open, intersecting ways, along which the quail roams at will, enjoying the slight shade. In the most sterile regions they are apt to come together in numbers about the few water-holes or moist spots that may be found, and remain in the vicinity, so that they become almost as good indication of the presence of water as the doves themselves. A noteworthy fact in their history is their ability to bear, without apparent AMERICAN GAME BIRD SHOOTING 96 inconvenience, great extremes of temperature. They are seemingly at ease among the burning sands of the desert, where for months the thermometer daily marks a hundred, and may reach a hundred and forty, ‘in the best shade that could be procured,’ as Colonel McCall says; and they are equally at home, the year round, among the mountains, where snow lies on the ground in winter. “The quail’s food is made up of various substances. Like the rest of its tribe, it is chiefly granivorous, eat- ing seeds of every description; but fruits and insects form a large portion of its fare. It devours insects of such sorts as it can capture, and particularly those kinds that infest plants. In the fall it gathers cherries and grapes, and other ‘fruits,’ properly speaking, as well as the various berries not usually so called. It visits patches of the prickly pear (Opuntia), to feed upon the soft, juicy tunas, that are eaten by everything in Arizona, from men and bears to beetles. Lophortyx gambeli. In the spring it shows fondness for the buds of different plants, particularly mesquite and willow; birds shot at this time are fre- quently found with sticky bits of the buds about their bills. But though they thus feed so extensively upon this substance containing salicine, I never noticed that the flesh acquired a bitter taste. There is, as yet, little cultivated grain in Arizona, but doubtless some future historian will have to add our cereals to the bird’s list, and speak of Gambel’s quail as frequenting old corn and wheat-fields and the neighborhood of hayricks, where a large share of its food is to be gleaned. Like GAMBEL’S QUAIL 97 other Galline, it swallows quantities of sand and gravel to facilitate, it is supposed, the trituration in the gizzard of the harder kinds of food. “T believe that the quail moults at least twice a year, “T believe that the quail moults at least twice a year, but the spring change is apparently less complete, and certainly more gradual, than that of the fall, the birds seeming rather to furbish up a part of their plumage than to furnish themselves with entirely new attire. By the latter part of summer (at Fort Whipple) the plu- mage is faded and worn with incubation and the care of the young, and the renewal begins as soon as the latest brood is reared. The process is a long one, and the birds are rarely found at any season in such poor condition as to be unfit for preservation, nor are they ever deprived of flight. No crest is occasionally found for a short time in early autumn, but new feathers gen- erally sprout before all the old ones are dropped. I think they are shed from behind forward, so that the front ones are lost the last. The fully developed crest is a striking and beautiful ornament, hardly to be sur- passed in stylishness and jaunty effect. It averages an inch and a half in length, and sometimes reaches two inches in the most vigorous males; in the female it is rarely over an inch. The male’s is a glossy jet black; the female’s has a brownish cast. The number of feath- ers composing it is variable; five or six is usual, but there may be ten. Lophortyx gambeli. They all spring from a single point on the top of the head—just behind the transverse white line that crosses the crown from eye to eye. The feath- ers are club-shaped, enlarged at the tip and curling over AMERICAN GAME BIRD SHOOTING 98 forward, together forming a helmet-shaped bundle. The webs are loose; they bend backward from the shaft so that this forms the front border of the feather. Each feather is thus folded or imbricated over the next suc- ceeding, and the whole are packed into a single fascicle in this manner. The crest is freely movable, and its motions are subject to voluntary control. It is usually carried erect, but sometimes drops forward, or oblique- ly, over one eye, and occasionally is allowed to hang backward, though it cannot be made to lie close over the occiput. The crest sprouts when the chicks are only a few days old, about the time that the first true feathers appear upon the wings and tail... . “All quail are Precoces, as already explained, and “All quail are Precoces, as already explained, and the chicks of this species are certainly precocious little things, if we may judge by their actions when they are disturbed. They run about as soon as they are hatched, though probably not ‘with half shell on their backs,’ as some one has said. In a few days they become very nimble, and so expert in hiding that it is difficult either to see or catch them. When the mother bird is sur- prised with her young brood, she gives a sharp warning cry that is well understood to mean danger, and then generally flies a little distance to some concealed spot, where she crouches, anxiously watching. The fledg- lings, by an instinct that seems strange when we con- sider how short a time they have had any ideas at all, instantly scatter in all directions, and squat to hide as soon as they think they have found a safe place, remain- ing motionless until the reassuring notes of the mother GAMBEL’S QUAIL 99 call them together again with an intimation that the alarm is over. Then they huddle close around her, and she carefully leads them off to some other spot where she looks for greater security in the enjoyment of her hopes and pleasing cares. Lophortyx gambeli. As long as they require the parents’ attention they keep close together, and are averse to flying. Even after becoming able to use their wings well they prefer to run and hide, or squat where they may be, when alarmed. If then forced up, the young covey flies off, without spreading, to a little dis- tance, often realighting on the lower limbs of trees or in bushes, rather than on the ground. As they grow older and stronger of wing they fly further, separate more readily, and more rarely take to trees; and some time before they are full grown they are found to have already become wary and difficult of approach. As one draws near where a covey is feeding, a quick, sharp cry from the bird who first notices the approach, alarms the whole, and is quickly repeated by the rest as they start to run, betraying their course by the rustling of dry leaves. Let him step nearer, and they rise with a whirr, scattering in every direction. “Newly hatched birds may be found all summer, and “Newly hatched birds may be found all summer, and incubation goes on from, say, early in May until the middle of August. Not that any single pair are en- gaged so long, but that different broods may be hatched during all this time. A greater number of old birds pair in April, and hatch their first brood some time during the following month. Most of them doubtless raise another. Others appear to defer incubation for AMERICAN GAME BIRD SHOOTING 100 a month or two, and have but one brood. The first chicks that I saw in the summer of 1865 were hatched in May, and I found others the same year only a few days old in August; but by this month almost all the birds of the year were well grown, and by September were in condition to offer legitimate sport. In Octo- ber a few are found not yet ready to be shot; but the great majority are as large as the parents, and nearly as strong of wing. Lophortyx gambeli. The season may, therefore, be said to begin in October and continue into March; but birds should not be pursued later than the middle of this month, for although few birds, if any, actually mate before April, it is cruel, as well as injudicious, to dis- turb them while they are preparing to do so. The be- ginning of the pairing season may be known to be at hand when certain peculiar cries, different from any usually emitted during the fall and winter, are heard. “These notes are a sign that the coveys are breaking up and mating is about to commence. They are analo- gous to the bobwhite of the eastern quail (Ortyx vir- ginianus), and are uttered, as with that species, more particularly in the breeding season. The note is a loud, energetic, two-syllable whistle, delivered in a clear, ringing tone. It is difficult to write down intelligently, but, once heard, is not likely to be afterward mistaken, except for one of the cries of the black-headed gros- beak. It sounds to my ears something like the forcible pronunciation of the syllables killink, killink, indefinite- ly repeated, sometimes in a rising and sometimes with a falling intonation. The old cocks, if they can be “AVAING JeosopoIg “SG *AQ 9Y} Jo UOTsstuIOd Ag ‘Tend sjequreg “SG *AQ 9Y} Jo UOTsstuIOd ‘Te sjequreg GAMBEL’S QUAIL IOI seen so engaged, are found strutting along some fallen log or gesticulating from the top of some broad rock or stepping with dignified air along the pathway under the bushes ; sometimes even perched in a tree or bush, without other motion than the heaving of the chest; but wherever they may be, they have one mind in com- mon, their only thought to secure the admiration and then the favor of birds more modest if not more fair. “A beautiful sight it is to see the enamored birds pressing suit with all the pomp and circumstance of their brilliant courtships—the firm and stately tread, with body erect, of comely shape, displayed to best ad- vantage ; the quivering wings, the motion of the plumes, that wave like the standard of knights errant, the flash- ing eyes—bespeak proud consciousness of masculine vigor. The beautiful bird glances defiance, and chal- lenges loudly, eager for a rival; but none disputes, and he may retire, his rights proven. Lophortyx gambeli. Only a gentler bird is near, hidden in a leafy bower, whence she watches, admiring his bearing, fascinated by the courage she sees displayed, hoping every moment that the next will bring him, dreading lest it may. As their eyes meet she trembles, and would turn to fly, but cannot; his glow again, but with a different light, even more in- tense than before. With an exultant cry he flies straight to her, and, like the true knight he is, for just one instant bends till his breast touches the ground, and is then erect again. He leans toward her, half spreads his wing over her, and pleads in an undertone. She AMERICAN GAME BIRD SHOOTING 102 listens, but draws away; she listens, but only stands irresolute; she listens, and, listening, yields. “They must prepare for new duties. With deep “They must prepare for new duties. With deep sense of responsibility and earnest solicitude the pair now cast about for a suitable spot for their home. They search through the tall, rank herbage alongside the stream, through the willow copses, among fallen moss- covered logs that are scattered around the glen, and at length make up their minds. Little more is needed than to fix upon the spot, for the nest is a simple affair, the work of a few hours, perhaps, scratching a suitable depression and lining it with a few dried grasses pressed together. Day by day eggs are laid, till a dozen or more fill the nest. They cannot be distinguished from those of the California quail. They measure an inch and a quarter in length by an inch in breadth, and are almost pyramidal in shape, the larger end flat- tish and very broad, the other narrow and pointed. The color is a buff, or rich cream, dotted and spotted all over with bright brown, and splashed here and there with large blotches of the same. When the fe- male is not pressed to lay, the pair ramble about to- gether in close company until the complement is fin- ished. Then she gives up all recreation, grown already quite sober and maternal, and resolutely sets about her long tour of duty. But she is not forgotten because she can no longer share the idle pleasures of her lord. Lophortyx gambeli. Mounted on a stump or bush near by, he stands watch, and continually solaces her with the best music he can make. It is not very harmonious, to be sure; in fact, GAMBEL’S QUAIL 103 his ditty at such times is a medley of odd notes, sound- ing rather lugubrious than hilarious, but it is presum- ably satisfactory to the one most concerned. So the long days pass, for two weeks or more, till feeble cries come from the nest; the mother dries and cuddles the curious little things, and the delighted birds, brimful of joy, lead their family off in search.of food. “From the number of eggs s “From the number of eggs sometimes found in a nest it becomes a question whether birds, hard pressed, may not occasionally deposit in nests not their own. We have no positive evidence that it may occur, but observation has rendered it highly probable, and such is the case with some other birds, as the rails andi, I think, the Virginia quail. However this may be, it is pretty certain that broods of young sometimes coalesce at a varying time after hatching. I do not remember to have myself seen a covey of more than twenty, but it is currently reported upon good authority that troops numbering as many as fifty partly grown birds, and including several old ones, may be met with. This raises, of course, the question of polygamy, so common in birds of this order, and something may be said in favor of the view. The same surmise has been made in the case of L. californicus, but I believe it remains to be proven. I am bound to observe that I have never witnessed anything supporting this view. . . . “Man is, I suppose, the quail’s w “Man is, I suppose, the quail’s worst enemy; what the White does with dog and gun the Red accomplishes with ingenious snares. The Indians take great num- bers alive in this way, for food or to trade with the AMERICAN GAME BIRD SHOOTING 104. whites along the Colorado, and they use the crests for a variety of purposes that they consider ornamental. I saw a squaw once who had at least a hundred of them strung on a piece of rope-yarn for a necklace. Lophortyx gambeli. But the birds have other foes : the larger hawks prey upon them, so also do the wolves, as I had good evidence upon one occasion, when hunting in a precipitous, rocky place near Fort Whipple. I heard a covey whispering about me as they started to run off in the weeds, and fol- lowed them up to get a shot. They passed around a huge boulder that projected from the hillside, and then, to my surprise, suddenly scattered on wing in every direction, some flying almost in my face. At the same instant a wolf leaped up from the grass where he had been hiding, a few feet off, intending to waylay the covey, and looking very much disappointed, not to say disgusted, at the sudden flight. We had marked the covey together, and were hunting it up from opposite sides, and neither of us could account for their flushing so unexpectedly. Then he caught sight of me, and it was a question which of us was most surprised. How- ever, I felt that I owed him a private grudge for get- ting in the way of the birds and spoiling my shot, so I fired both barrels in quick succession. With nothing but mustard seed in my gun, I hardly expected to more than frighten the beast, but he was so near that he rolled over quite handsomely, his hindquarters para- lyzed with the charge, which took effect in the small of the back. I kept his skin as a trophy, and since that time have had unlimited confidence in small shot.” GAMBEL’S QUAIL 105 This account was written, I believe, as long ago as 1872, or 1873, but it remains the best description of Gambel’s partridge that has been writtten. The birds appear t The birds appear to be as numerous in their range as ever they were, and with the increase of water which goes with the progress of irrigation in Arizona and New Mexico they are likely to increase still further. Captain Bendire cites an obse Captain Bendire cites an observation of Mr. Herbert Brown, of Tucson, Arizona, which shows that the Gila monster (fZeloderma) eats the eggs of this species. MASSENA QUAIL. Cyrtonyx montezume mearnst. Cyrtonyx montezume mearnst. This handsome and highly-colored bird is of the southwest, and until very recently has never been pur- sued for sport, though often killed for meat. In ap- pearance it is unlike any of our United States par- tridges, having large feet, a full, soft crest, and being more or less dotted below with large round spots of pure white. The sides of the head are marked with black and white stripes; the chin, throat, and a band across the fore neck, are all black ; the crest is brownish; the upper parts are brown, barred and spotted with black and streaked with white, buff or reddish. The wing quills are spotted with white on their outer webs, while the thighs, belly and under tail coverts are black. The flanks and sides are lead-color, marked with many round spots of pure white, and the middle line is chest- nut. The female is paler everywhere, but has the upper parts streaked and barred as in the male. The head lacks the distinctive white and black stripes, and the sides are slightly barred with black. Length, 8% to g inches; wing, about 5, or a little more. This quail is found in western Texas, New Mexico, Arizona, and well down into western and central Mexico. The typi- 106 106 106 MASSENA QUAIL 107 cal montezume and other species occur in Mexico. One found in Guatemala and southern Mexico has the flanks chestnut instead of lead-color. This bird was described as long ago as 1830, yet This bird was described as long ago as 1830, yet for many years thereafter little or nothing was known about it, and it was only in 1890 that the eggs were found and described. MEARNS, OR MASSENA QUAIL MEARNS, OR MASSENA QUAIL The Massena quail, or, as it is now called, the Mon- tezuma quail, is notable for its odd and beautiful col- oring and for its extraordinary simplicity and lack of suspicion, a quality which is reflected in the name “fool quail,” given it in some sections. This title, commonly enough applied to certain unsuspicious grouse and quail, is an unintentional testimonial by man to his own bloodthirstiness, declaring in effect that any bird or ani- mal must be a fool that does not know that civilized AMERICAN GAME BIRD SHOOTING 108 man is a natural butcher. Cyrtonyx montezume mearnst. It takes wild things some time to realize that there are no gentle men, and obvi- ously this lesson is learned more slowly by birds or animals living in places unfrequented by man than in regions where he is more numerous. Even the ruffed grouse, which, in thickly settled regions, is the wisest, wildest and most wary of birds, is in some localities so unafraid that the passer-by who will tie a noose of cord on the end of a six-foot pole, may pass the loop over the bird’s head and drag it from its perch. The Mearns quail is reported to be less graceful in The Mearns quail is reported to be less graceful in carriage and less elegant in shape than some of its rela- tives which inhabit the same region, but may not this be only another way of stating that it is unsuspicious -and not easily alarmed? The wariest of gallinaceous birds, if ignorant of the presence of an enemy, carries its feathers more or less loosely, walks with a short neck, and has a rounded back, thus presenting an ap- pearance very different from the same bird when it is startled or alarmed and about to take to flight. Then the neck is stretched upward, the bird stands high on its legs, all its feathers are pressed close to its body, its crest is raised, and it stands there alert and pre- pared to dart away at a second’s warning. In Texas this bird is known as the black quail, or In Texas this bird is known as the black quail, or the black-bellied quail, while, as said, in Arizona, on account of its gentle nature it is called fool quail. It is said to frequent rocky ravines heading well up into the mountains, but of recent years has come to the ranches, and is found feeding in the green fields. Cap- Mearns’ Quail. Mearns’ Quail. MASSENA QUAIL 109 tain Bendire says that in Mexico he has several times seen them living contentedly in cages. Capt. W. L. Carpenter saw the Massena partridge in Capt. W. L. Carpenter saw the Massena partridge in the Rio Grande Valley, near Taos, Mexico, and on the headwaters of the Black and White rivers, where he believed it bred. Cyrtonyx montezume mearnst. In the breeding season, in Arizona, they are said to frequent live-oak scrub and patches of rank grass, at an altitude of seven to nine thousand feet. “Here,” according to Mr. John Swinburne, writ- ing to Captain Bendire, “they are summer residents, only descending to much lower altitudes in winter. They lie very close at all times, allowing one to almost step on them before they move. I have seen this species on the White Mountains during the breeding season, and saw young birds of the year shot there. Even the adults seemed very stupid when suddenly flushed, and after flying short distances would alight and at- tempt to hide in most conspicuous places. I have seen men follow and kill them by throwing stones.” Captain Bendire also quotes Mr. G. W. Todd, as follows: “T first met with the Massena partridge in Bandera County, Texas, in 1883, where they are very scarce, and I learned but little of its habits for a long time. They are very simple and unsuspicious, and apparently live so much in barren and waste places that they do not see enough of man to make them afraid. On see- ing a person they generally squat at once, or run a little way and hide. They will hardly fly until one is almost on them, but when they finally do fly they go much AMERICAN GAME BIRD SHOOTING T10 further than either the Texan bobwhite or the scaled partridge, and on alighting they run rapidly for a lit- tle distance and then squat again, generally flushing easier the second time. It is rare to see more than six together; two or three are more often met with. In the fall of 1886 I found a covey of five, on a wet and misty day, and killed three of them with a Win- chester rifle before the remaining two flew. I never found their eggs nor met with small young until this year. I saw but a single bird this season, and this seemed to be entirely alone. They are not very abun- dant here, and are always found in the most barren places, among rocks and wastes where even prickly pear is stunted and no bush grows over three feet high. Cyrtonyx montezume mearnst. When scared they utter a kind of whistling sound, a curious combination between a chuckle and a whistle, and while flying they make a noise a good deal like a prairie hen, though softer and less loud, like chuc-chuc-chuc, rapidly repeated. “The only nest of this species I have ever seen was situated under the edge of a big bunch of a coarse spe- cies of grass, known as ‘hickory grass.’ This grass grows out from the center, and hangs over on all sides until the blades touch the ground. It is a round, hard- stemmed grass, and only grows on the most sterile soil. According to my observations, the Massena partridge is seldom seen in other localities than where this grass grows. I was riding at a walk up the slope of a bar- ren hill, when my horse almost stepped on a nest, touch- ing just the rim of it. The bird gave a startled flut- MASSENA QUAIL II! ter, alighting again within three feet of the nest, and not over six feet from me; then she walked away with her crest slightly erected, utering a low chuckling whistle, until lost to view behind a Spanish bayonet plant (Yucca) about thirty feet off. 1 was riding a rather unruly horse, and had to return about thirty yards, to tie him to a yucca, before I should examine the nest. This was placed in a slight depression, pos- sibly dug out by some animal, the top of the nest being on a level with the earth around it. It was well lined with fine stalks of wire-grass almost exclusively, the cavity being about five inches in diameter and two inches deep. At the back, next to the grass, it was slightly arched over, and the overhanging blades of grass hid it entirely from sight. The nest was more carefully made than the average bobwhite’s nest, and very nicely concealed.” The food of the Massena quail, in western Texas and New Mexico, appears to consist of small bulbs, to se- cure which the birds often dig or scratch out holes to the depth of two inches. In the mountains they also eat acorns, mountain laurel, tubers of nut grass, cedar and other berries. Cyrtonyx montezume mearnst. The nest is likely to be built on a hillside, sometimes The nest is likely to be built on a hillside, sometimes well hidden, and often sunk quite deeply in the ground. The eggs seem to run from 8 to 10 in number. They are pure white in color, and, while often glossy, are sometimes more or less rough and granulated. As already stated, the Massena quail is scarcely pur- As already stated, the Massena quail is scarcely pur- AMERICAN GAME BIRD SHOOTING | 112 sued for sport, and on this point Mr. Herbert Brown, of Tucson, Arizona, writes me as follows: “The Massena quail, or, as it is now called, Monte- zuma quail, will lie well to a dog, but, practically speak- ing, they are not hunted as a game bird; in fact, not hunted at all. They are mountainous in their habits, and are not commonly killed. “This bird, among the Mexicans, is known as ‘suinea,’ and also as ‘chacalaca.’ The latter word is commonly applied to persons of garrulous or talkative dispositions. As the male bird, when strutting among the females, keeps up a continual clatter, the Mexican people, with their readiness for characterization, call them ‘chacalaca.’ “Tt is among the most peculiar of our game birds, and I much regret that it is so little known. Of all the quail it is the most easily domesticated. Some years ago a young female was sent to me from Sonora. As I then had no means of keeping it, I gave it to a friend. It was raised about the house, and was as tame as a pet chicken. A swinging door led into the kitchen, and it was always careful to await its opportunity, and make its run through as the door swung open. Some years ago I heard of a small bunch on the Sonoita that fed regularly with the chickens at the ranch house. At a ranch house on the Sopre two adult birds took up with the chickens, and in the spring following raised a brood of little ones, some four or five of which grew to ma- turity. Eventually they fell into disfavor and were driven away or killed. This was due to the pugnacity MASSENA QUAIL 113 of the male bird. They appeared to have no use for the chickens, and were continually fighting them with- out provocation. of the male bird. They appeared to have no use for the chickens, and were continually fighting them with- out provocation. The big ones could stand it, but the little ones were the sufferers,” Cyrtonyx montezume mearnst. The big ones could stand it, but the little ones were the sufferers,” Tetraonide. The differences between the American grouse and quail have been pointed out in two earlier chapters, but, nevertheless, may be repeated here. The feet of all the grouse are covered with hair-like feathers. In this group a tuft of small feathers runs forward from the forehead along each side of the bill and covers the nostrils, while in the quails the nostril is naked and is protected by an overhanging scale. All the grouse have on head over the eyes narrow patches of naked skin, and most or all of them have on the sides of the neck bare patches, which are often inflatable. The pectinations on the toes of the grouse are shed -annually. Mr. Manly Hardy of Brewer, Me., informs me that in the ruffed grouse the whole scaly envelope of the foot is shed each year. The grouse are usually large birds, while the quail The grouse are usually large birds, while the quail are small, and in the grouse the tail is usually long, while in all the quail it is short. The outline drawings, showing the bill, foot, wing The outline drawings, showing the bill, foot, wing and tail of the various grouse and quail will assist those who desire to learn these characters. 114 114 114 DUSKY GROUSE. Dendragapus obscurus. Dendragapus obscurus fuliginosus. Dendragapus obscurus richardsom. Dendragapus obscurus sierre. The dusky grouse is the largest of the American wood grouse, sometimes weighing four pounds. Above, the male is dusky gray or dull black, usually more or less waved with fine, blacker lines. Sometimes there is a variety of light and dark color on the back and wings, producing a mottled effect. The tail is black with a gray band across the end, usually from half an inch to an inch wide. The under parts are slaty gray, somewhat streaked with white on the flanks. The bird is from twenty to twenty-three inches long, and is stout and graceful. On the side of the neck there is a patch of white surrounding a naked, inflatable bare space which is hardly, or not at all, to be seen, except during the breeding season. The throat is also white or very pale. The much smaller female is grayish or brownish in The much smaller female is grayish or brownish in color, marked with spots and bars of blackish. This is the typical dusky or blue grouse, and is This is the typical dusky or blue grouse, and is found in the Rocky Mountains, west as far as they ex- tend, east to the Black Hills, south to the Mogollon 115 115 AMERICAN GAME BIRD SHOOTING 116 Mountains of western New Mexico and the White Mountains of middle eastern Arizona. The sub-species, known as the sooty grouse, is found on the northwest coast in the region of great precipitation, north to an ae 19159 aN =— Ee cal Aa TTT TL LS SG if ; Lx LAL Ll hy Ce OLILL LP Pr DUSKY GROUSE DUSKY GROUSE southern Alaska and southern Yukon. It is darker colored, with a narrower tail band, lacks the whitish space on the side of the neck and has the throat darker. Often or usually the scapulars or shoulder feathers are without distinct white streaks or spots. southern Alaska and southern Yukon. It is darker colored, with a narrower tail band, lacks the whitish space on the side of the neck and has the throat darker. Often or usually the scapulars or shoulder feathers are without distinct white streaks or spots. DUSKY GROUSE. DUSKY GROUSE 17 The adult female is much darker than the typical dusky grouse, and often has the upper parts distinctly reddish, instead of being merely gray or buffy. Richardson’s grouse, found in the northern Rocky Richardson’s grouse, found in the northern Rocky Mountains, has the tail without the terminal band of gray; otherwise it is much like the dusky grouse, but usually the tail feathers are broader and the tail more even. There are certain places in the Rocky Moun- tains—for example, in northwestern Montana—where the dusky grouse and Richardson’s grouse intergrade, and often it is impossible to say to which form a speci- men belongs. In this latter region the female is often gray, black mottled, with little or no tinge of buff. The sierra grouse, with a narrower tail band than The sierra grouse, with a narrower tail band than the typical form, is found in the region from Fort Klamath, Oregon, south, through the mountains of California, to Mount Pifios, near old Fort Tejon. The dusky grouse, although not at all known by east- The dusky grouse, although not at all known by east- ern sportsmen, is yet easily the finest of our American species. It is exceeded in size only by the sage grouse, but in beauty, in edible qualities and in the sport it furnishes it far excels that species. The dusky grouse is found in all the mountain re- The dusky grouse is found in all the mountain re- gions of the farther West, from Alaska south as far as the White Mountains of Arizona. Naturalists sepa- rate it into four forms, as given above; but in habits, and in the qualities which interest the gunner, all are much alike. It is not a migratory bird, but remains essentially in one locality throughout the year, al- though, like many mountain-dwelling birds and mam- AMERICAN GAME BIRD SHOOTING 118 mals, the altitude at which it lives changes with the changing season. One of the earliest spring sounds in the country where the dusky grouse are found is the hooting of the male, a booming or blowing sound analogous to the grating song of the sage grouse or the tooting of the pinnated grouse. The call possesses a certain ven- triloquial quality, which makes it exceedingly difficult to follow, and it is often hard to locate the bird that is uttering it. DUSKY GROUSE. A similar difficulty is usually found in attempting to follow up the drumming of the ruffed grouse. This call and the accompanying mating ac- tions were well described by a correspondent, “Stand- stead,” in Forest and Stream twenty years ago. He wrote: “While driving near the city. [ Victoria, B. C.] with the veteran shot, R. Maynard, we saw a pair of blue grouse quite near the trail, and the cock bird gave us a most entertaining exhibition of the charms that he displays in wooing his mate. Like a turkey cock he strutted about with his wings trailing on the ground, his tail feathers erect and spread out fart-like to their fullest extent, his neck distended, and on each side of his neck the feathers were turned out so as to resemble a pair of round white rosettes, nearly three inches in diameter, with an oblong red spot in their center, where the skin of the neck was exposed. His head seemed to be crowned with a fiery red comb. Excepting the rosettes, he was in appearance a miniature turkey gob- bler. Every few seconds he would strut up to his de- mure but sleek-looking mate, puff out his neck and *& e > i Be em a aL Sierra Dusky Grouse and young. From group in American Museum of Natural History. Sierra Dusky Grouse and young. From group in American Museum of Natural History. DUSKY GROUSE 119 with a jerky movement of his head utter his boom, or hoot, boom-boom-boom. As he grew more and more demonstrative in his actions, his modest mate flew up to an overhanging limb to escape his familiari- ties, and we drove away, leaving him still strutting on the ground underneath the tree where his mate sat perched.” Another writer refers to the sound uttered at this season by the male dusky grouse as “growling” or “groaning,” and notes, as have many others, the pe- culiarity that when near it often seems quite distant, and when distant it sometimes seems near, appearing to come from every direction but the true one. The female usually makes her nest in the open at the foot of the mountains, quite a little way from the timber, perhaps under some little clump of brush at the foot of a steep bluff, partly clad with pines, or perhaps among the aspens in some mountain valley. DUSKY GROUSE. The eggs vary in number from seven to fifteen, and perhaps are oftenest nearer the smaller number. They are buffy in color and are’usually more or less thickly spotted with fine dots of reddish brown or even choco- late. These dots and spots are usually quite distinct and seldom or never run into blotches and cloudings, as is usually the case with the eggs of the ptarmigan. After the nest is constructed and the eggs are laid, the male leaves his mate and by slow stages betakes himself to the mountain-tops, where, in midsummer, old cocks and barren hens are found in the extreme edge of the timber, and very often on the alpine mea- AMERICAN GAME BIRD SHOOTING 120 dows and amid the rocks above timber line. The female all through the summer, leads her brood of little ones about where insects, berries and seeds are most accessible. Like many other grouse, if her young are closely approached she feigns to be hurt, and runs off with dragging wings and seemingly feeble footsteps to lure the enemy to pursue her; but, after she has been followed for a little distance, takes wing and flies off up the mountain, to return on foot when the danger is past. By the middle of July the young are usually somewhat larger than quails, and at this time, instead of immediately hiding when approached, they take wing for a short flight, and, immediately on reach- ing the ground, hide, lying as close as any birds that I have ever seen. At such times, if the ground be fairly open, it is not difficult to see them as they crouch close to the earth, but as soon as they recognize that your eye has caught them, they spring into the air for another flight. I recall one morning when, traveling along a little valley, with a companion, we started several broods of well-grown young, of which we killed seventeen by shooting their heads off with our rifles. The birds were needed for food for the camp. As the young birds grow larger and more hardy, the mother leads them higher up the mountain, and by the end of August or first of September they are feeding among the green timber, at which time their principal food in some localities is a species of low- growing red huckleberry. DUSKY GROUSE. By September or October the birds are nearly as large as the parent, and long be- DUSKY GROUSE I21 fore this it is their practice when disturbed to fly up into the branches of the pines, where they stand erect with feathers pressed close to their bodies, each looking for all the world like a broken-off dead branch. Often in August or September broods of these birds may be found on the bare mountain-side, feeding on the huckleberries, or among low sarvis trees. In such a case splendid and very easy shooting may be had at these great birds, but if a flock be scattered in the tim- ber, or among thick alders, the shooting is quite as difficult as that of the eastern ruffed grouse. Although these birds can know little about the shotgun, they are often quite as acute as the partridge in putting the trunk of a tree or a clump of bushes between the gun- ner and themselves. On the other hand, when they take refuge in the branches of a tree, and stand there watching you, they will often permit half-a dozen shots to be fired at them without moving, unless hit. In Captain Bendire’s admirable account of the sooty In Captain Bendire’s admirable account of the sooty grouse, the northwestern form of D. obscurus, he quotes a letter from Captain (now General) T. E. Wilcox, in which the writer says: “I once caught a mother grouse with a fish hook. I had my rod on my shoulder and suddenly came upon a covey about the size of quails and caught one with my hands. This made the old bird frantic; she attacked me, and alight- ing on my rod, the hook pierced her foot. I was pulling her in when my leader broke and she flew off. Of course, I released her chicken. I killed a male in the Boise Mountains, December 2, 1879, which AMERICAN GAME BIRD SHOOTING r22 weighed three pounds ten ounces; but some killed by me in the Cascade Mountains seemed to be much larger. While on Lake Chelan in 1883, hunting white goats, I flushed a covey of grouse and here heard for the first time the call note of the female for her young. It was low, but distinct, something like that made by the bobwhite just before it flushes. DUSKY GROUSE. At this time, the last of August, the birds were well grown. I have always found these birds near water. In 1881, while going to Indian Valley, Idaho, I rode past some, one being near enough to touch with a switch I had in my hand, yet they all walked out of the trail as quietly as domestic fowls would have done and then resumed their dusting.” Richardson’s grouse, the form of the dusky grouse found in the northern Rocky Mountains from southern Montana, north far into British America, resem- bles in habits the other forms and is quite abundant. About the Continental Divide, specimens are some- times seen which show approach to obscurus, or to the sooty grouse. In many places, where seldom disturbed by man, they are exceedingly gentle and unsuspicious, and a brood will walk slowly along before one, feed- ing and calling to one another in a particularly inno- cent and attractive way. It is stated that the birds spend their winters largely It is stated that the birds spend their winters largely among the branches of the pine trees, and that they feed to a very great extent on the pine needles. Mrs. Florence Merriam Bailey says of the winter Mrs. Florence Merriam Bailey says of the winter habits of the dusky grouse in New Mexico: ‘asnoig Aysng ‘as Aysng DUSKY GROUSE 123 “Near our camp by the foot of Pecos Baldy, Mr. Bailey discovered a winter roosting tree of the grouse. The tree was on a sheltered part of the wooded slope and was so densely branched that after a prolonged rain the ground beneath was perfectly dry. The earth was strewn with winter droppings, composed entirely of the leaves of conifers. Conifer needles had also been eaten by three of the grouse that were taken... in July and August, but at this season the birds were living principally on such fresh food as strawberries, bearberries (Arctostaphylos uva-urst), sheperdia ber- ries, flowers of the lupine and paint brush, seeds, green leaves, grasshoppers, caterpillars, ants and other in- sects. DUSKY GROUSE. But one crop contained twenty-seven strawber- ries, twenty-eight bearberries and twelve sheperdia berries, besides flowers, leaves and insects, while the accompanying gizzard was filled with seeds, green leaves and insects.” In some sections of the West the dusky grouse are In some sections of the West the dusky grouse are reported to pack in the autumn, forming very large flocks, but I have never seen anything of the kind. Usually, in my experience, only the members of a single brood, ranging in numbers from eight to twenty, are found together. Sometimes, however, in summer, on the high peaks, flocks of a dozen old males are found associated together. It is said that in Colorado and parts of Montana, It is said that in Colorado and parts of Montana, where grain is grown adjacent to the mountains or the timber occupied by the dusky grouse, the birds are frequently found feeding on the stubble fields. If this AMERICAN GAME BIRD SHOOTING 124 is the fact they must give great shooting in such situations. | It is readily conceivable that this bird might be in- troduced into the mountains of the Eastern States— for example, in New England, Pennsylvania, Virginia and North Carolina, and in these sections, if for a time protected, might establish itself. In the West it is usually found at altitudes much higher than these, though, in summer, females and young broods of the dusky grouse inhabit essentially the same territory as the ruffed grouse. The dusky grouse has but three vernacular names The dusky grouse has but three vernacular names commonly in use—blue grouse, pine grouse and gray grouse. Dusky grouse and blue grouse are the two most common. ‘CANADA GROUSE, SPRUCE PARTRIDGE. Canachites canadensis. Canachites canadensis osgoodi. Canachites canadensis canace. Two small wood grouse, notable for their extreme simplicity and lack of fear of man, are found in the evergreen country, Canadian life zone, of the east, north and west. Of these the first is the Canada grouse, a small, blackish bird, variously marked below with spots of white and with the tail tipped with rusty red- dish. The male above is black, waved with dark gray, and beneath black; its throat bordered with white, many of the feathers tipped with white; the sides and the flanks marked by long white streaks. The female is barred with black and rusty, but has the sides and the scapulars or shoulder feathers streaked with white. The tips of the tail feathers are bright rusty. Length, 14% inches; wing about 7 inches and tail 5 inches. It is found in northern New England, New York, Michi- gan, Minnesota and through Canada to Alaska. The three forms of the Canada grouse are very The three forms of the Canada grouse are very closely related, and, from the sportsman’s viewpoint, hard to separate. True canadensis is restricted to the northern part of the range—eastern flanks of the Rocky Mountains west of Edmonton, Alberta, easterly to Labrador Peninsula, and Alaska from Bristol Bay 125 The three forms of the Canada grouse are very closely related, and, from the sportsman’s viewpoint, hard to separate. True canadensis is restricted to the northern part of the range—eastern flanks of the Rocky Mountains west of Edmonton, Alberta, easterly to Labrador Peninsula, and Alaska from Bristol Bay 125 125 AMERICAN GAME BIRD SHOOTING 126 to Cook Inlet and Prince William Sound; osgoodi to Mount McKinley Range and Yukon region, east to Great Slave and Athabaska Lakes; canace to Mani- toba, southern Ontarie and New Brunswick, south to northern Minnesota, Wisconsin, Michigan, New York and New England. Another race, atratus, has been de- scribed from the coast region of Alaska. The Canada grouse is not a familiar bird to the TAIL OF CANADA GROUSE TAIL OF CANADA GROUSE sportsman, and is scarcely ever shot over dogs, but the angler and traveler through the wilderness of the North often meets it in the depths of the forest. There is little to be said about shooting it, but in its life his- tory there is much that is worth recording. ‘CANADA GROUSE, SPRUCE PARTRIDGE. Most of this has already been written down in works on natural history, but as many of these are not easily accessible to sportsmen, it is well to repeat some of these facts. This is one of the most unsuspicious and least fear- ful of man of all the grouse. Often it may be killed by stones or sticks thrown at it, or a noose of twine sportsman, and is scarcely ever shot over dogs, but the angler and traveler through the wilderness of the North often meets it in the depths of the forest. There is little to be said about shooting it, but in its life his- tory there is much that is worth recording. Most of this has already been written down in works on natural history, but as many of these are not easily accessible to sportsmen, it is well to repeat some of these facts. This is one of the most unsuspicious and least fear- This is one of the most unsuspicious and least fear- ful of man of all the grouse. Often it may be killed by stones or sticks thrown at it, or a noose of twine CANADA GROUSE, SPRUCE PARTRIDGE 127 tied to the end of a stick may be passed over its head as it sits on a limb, and it may thus be dragged from its perch. A bird of such confiding disposition would not afford much sport, and besides this the deep forests which this species inhabits are far from the usual haunts of the gunner. The range of the Canada grouse extends from The range of the Canada grouse extends from northern New England, north through Labrador, west to northern Minnesota and northwestward to Alaska. We have seen them on the shores of Prince William Sound and Cook Inlet in that territory. Its northern limit, according to Swainson, extends to the parallel of 67°. It is not migratory, and breeds wherever found, and its favorite home is in the dense swamps and ever- green thickets of the north land. It is upon the buds and leaves of these evergreens that it chiefly feeds, and from this food its flesh often takes a resinous taste that is not agreeable. The breeding season is in May or June, according to latitude. The mating actions of the male—his drum- ming—differ much from those of other grouse. ‘CANADA GROUSE, SPRUCE PARTRIDGE. Major Bendire quotes a correspondent as saying: “After strutting back and forth for a few minutes the male flew straight up as high as the surrounding trees, about fourteen feet; here he remained stationary an instant, and while on suspended wing did the drumming with the wings, resembling distant thunder, meanwhile dropping down slowly to the spot from where he started, to repeat the same thing over and over again.” Mr. Everett Smith says in Forest and Stream: ‘The Mr. Everett Smith says in Forest and Stream: ‘The AMERICAN GAME BIRD SHOOTING 128 Canada grouse performs its drumming upon the trunk of a standing tree of rather small size, preferably one that is inclined from the perpendicular, and in the following manner: Commencing near the base of the tree selected, the bird flutters upward with some- what slow progress, but rapidly beating wings, which produce the drumming sound. Having thus ascended fifteen or twenty feet, it glides quietly on wing to the ground and repeats the maneuver. . Favorite places are resorted to habitually, and these drumming trees are well known to observant woodsmen. I have seen one that was so well worn upon the bark as to lead to the belief that it had been used for this purpose for many years. This tree was a spruce of six inches diameter, with an inclination of about fifteen degrees from the perpendicular, and was known to have been used as a drumming tree for several seasons. The upper surface and sides of the tree were so worn by the feet and wings of the bird or birds using it for drumming, that for a distance of twelve or fifteen feet the bark had become quite smooth and red as if rubbed.” Major Bendire quotes Manly Hardy as saying: “My father, who had opportunities to see them drumming, told me they drum in the air while descending from a tree. They would fly up on a tree and then start off and drum on their way to the ground like a Quaker grasshopper. When on the ground they scratch a great deal more than other grouse do.” CANADA GROUSE, SPRUCE PARTRIDGE 129 Thus there seems to be a wide individual variation in the practice of this nuptial performance. ‘CANADA GROUSE, SPRUCE PARTRIDGE. The number of eggs laid by the Canada grouse varies The number of eggs laid by the Canada grouse varies from nine to thirteen, though rarely two or three more may be found ina nest. The ground color of the eggs is buff, and they are irregularly spotted and blotched with reddish brown, which sometimes is very dark. Speci- mens are rarely seen with few or no markings. A number of years ago, Mr. Watson L. Bishop, of A number of years ago, Mr. Watson L. Bishop, of Kentville, Nova Scotia, was successful in domesticat- ing some Canada grouse, and he contributed to Forest and Stream many interesting observations on the hab- its of these birds in confinement. He says: “As the nesting season approaches I prepare suitable places for them by placing spruce boughs in such a way as to form cozy little shelters, where the birds will be pretty well concealed from view. I then gather up some old dry leaves and grass and scatter it about on the ground, near where I have prepared a place for the nest. The bird pays no attention to this until she wants to lay. She will then select one of these places, and after scratching a deep cup-shaped place in the ground, de- posit in it her eggs. When the hen is on the nest she is continually making a kind of cooing sound, which I have never heard them make on any other occasion. If there should be sufficient material within easy reach of the nest, the bird will sometimes cover the eggs up, but not in all cases. “No nesting material is taken to the nest until after “No nesting material is taken to the nest until after three or four eggs are laid. After this number has AMERICAN GAME BIRD SHOOTING 130 been deposited, the hen, after laying an egg, and while leaving the nest, will pick up straws, grass and leaves, or whatever suitable material is at hand, and throw it backward over her back as she leaves the nest, and by the time the set is complete, quite a quantity of this litter is collected about the nest. She will then sit on her nest and reach out and gather in the nesting ma- terial and place it about her, and, when completed, the nest is very deep and nicely bordered with grass and leaves. ‘CANADA GROUSE, SPRUCE PARTRIDGE. “So strong is the habit or instinct of throwing the nesting materials over the back, that they will fre- quently throw it away from the nest, instead of to- ward it, as the hen will sometimes follow a trail of material that will turn her right about, so that her head is toward the nest, but all the time she will con- tinue to throw what she picks up over her back. This, of course, is throwing the material away from the nest. Discovering her mistake, she will then right about face and pick up the same material that an instant before was being thrown away, and throw it over her back again, toward the nest. “The way they will steal eggs from one another would do credit to a London pickpocket. Two hens had their nests near together, perhaps two feet apart, and as each hen laid every other day, one nest would be vacant while the other would be occupied. The hen that laid last would not go away until she had stolen the nest egg from the other nest and placed it in her own. I once saw a hen attempt to steal an egg ‘aqe[q Ss uoqnpny wo1g ‘asnolg epeurg ‘as epe CANADA GROUSE, SPRUCE PARTRIDGE 131 from another nest that was twenty feet away. She worked faithfully at it for half an hour or more, but did not succeed in moving the coveted egg more than about eight feet, it being uphill. The egg so fre- quently got away from her, and rolled back a foot or more each time, that she at last got disgusted and gave up the task. “On going to the pen one evening I found one of “On going to the pen one evening I found one of the hens on the nest, and I knew she was beginning to set, as all the others had gone to roost. Slipping my hand under her, I found three eggs—the nest egg, the one just laid, and the one stolen from the other nest. ‘CANADA GROUSE, SPRUCE PARTRIDGE. I picked two of them up and held them before her, when she all at once placed her bill over the one held between my thumb and fore finger and tried to pull it out of my hand; I did not let her have it, however, and she immediately stepped upon the side of the nest, and placing her bill over the remaining egg, drew it up out of the nest and pushed it back out of sight, as much as to say: “You have two, and that is all you can have.’ I must confess that it was with great reluctance I took these eggs from her, she pleaded so hard for them. “The male bird begins to strut in March. I re- member very well the first time I saw one strutting. I had obtained the bird in the fall, and he used to sit about bunched up almost in a round ball, as the female did, until one morning, when I went to feed them, I found him strutting. His attitude was so different AMERICAN GAME BIRD SHOOTING 132 that one would scarcely have known it was the same bird. “T will describe as nearly as I can his conduct and at- titude while strutting. The tail stands almost erect, the wings are slightly raised from the body and a little drooped, the head is still well up, and the feathers of the breast and throat are raised, and standing out in regular rows, with the feathers of the nape and hind neck well back, forming a smooth kind of cape on the back of the neck. ‘This smooth cape contrasts beauti- fully with the ruffled black and white feathers of the throat and fore breast. The red comb over each eye is enlarged, until the two nearly meet over the top of the head. This comb the bird is able to enlarge or reduce at will, and while he is strutting the expanded tail is moved from side to side. The two center feathers do not move, but each side expands and contracts al- ternately with each step as the bird walks. This move- ment of the tail produces a peculiar rustling like that of silk. This attitude gives him a very dignified and even conceited air. ‘CANADA GROUSE, SPRUCE PARTRIDGE. He tries to attract attention in every possible way by flying from the ground up ona perch, and back to the ground, making all the noise he can in doing so. Then he will thump some hard sub- stance with his bill. I have had him fly up on my shoulder and thump my collar. At this season he is very bold and will scarcely keep enough out of the way to avoid being stepped on. He will sometimes sit with his breast almost touching the earth, his feathers erect as in strutting, and making peculiar nodding and CANADA GROUSE, SPRUCE PARTRIDGE 133 circular motions of the head from side to side; he will remain in this position two or three minutes at a time. He is a most beautiful bird, and shows by his actions that he is perfectly aware of the fact. “As the spring and summer advance, the food given these Canada grouse must be changed with the season, and it is only with a perfect knowledge of their wants, and with constant care, that they can be safely carried through the heat of the summer and the moulting sea- son. In the nesting season the females are very quar- relsome, and at this time more than two or three can- not be kept in the same pen, but in July they may be all turned together again and they will agree very well until the following March.” The winter food of the Canada grouse is chiefly leaves and buds of spruce and tamarack. In spring, when insects make their appearance, no doubt they feed largely on them and on the leaves of various plants, while at the ripening of the berries these are eaten. Little or nothing is said by writers generally about the gathering together of these birds in great flocks, yet, like many other species of our grouse, at certain seasons of the year they seem to unite in great com- panies. Major Bendire quotes Manly Hardy, of Brewer, Maine, as saying: “A Micmac Indian, whom I consider reliable, tells me of having seen a pack of many thousands, somewhere east of Halifax, Nova Scotia, on which their whole village lived for weeks, moving after them when they moved. The males greatly preponderate over the females, at least two to AMERICAN GAME BIRD SHOOTING 134 one. ‘CANADA GROUSE, SPRUCE PARTRIDGE. They feed almost entirely on the needles of spruce and fir, also hackmatack and berries in summer. They show a preference for some fir trees over others, as J have seen them return to the same tree until it was nearly stripped. When disturbed they always take to the trees, walking about in them.” Canachites franklini. Extremely similar to the foregoing, but with the tail black to the end, or bordered with white; its cov- erts white-tipped. The female has the tail feathers tipped with white and the upper tail coverts streaked with white, as also have the full-grown young of both sexes which have not yet assumed the plumage of the adult. It is slightly larger than the Canada grouse: length, over 15 inches; wing, 7% inches; tail, 5 inches or over. Franklin’s grouse is found in southern Alaska, cen- tral British Columbia and west central Alberta, south to western Montana, central Idaho and northern Oregon east of the coast range. At many points in northwestern Montana, northern and central Idaho it is abundant. It is the bird commonly called “fool hen” in the western mountains, though this, after all, is a general term applied to any grouse or quail that has not yet learned that man is an enemy to be dreaded. The differences in plumage between Franklin’s The differences in plumage between Franklin’s grouse and the Canada grouse are so slight that they are not likely to be noticed by the average gunner, though at once obvious to the ornithologist. So far 135 135 135 AMERICAN GAME BIRD SHOOTING 136 as is known, their habits are closely alike, though about the present species not very much has been written. Unless they should greatly change in habits, neither Unless they should greatly change in habits, neither the Canada grouse of the East nor the Franklin’s grouse of the West is likely ever to offer much sport to the gunner. These little birds are so gentle and confiding that they hardly take the trouble to move out of the intruder’s way; they will stand on the ground TAIL OF FRANKLIN’S GROUSE TAIL OF FRANKLIN’S GROUSE or a limb to have a dozen shots fired at them, or may be pelted with stones and sticks without manifesting great alarm. I knew of one at which several rifle shots were fired, the last of which cut off one of the bird’s feet. It staggered on its perch, and then, re- gaining its balance, stood there on one foot, apparently wondering what had happened. It is often possible to capture these birds by means of a noose tied to a pole six or eight feet long, by means of which the noose can be slipped over the head. Canachites franklini. It used to be not unusual, and no doubt in many lo- or a limb to have a dozen shots fired at them, or may be pelted with stones and sticks without manifesting great alarm. I knew of one at which several rifle shots were fired, the last of which cut off one of the bird’s feet. It staggered on its perch, and then, re- gaining its balance, stood there on one foot, apparently wondering what had happened. It is often possible to capture these birds by means of a noose tied to a pole six or eight feet long, by means of which the noose can be slipped over the head. It used to be not unusual, and no doubt in many lo- It used to be not unusual, and no doubt in many lo- FRANKLIN’S GROUSE 137 calities still is so, for a brood of these birds to fly into a tree and sit there unalarmed until the heads of half or all of their number have been cut off by the rifle balls of some passing traveler. The flesh is excellent eating, and in places where fresh meat is not easily to be had, many of these birds are killed along the trails. On the other hand, it is to be remembered that the country in which they live is sparsely inhabited by man, and that those which he kills constitute only a very small fraction of the whole number in a district. On the other hand, it is difficult to comprehend how these birds escape their natural enemies, and heavy toll of them must be taken by lynx, fox and wolf. With the destruction of the forests, which now threatens us, the numbers of both these forms of grouse must constantly diminish, but before the birds are too greatly reduced in numbers some means should be devised for their preservation. Franklin’s grouse is still more gentle and unsuspi- cious than the Canada grouse, its eastern cousin. One may frequently walk up to within fifteen or twenty feet of these birds as they sit on the ground or on the low limbs of trees without apparently being noticed by them. When they discover the approach of man they seem at first but little interested, but as one draws nearer, they are likely to stretch up their heads and look more carefully. Canachites franklini. If really alarmed or uneasy they stand very erect, holding the neck outstretched, and often raise the feathers on the sides of the neck just below the head. This habit is common to this species and to AMERICAN GAME BIRD SHOOTING 138 the dusky grouse, and is seen to a greater or less extent in other members of the family, as the ruffed, pinnated and sharp-tailed grouse. Franklin’s grouse is commonly found in the summer Franklin’s grouse is commonly found in the summer in timbered valleys, often close to the water, but some- times ‘also on timbered plateaus much higher in the mountains. I have not observed that it seems especially to prefer swampy lands, as the Canada grouse is sup- posed to, but rather a thickly timbered country, not far from water. Even if alarmed and forced to take to wing, they do nothing more than fly up into the branches of the tree immediately above them, where they always either huddle down on a branch, as if to rest again, or, if seriously frightened, stand with head erect, stretching the neck out, first in one direction and then in another, until the cause of alarm has passed, or their suspicion has become allayed. Contrary to what might be expected, the spruce partridge, found in west central Alaska, is not Franklin’s grouse, but a form of the Canada grouse. RUFFED GROUSE—PARTRIDGE. Bonasa umbellus. Bonasa umbellus togata, Bonasa umbellus umbelloides, Bonasa umbellus sabini. The ruffed grouse is peculiar to North America, and is found mainly in the Transition and Canadian life zones. Ornithologists recognize four geographical races in different sections of the continent. Of these four forms, the typical species (Bonasa umbellus) in- habits the eastern United States as far north as north- ern Massachusetts, thence westward to and beyond the Mississippi River; in southern Vermont, southern New York, through Ohio, Michigan, Wisconsin and southern Minnesota, touching the eastern portions of North and South Dakota, eastern Nebraska, eastern Kansas and Missouri, northern Arkansas, Tennessee, Kentucky, and in the Alleghanies to Georgia. The ruffed grouse of northern New England, northern New York, northern Michigan and eastern Oregon, known as B. umbellus togata, the Canada ruffed grouse, is found also northward to Nova Scotia, Manitoba, cen- tral Keewatin, southern Ungava and British Columbia. The gray ruffed grouse (Bonasa umbellus umbelloides) occurs in the central Rocky Mountains, from Colorado, 139 139 AMERICAN GAME BIRD SHOOTING 140 Utah and western South Dakota to Alaska, along the Yukon and Mackenzie rivers, throughout much of Brit- ish North America, east as far as Manitoba. Another darker race (Bonasa umbellus sabini) inhabits the wooded country of the northwest coast region of south- ern Alaska, to Humboldt County, California. 32312 < sash on / RUFFED GROUSE RUFFED GROUSE All these forms are very similar in appearance, and it is not unusual to find in one locality a bird which, in color, may closely resemble those of some far distant locality. The most that can be said for these races is that they average lighter or darker—as the case may be—than certain other relations, the center of whose abundance may be far distant. RUFFED GROUSE—PARTRIDGE I4I Bonasa umibellus is above, reddish brown or grayish brown, varied with black, brown and gray in different shades, the scapulars and wing coverts streaked with whitish or cream color, the rump and upper tail cov- erts with long streaks or spots of grayish or yellowish. Tail long and wide, gray or reddish, more or less band- ed with paler, each pale band bordered by a narrow, irregular blackish edge. A broad sub-terminal band of black or dark brown near the end, followed by a nar- rower terminal band of grayish. RUFFED GROUSE—PARTRIDGE. Feathers of the tufts on the side of the neck—the ruff—usually broadest at the ends, and black in color, sometimes with green- ish reflections. Occasionally the ruff, instead of being black, is dark brown, or even pale chestnut. The throat is pale buff, sometimes dotted with darker. The lower parts are whitish or yellowish, barred with dull brown, broadest and darkest on the flanks. The lower tail coverts are white-tipped. The female is similar to the male, but smaller, and with the plumage slightly paler. At the same time the plumage often fails to give any suggestion of sex. There is a high-pointed crest on the head. The lower portion of the tarsus—that is, of the foot—is naked. This is a general description of the ruffed grouse. This is a general description of the ruffed grouse. The typical uwmbellus is described as mostly reddish above. The sub-species, B. umbellus umbelloides, is mostly gray, and has the tail always gray. It is re- garded as the Rocky Mountain form. B. wmnbellus togata, the Canada ruffed grouse, is B. wmnbellus togata, the Canada ruffed grouse, is AMERICAN GAME BIRD SHOOTING 142 mostly grayish, and the tail is usually gray, but some- times tinged with reddish. B. umbellus sabini is dark reddish, with little or no gray. This is the northwest coast form, found in the region of great precipitation, and by many is consid- ered the most beautiful of the ruffed grouse. The descriptions of these sub-species do not always The descriptions of these sub-species do not always give a very clear idea of the grouse of the regions the various forms are supposed to inhabit. The center of such a region, no doubt, will be occupied by birds of the typical form. On the border lines, however, where the sub-species tend to meet, individuals may some- times be found that are typical of some taken thousands of miles away. The length of the ruffed grouse varies from 16 to 19 inches. The wing measures from 7 to 7% inches, and the tail from 514 to 7 inches. The extent of wing varies from 22 to 25 inches, and the bird commonly weighs from 21 or 22 up to about 30 ounces. Grouse are often reported to weigh 2% or 2% pounds; but it may be doubted if birds so heavy are ever found. RUFFED GROUSE—PARTRIDGE. The ruffed grouse is the best known and most highly The ruffed grouse is the best known and most highly esteemed of the game birds of the North, and in Can- ada, New England, northern New York, Pennsylvania, Michigan and Wisconsin it 1s now the most important of the upland birds. In the South the bird is called “pheasant,” and in the North, especially in the Northeast, “partridge’”— both misnomers, since it is neither. In parts of Canada RUFFED GROUSE—PARTRIDGE 143 it is called ‘birch partridge,” in contradistinction to the Canada grouse, called “spruce partridge.” The grouse spend the winter in thick, deep swamps, or else on steep hillsides in sections where evergreens grow, which give protection from the snow, in dense runs, and white birch and other thickets. Life is easy for the birds, which wander about over their limited range, scratching, when the snow is not too deep, for the fruit of the skunk cabbage, for the fruit and leaves of wintergreen and partridge berry and arbutus, for hibernating insects, for nuts overlooked in autumn by themselves and the squirrels; or, if the ground is deeply snow-covered and ice-bound, taking to the tree-tops, where they glean a plenteous harvest of buds, and usually come out in spring strong and well nourished. At Lake George, New York, the grouse feed extensively on the buds of ironwood, poplar, birch and apple, from late October to early April, irrespective of the snowfall. An hour or two before sunset they fly to the trees and gorge on the buds and catkins. In New England and the Middle States the mating time comes in early April, after the weather has grown warm and the grass and flowers have begun to spring. By this time the grouse has begun to sound his drum- ming call. In a country where grouse are plenty you may hear the sound from a hundred hills, but you will find it a difficult matter to trace it to its source, and when you approach the place from which you thought it came, you will find it is no longer heard there, and when it next reaches your ear it seems a long way off. AMERICAN GAME BIRD SHOOTING 144 In the Rocky Mountains, in September, a few years ago, I saw a grouse cross the trail along which I was walking and disappear in the bushes to the left. RUFFED GROUSE—PARTRIDGE. The clump of bushes was a small one, not more than twenty yards across. I watched the bird while it was walking perhaps ten yards, and then went on, but had hardly come to where the bird had crossed the path when I heard it drum. The sound was not loud and gave the impression of being quite distant. I stood still for a moment, trying to see the bird, but without success, and after a little while the drumming began again, still sounding as distant as before. I then very quietly made my way into the bushes, advancing six or eight yards toward the point where I had last seen the grouse, but the drumming was not resumed, and after a few mo- ments I withdrew, but had hardly reached the trail when I again heard the sound. A second excursion into the bushes was no more successful than the first had been, but somewhat later, on my return, hearing the drumming, I again went into the bushes, and just beyond where I had seen the grouse disappear I saw it walk away among the brush. This is mentioned only to indicate how very faint the sound of the drumming may be, although it is quite close at hand. Few subjects have been more discussed by American Few subjects have been more discussed by American naturalists and sportsmen than this same drumming, a sound so familiar to outdoor men as hardly to need description. It is commonly thought to be a mating call, though by no means sounded only at the pairing season in spring; for the roll of the drumming, sound- ‘aspoyH “Wa ‘<O Aq poeydeis0j0ydg *Zur}jnIys ‘asnorn peyny ‘a “Wa ‘<O Aq poeydeis0j0ydg *Zur}jnIys ‘asnorn peyny RUFFED GROUSE—PARTRIDGE 145 ing like far-off thunder, is often heard in late summer and autumn. It is possible that this autumnal drum- ming is made by the young males, just as sometimes in autumn a young male songbird may be heard to prac- tice in low tones the lay which it will utter during the mating season of the following spring. A great variety of explanations have been given as to the method by which the drumming is produced. RUFFED GROUSE—PARTRIDGE. It is made by the wings, and the observations of ornithologists have es- tablished the fact that the sound is caused by the out- spread wings of the bird rapidly beating the air with- out striking the bird’s body or any other object. Pho- tographs taken by Professor Hodge show the wings beating in front of the breast as the bird sits upright during the act. The belief that the resonance of the sound is caused in part because, while drumming, the bird stands on a hollow log, was long ago abandoned. One of the best descriptions of the process of drum- ming is that given by Mr. Manly Hardy, of Brewer, Maine, who says: “The cock grouse usually selects a mossy log near some open hedge, clearing, or woods road, and partly screened by bushes, where he can see and not be seen. When about to drum he erects his neck feathers, spreads his tail, and with drooping wings steps with a jerky motion along a log for some distance each way from his drumming place, walking back and forth several times and looking sharply in every direc- tion. Then standing crosswise, he stretches himself to his fullest height and delivers the blows with his wings fully upon his sides, his wings being several inches clear AMERICAN GAME BIRD SHOOTING 146 from the log. After drumming he settles quietly down into a sitting posture and remains silently listening for five or ten minutes, when, if no cause for alarm is dis- covered, he repeats the process.” Mr. Ernest E. Thompson, in his “Birds of Mani- toba,”’ describes the drumming as he saw it. It is said that if, during the drumming, another male grouse makes its appearance near the drumming place, the birds fight with much spirit. An account of such a bat- tle is given by a correspondent of Forest and Stream, who writes from Schenectady, New York, and signs himself ‘“Dorp.” He says, in substance: “T was walking along a country road, about the first “T was walking along a country road, about the first of June, when I heard from the depths of an adjacent wood an unusual rustling sound for which I could not account. The sound stopped, began again, again stopped, and was repeated at intervals of a minute or two. RUFFED GROUSE—PARTRIDGE. Not knowing what caused the sound, I crossed the fence and cautiously approached the place. After I had gone about fifty yards, screening myself as much as possible behind trees, I came up to within twenty yards of a large dead log, upon which stood two ruffed grouse in battle array. They stood something like six feet from each other, with the black feathers around their necks raised till they almost pointed toward their heads, which were lowered, and which they were shak- ing at each other in defiance. In a few moments they gradually approached each other, and when about two feet apart they rushed together, the momentum and their wings carrying them into the air about a foot RUFFED GROUSE—PARTRIDGE 147 above the log. While there they struck repeated blows and then fell back to the log, where a short tussle took place, when they separated and moved back to a dis- tance of about six feet from one another. “After an interval they advanced toward each other “After an interval they advanced toward each other again and repeated the same performance as before. The desperate battle went on by ‘rounds’ till several were fought. How long the fight had been going on before I came upon the scene I had no means of know- ing, but after they had come together several times in my presence, the bird that seemed the smaller moved backward, still presenting a defiant attitude, with his head lowered, till he came to the end of the log, when he jumped off and disappeared. “Upon this the victor raised himself and stretched “Upon this the victor raised himself and stretched up until he nearly stood on his toes, expanding his wings at the same time. It looked for a moment as if he would crow. Then settling himself back, he brought his wings down against his breast with a sound like that produced by forcibly striking a bass drum. Again he raised his wings and repeated the sound. Still continuing to raise and lower his wings more and more rapidly, the sound finally rolled away ‘in one continu- ous murmur and was still.’ The sound was indescrib- able. As soon as he had drummed he jumped off the log and disappeared. RUFFED GROUSE—PARTRIDGE. Wishing to know if the female, the probable cause of the conflict, was near by, I ad- vanced rapidly and started her, together with the vic- tor, and a short distance away the defeated bird.” An interesting account of a case of drumming in An interesting account of a case of drumming in AMERICAN GAME BIRD SHOOTING 148 the autumn, with its possible cause, is given by this same correspondent of Forest and Stream, who says: “On one occasion, while out shooting in Clifton Park, I fired at a grouse, which fell to the ground and fluttered over it for some distance, but before I could reach him rose again and, flying high, disappeared over the tops of the trees in the woods. Pursuing, I looked the ground well over, but was unable to find him. It so happened that two or three days after this occurrence I was again on the same ground, and re- membering the wounded grouse, went in search of him. I had not gone far when I heard a grouse drum, and moving cautiously I finally got behind an old moss- covered stone wall that ran through the woods. I had often heard grouse drum, but I had never seen one per- form the act, and I was very desirous to do so. After a while he drummed again, and I moved along the fence, nearer, having by this time got his bearings. Upon looking over the wall I saw the bird standing on what I supposed to be a stone about five or six inches in height. After he had drummed he pecked at the supposed stone and then bobbed his head and tail up alternately, the latter being spread out. Then he jumped off the object and walked in a wide circle around it, after the manner of the turkey cock, with wings lowered. After an interval he got on the stone again and drummed and performed as before. About this time something alarmed the drummer and he dis- appeared into the covert. “Upon going to the spot where he had drummed I “Upon going to the spot where he had drummed I Faster The roll The drumming Ruffed Grouse. Photographed by C. F. Hodge. The roll Faster The drumming Ruffed Grouse. Photographed by C. F. Hodge. RUFFED GROUSE—PARTRIDGE. RUFFED GROUSE—PARTRIDGE 149 was astonished to find what I supposed a stone to be a dead male grouse. Upon it the drummer had stood and drummed, and about it he had paraded as described. The dead bird had one shot only, through his head. It was the same I had shot a couple of days before, which had flown there and fallen dead. “T have read that the drumming of the ruffed grouse is a demonstration of love and courtship, but from this incident I conclude that it is also either one of rivalry and triumph, or sounding a knell or requiem of a departed mate or friend.” With this should be compared Audubon’s account of the actions of a wild turkey cock after a fight in which he has killed his rival. This same correspondent of Forest and Stream, an observant man, has expressed the view that the grouse drums to announce the approach of day at dawn, to announce any approaching unusual atmospherical change during the night or day, to make his where- abouts known to the female, to celebrate her coming, to announce a triumph over a rival, to sound a requiem over a dead mate or friend, to amuse himself. Most of these motives are precisely those which are supposed to actuate the domestic cock in his crowing, or in the flapping of his wings which commonly accompanies the crowing. It is certainly true that the grouse drums at night as well as by day, and that he frequently drums just before daylight and often just before “a change of weather.” Although so excellent an observer as Captain Ben- Although so excellent an observer as Captain Ben- AMERICAN GAME BIRD SHOOTING 150 dire thinks that the drumming cannot be considered a love note because, as he says, “It may be heard almost every month in the year and sometimes in the night as well as in the day time,”’ I am disposed to think that it is really in part a mating call. Other grouse perform certain operations usually thought to be connected with the mating time in autumn as well as in spring. The sharp-tailed grouse holds its dances in autumn, and I have been told that the dusky grouse hoots in the autumn as well as in spring, though by no means so vigorously. RUFFED GROUSE—PARTRIDGE. Certainly we may believe that at the proper season of the year it possesses an attraction for the female, and S. T. Hammond, in his capital book, “My Friend the Partridge,” gives some testimony to this effect. The nest of the ruffed grouse is built almost any- where, yet perhaps most often on a hillside more or less steep, overlooking a swamp or a piece of woods. I have found nests among thick cedars on a hillside, or perfectly open and exposed, at the foot of a cedar tree in a mowing lot close to a fence, and again between two cedars in an open piece of cedar wood, where there was no apparent cover whatever. The eggs are laid usually in April or early May, and by the middle of that month the clutch of twelve to seventeen is usually completed. The eggs are cream color or buffy, rarely spotted with brown at the larger end. If the female is startled from her nest she leaves it with a roar of wings, whose rapid beats often spread over the precious eggs the dry leaves by which the nest is likely to be surrounded. On a num- ‘asno payn jo ison ‘as pay jo iso RUFFED GROUSE—PARTRIDGE I51 ber of occasions I have seen the eggs almost wholly hidden by this simple means. Most sportsmen know how very difficult it is to Most sportsmen know how very difficult it is to see game birds when they are not in motion. The bird’s feathers harmonize so admirably with its sur- roundings—whether these be the leaf-strewn ground of October, the bare branches of December, or the yel- lowing grass and weed stems of July—that it is often almost impossible to detect the bird, even though one knows precisely where it is. I recall an occasion when, happening to cross a fence which separated a road from a mowing lot, I almost stepped on a female grouse as I sprang to the ground. She was sitting on a nest with thirteen eggs, at the foot of a large cedar. Often after that I used to go down to the lot and slowly approach the place to look at the bird as she sat on her eggs. She soon became so accustomed to me that she mani- fested no alarm, and I could approach quite close to her. RUFFED GROUSE—PARTRIDGE. It always took me some little time to see the bird, though she sat in plain sight, with only half a dozen slender grass stems between her head and me. After looking for some time at the spot where I knew she sat, the shape of her head, her markings, and above all her bright eye, would gradually grow out of the con- fusion of the grass stems in front of her and the cedar bark beyond, and I could see the whole bird plainly. Yet if I turned my eyes away it again took a little time to find and recognize her. The eggs hatch from the first to the middle of June, The eggs hatch from the first to the middle of June, and the young often number as many as a dozen or AMERICAN GAME BIRD SHOOTING 152 fifteen. They leave the nest at once and nimbly follow the careful mother, as her chickens follow a domestic hen. She leads them quietly through the woods, teach- ing them the while how to live their lives and how to keep themselves safe from their enemies. At her call of alarm each chick sinks down on the ground and dis- appears, looking like a leaf, a bit of stick or a pebble. No one can recognize them as living things, and their only danger is that some clumsy person may step on one of them. Meantime the mother, with feathers erect and trailing wings, is limping in front of the intruder, falling down, pushing herself along on her breast, pant- ing as if in the very agony of death, often “growling” or “whining” in the effort to lure the enemy away from the brood. Usually she succeeds. No dog, and few boys and men, can resist the temptation to catch a partridge. The pursuer runs forward and almost grasps her, but his approach seems to give her a little strength and she flutters feebly forward. A few steps more and she will be his; but still she evades him, and presently, after having gone thirty or forty yards, she rises on strong wing and, swift as a bullet, darts off among the tree trunks. RUFFED GROUSE—PARTRIDGE. If the pursuer returns to the place where he first saw her, sits down and remains there quiet, after a time he will see her return on foot, call together her little brood and start off again on her travels. In Captain Bendire’s admirable work, so often cited, In Captain Bendire’s admirable work, so often cited, Manly Hardy, after describing the actions of the mother bird, says: RUFFED GROUSE—PARTRIDGE 153 “T have once seen the old cock with the brood, and on this occasion he gallantly defended the rear until the rest made good their escape. He stood with wings raised and tail spread, ready to fight the intruder. I have seen the young fly into a tree when still in the yel- low down, and when not larger than a pine grosbeak they will fly long distances, giving the alarm note of ‘quit, quit, just like an old bird. The young a few days old are shyer than the wariest adult. The noise made by the ruffed grouse in flying ‘is made on purpose’ to alarm others in the vicinity; they can fly as quietly as any bird if they choose. “The males never congregate during the breeding “The males never congregate during the breeding season or after, and I never but once saw two adult males within one-fourth of a mile of each other between April and September. I consider that the drumming is not a call to the females, as they drum nearly or quite as much in the fall as in the spring, and I have heard them drumming every month in the year. I have never seen the least evidence that the ruffed grouse is polygamous.” With this last statement I quite agree. I know of no evidence to justify the common assumption that this grouse is polygamous. When the young birds are two or three weeks old When the young birds are two or three weeks old their wing feathers have grown enough to enable them to fly, and sometimes, if suddenly surprised, some of them may leave the ground and fly up into low branches, where they stand motionless with their necks AMERICAN GAME BIRD SHOOTING 154 stretched upward and may be so closely approached that one may almost take them in the hand. RUFFED GROUSE—PARTRIDGE. By the middle of August the young are almost fully By the middle of August the young are almost fully grown, and as cool weather approaches they begin to separate, no longer keeping in a close flock, but wan- dering off by twos, threes and fours. As the weather grows colder they become still more solitary in their ways, although up to midwinter two or three may often be found together. This has been my experience; but other observers, This has been my experience; but other observers, whose opinions are entitled to the greatest respect, be- lieve that the broods keep together until midwinter, unless broken up and scattered by the gunners. But if this is true, how do the birds get together after the “crazy season’? Dr. A. K. Fisher expresses the opinion that com- paratively few grouse become “crazy” birds. He is inclined to believe that the “‘crazy”’ birds are lost birds —1t.é., individuals that for some cause or other have been driven from their homes, and do not know how to return. It is during late September and October that the grouse for a short time wander about in a most eccen- tric manner and indulge in such unusual performances that this is sometimes called their ‘‘crazy season.” Having separated, they seem to be searching for new homes or perhaps for winter quarters, and wander about in seemingly aimless fashion, so that they are often found in most unusual places. Such are vacant buildings, village streets, the lawn of a country place, RUFFED GROUSE—PARTRIDGE 155 or even within a covered country bridge. At this time, too, they often fly against houses or fences, or through windows, opened or closed, and in this way many birds are killed. I have personal knowledge of three birds flying against houses, one against a fence and two through windows, opened or closed, on a single farm in Connecticut. : A correspondent of Forest and Stream says: “Once while walking along the street I saw a g “Once while walking along the street I saw a grouse dusting in the roadway. At first I supposed it was a hen, but as I approached, ittrose, flew straight down the street and in at the opening of the large covered bridge that spanned the Mohawk. The bird lit on a beam near the roof. Getting up to the beam I took the bird off. RUFFED GROUSE—PARTRIDGE. He remained perfectly motionless, as is their habit when approached by man. This grouse when he rose in the roadway made comparatively little noise. . . .” The same writer says: “I recollect an incident that The same writer says: “I recollect an incident that occurred in our city where a grouse flew into a small barn and perched on a beam. The owner of the prem- ises having invited a gentleman into the barn on some business, the guest said to the owner: ‘I see you keep poultry.” “No,’ said the owner, ‘I keep no poultry, but my neighbors do, and I wish they would come and take that pullet away. She has been here now three or four days and I am tired of feeding her.’ The visitor looked a little more sharply and said: ‘That is not a pullet; that is a partridge’; and so it was, to the great surprise of the other.” The food of the ruffed grouse is very varied. In The food of the ruffed grouse is very varied. In AMERICAN GAME BIRD SHOOTING 156 summer—and especially for the young birds—insects, notably grasshoppers and crickets, make up a portion of its diet. It is fond of berries of all sorts, and black- berry patches and wild grape vines are often visited by it when the fruit is ripe. Indeed, from midsum- mer until early winter there are always berries for them to feed on. In autumn also they feed on fallen apples, and wild apple trees in the midst of woods are a fa- vorite resort for them morning and evening. They eat some grass and the leaves of many plants. They feed on the fruit of the skunk cabbage (Symplocarpus) ; early in the winter tearing away the pithy covering that holds the seeds and picking them out from their spongy bed, or later gathering them from the ground. Most of all they like nuts, such as chestnuts, acorns and beechnuts. I have taken from the crops of grouse two or three pignuts, a double handful of chestnuts and as many beechnuts as I could hold in one hand. There is a record of a small snake having been taken from a grouse’s crop. The Biological Survey has shown that over Io per The Biological Survey has shown that over Io per cent. RUFFED GROUSE—PARTRIDGE. of the food of the ruffed grouse is animal and 89 per cent. vegetable matter. The vegetable food is seeds, more than 11 per cent.; fruit, more than 28 per cent. ; leaves and buds, more than 48 per cent. Most of the insects eaten are injurious; either those that prey upon the growing crop or borers destructive to the forest. Every ruffed grouse that is killed, if he had lived out his time would have destroyed a great mul- RUFFED GROUSE—PARTRIDGE 157 titude of harmful insects and so would have done good service to the farmer. Although grain has not been found in the crops of Although grain has not been found in the crops of any of these grouse, there is no doubt that it is eaten when obtainable. The birds are often seen scratching in the roads, presumably for oats dropped by the horses, and I have vivid recollection of a partridge which one winter could be started two or three times a day at the head of a small pond where corn had been scattered along the edge of the water to attract the black ducks. This grouse frequented the place all through the winter to feed on the scattered corn. In autumn, winter or spring it resorts to orchards to In autumn, winter or spring it resorts to orchards to feed on the buds of the trees, but it probably does no harm in this way. Buds and the leaves of poplar, birch, ironwood and willow form a large portion of the food of the grouse. It is said that the buds of the black birch are sometimes eaten so largely that they impart a taste to the bird’s flesh. In Alaska, E. W. Nelson found the grouse feeding on spruce buds and declared that the flesh had become bitter from this diet. There was long a belief that the flesh of the ruffed grouse was sometimes poisonous because it ate the buds of the laurel. The statement was made by Alexander Wilson, and cases are quoted by Mr. Judd to show that there is a foundation for the belief. On the other hand, grouse which had fed on laurel have been eaten without any resulting harm. RUFFED GROUSE—PARTRIDGE. It is quite possible that where injury has resulted, it came from leaving the bird undrawn for a long time after death, when the AMERICAN GAME BIRD SHOOTING 158 poison from crop and intestines spread itself through the flesh. Grass, flowers and especially red clover are often found in the crop of the ruffed grouse, and many other plants in small quantities. A grouse shot at Lake George had its crop distended with the leaves of the spearmint. Few birds are more local in their habits than the Few birds are more local in their habits than the ruffed grouse, and one who is familiar with their ways can usually visit the same spot in the woods at the same time of the day and be quite sure of starting a bird. A brood of birds started and scattered in all di- rections will be found a few hours later within a few hundred yards of the place from which they flew. I believe that a circle with a diameter of half a mile will cover the range of a group of ruffed grouse during the summer or during the winter. In spring and autumn, of course, there are marked changes of location, the causes of which are not well understood. We merely know that these changes take place, and that they shift their ground with the seasons, usually being found in different places in summer, autumn and winter. The bird has a certain place for resting—basking in the sun or wallowing in the dust—another for feeding and another for drinking. Very often it passes between these different places on foot, but not infrequently it flies. It is perhaps during their flights in new and un- familiar localities that they dash themselves against houses or other obstacles. Occasionally they are killed by flying against telegraph wires. When a grouse rises, it is likely to fly swiftly and When a grouse rises, it is likely to fly swiftly and When a grouse rises, it is likely to fly swiftly and RUFFED GROUSE—PARTRIDGE 159 straight at a distance of not more than from three to thirty feet above the ground. As it approaches the place where it wishes to alight it sets its wings and sails straight for the place and there alights on the eround. RUFFED GROUSE—PARTRIDGE. I do not think that it ever turns up in the air as a quail does, but on the other hand I have never seen a bird actually alight. In winter I have, however, seen where they had come to the ground after a short flight, and the impression of the bird’s breast in the snow two or three inches deep. The breast evi- dently came down very close to the ground, sweeping away the snow, and the grouse came to its feet at once. I have seen a bird fly from the top of a high hill to a piece of woods much lower down. It came straight until it was almost over the woods, and then, making a wide half circle, swiftly lowered and alighted among the dry leaves not more than twenty steps from me. The various methods by which the grouse avoid ob- The various methods by which the grouse avoid ob- servation are little known and are seldom observed by those who are only occasionally in the woods and who, when there, are possessed with the idea that they must kill as many birds as possible in the time at their dis- posal. The ones more likely to see such things are the men who spend much time in the woods and have the leisure to sit down and observe, watching the ways of the wild creatures, which, after a time believing that the intruder has gone away, emerge from their hiding places and resume their usual pursuits. But if the ob- AMERICAN GAME BIRD SHOOTING 160 server makes a movement and the bird sees him, it will stop for a moment, look carefully, and then, turning, will seem to melt out of sight. It is most difficult to tell when it disappears. Under such circumstances a bird will sometimes fly, but more often will run for two or three yards and disappear, and then springing from the ground at a greater distance will fly off low, not being seen after it takes to wing. Rarely one may see a frightened grouse on the Rarely one may see a frightened grouse on the ground and may get up close to it, but if he does this and wishes to observe the bird, let him avoid looking directly at it. RUFFED GROUSE—PARTRIDGE. If it catches the observer’s eye, it is al- most sure to fly, and when it flies the very fact that you are so close to it may make the shot a difficult one. On one occasion many years ago, after shooting two or three times at a grouse, the dog pointed it at the foot of a great oak tree. The other dog backed, and my two brothers and myself, realizing where the bird must be, surrounded the foot of the tree. Presently we saw it crouched on the ground between two roots, looked squarely at it and talked about it. That grouse must have had an unhappy time, for it feared to fly. For some little time we looked and talked of it, and then, fearing that the pointing dog might try to seize the bird, which was almost under his nose, one of us stepped forward and grasped his collar. As this was done, the grouse took wing, twisted around the root of the tree, passing within two feet of one of my broth- ers, dodged away and up and over a little rise of Daydreaming. Photographed by C. F. Hodge. Daydreaming. Photographed by C. F. Hodge. RUFFED GROUSE—PARTRIDGE 161 ground twenty yards from us, escaping untouched, not- withstanding the fact that three shots were fired at it. The grouse sometimes takes refuge in the snow in cold weather, and is credited with diving into drifts at nightfall and passing the night there. A friend walk- ing through the deep snow along a little swale, through which passed a brook, saw sticking up out of the snow what he supposed was an odd-looking stick. He de- clared to himself that it looked just like a partridge’s head, and when he had come within a few feet of the place a bird rose out of the snow like a whirlwind and flew away to a near-by swamp. No doubt these birds often inspect the gunner when he is little aware of their observation. Dorp tells of a case of that kind in the following words: “T turned to resume my walk, and as I did so I cast “T turned to resume my walk, and as I did so I cast my eyes upward, and there on a plateau covered with moss and projecting nearly over me stood a ruffed grouse looking intently down upon me. RUFFED GROUSE—PARTRIDGE. He was twelve feet from me, as I afterward measured it. He was partly turned sideways, with the neck stretched and head bent down, and made a beautiful picture with background of evergreen. “He remained motionless, as I did also, for about a half minute, when he slowly turned and was quickly lost to sight. This was the closest I remember ever to have been to this wild bird in his native state when seen. I have been closer, but then I did not know it until he flew. “This bird had heard me walking and his curiosity AMERICAN GAME BIRD SHOOTING 162 had prompted him to come to the edge and look down to discover the cause of the noise. Perceiving that my back was toward him he knew that I could not see him and so remained. Perhaps he had not seen enough of me to satisfy him, and when I turned around he knew I could not reach him. He had probably never been shot at and knew nothing of the power of the gun which I held in my hand, or of man’s ability to harm at a distance.” In a country where it is much pursued by man or dog, the ruffed grouse, if unexpectedly approached closely, is likely to walk or run away rather than to fly. A good grouse dog follows the trail of the bird slowly and not much faster than the bird itself can run. Those of us who have possessed good grouse dogs, which are always scarce enough, have often seen them leave the trail, make a wide circle and come up on the other side of the bird to head it off and stop it. Unless some- thing like this is done, the bird may readily enough be followed to the edge of the cover, where it will presumably take wing and disappear. If a dog is alone—especially if it be a dog of red or yellow color, suggesting a fox—the bird is likely merely to hop up on a limb well out of reach and remain there looking down at the enemy; but if a man follows the dog, the bird, if he gets into a tree, is likely to stand close to the tree trunk and remain absolutely motionless. RUFFED GROUSE—PARTRIDGE. Ruffed grouse are subject to more or less unex- Ruffed grouse are subject to more or less unex- plained periods of scarcity. During one season the woods will contain their usual number of the birds, and RUFFED GROUSE—PARTRIDGE 163 the following year very few may be found. After that it may take some years for the birds to re- cover themselves and again to become reasonably abun- dant. Whenever such a period of scarcity occurs, sportsmen very naturally endeavor to assign reasons for the reduced numbers of the birds. Among the causes suggested are these: that they Among the causes suggested are these: that they have been swept away by an epidemic disease, that they have been destroyed by insect enemies, that they have been killed by hawks, owls and foxes, that the breeding season has been unfavorable, that the winter’s snow and cold have killed them, while many men believe that over-shooting furnishes the best reason of all. None of these explanations appear to fit all cases. The birds may succumb to disease, but there appears to be no evi- dence that they do so. The young chicks in traveling through the woods and swamps undoubtedly occasion- ally pick up wood ticks which suck their blood, and occasionally a young and weakly bird may perish from this cause. Those who attribute the scarcity of grouse at any time to hard winters—to their being covered up and frozen in under the snow—cannot know much about grouse nature. The bird is found far up in the north, where it is exposed to weather far more rigorous than it can ever experience in temperate climes, and if it had been so tender as to be killed by the winter, it would long ago have been exterminated in the moun- tains of Alaska, along the Mackenzie River and south- ern Ungava. It seems more probable that over-shoot- ing must have much to do with these disappearances AMERICAN GAME BIRD SHOOTING 164 of the grouse, not directly, perhaps, but because by constant attacks the breeding stock is kept down so low that when conditions arise even slightly unfavorable to the species, and a few birds are swept away, the breed- ing stock is so reduced that not enough birds are reared the next season to replenish the covers. RUFFED GROUSE—PARTRIDGE. The widest differences of opinion about this matter The widest differences of opinion about this matter exist between sportsmen and naturalists of experience. So good a field naturalist as Nap. A. Comeau, in his recent book, entitled “Life and Sport on the North Shore,” says of the ruffed grouse: “In some years they are abundant for a time and “In some years they are abundant for a time and then disappear. I have noticed that heavy sleet in winter will sometimes drive them away from certain tracts of country. Since 1905 they have been pretty scarce all over the country (the north shore of the Gulf of St. Lawrence). I think this must be due to some kind of contagious disease, something similar, proba- bly, to the ‘grouse disease’ of Scotland. There is no other way of explaining their scarcity over such an immense extent of territory. Where the country has opened up, and there are only patches of wood here and there, it would be reasonable to suppose that they might have been exterminated by over-shooting and snaring; but where there are thousands of miles of forests, and not one in a hundred shot over, it cannot be put down to excessive shooting. As to natural enemies, they do not seem to have been any more numerous here than elsewhere. Last year (1908) I was over six weeks in the woods with two of my boys, and we only saw six. *jSoU UO BsnoID payny - 5. ple , ae 3 = *jSoU UO BsnoID payny RUFFED GROUSE—PARTRIDGE 165 From various points throughout the country, both in- land and along the coast, I received the same reports— no grouse.” Though constantly pursued by man during the open season and exposed to the attacks of a multitude of natural enemies, the ruffed grouse in many of our covers seems still almost to hold its own. There are seasons of abundance, when the birds are more numer- ous than usual, and others of scarcity, when sportsmen fear that they are about to disappear forever from par- ticular localities; but they continue to exist, and will long exist over much of the wooded country of the Eastern United States. The cutting off of the forests constitutes the gravest danger to which they are ex- posed. RUFFED GROUSE—PARTRIDGE. Where this is done the birds disappear, but, even after the heavy timber has been cut off, a period of ten or twelve years often results in the reforestation of the tract, at first only with underbrush and saplings, but later with larger trees. Then the ruffed grouse tend to come back again. For the ruffed grouse is a dweller in thickets. It seldom frequents the open land, except that it may venture out a little way from the edge of swamp or forest to pick up the grain in a cultivated field, or to eat the blackberries, huckleberries or wild grapes which ripen in some opening at the edge of the woods. For the most part, however, it is found in cover, sometimes quite open, among tall tree trunks of great size, or again in the most tangled swamp, among thickets of alder, blackberry, catbrier and grape vines. Wherever AMERICAN GAME BIRD SHOOTING 166 found—in a country that has been much gunned—it is well able to take care of itself. By much training it has acquired a great variety of tricks and stratagems which it practices to the utter discomfiture of many gunners. It may rise far ahead of the dog and out of shot and fly straight up a mountainside out of sight, so that it is impossible to mark it down; or if for some good reason of its own it continues to lie, it will very likely let man and dog pass it, and then when the man is tangled up in difficult brush and is trying to push his way ahead, the partridge with thunderous roar will rise behind him and disappear before he can free him- self from his fetters and bring the gun to his shoulder. Very commonly the partridge runs rapidly ahead of the dog, sometimes in a straightaway course, apparently to make sure that it will be well out of gunshot before it rises, or, again, it may run straight away, and then, bending off to right or left, may come around nearly to its trail again so that its pursuers will pass it. This is the precise trick played by the moose and sometimes by deer and bear when the conditions are favorable for tracking them. RUFFED GROUSE—PARTRIDGE. A favorite device is to rise behind a tree trunk, a clump of brush, a great rock or even a stone wall, and to keep this barrier between itself and the gunner until safely out of range. The flight of the grouse is very swift, and though The flight of the grouse is very swift, and though when well under way usually flying straight, yet often it rises on a curve, so that one may easily shoot behind it. Although often rising from the ground with a thunderous roar of wings, which may upset the nerves RUFFED GROUSE—PARTRIDGE 167 of its pursuer, the grouse does not always do this. Fre- quently it takes wing as quietly as the smallest bird, so that unless the shooter happens to be looking in the bird’s direction he may not be aware that it has flown. Sometimes, too, it will merely hop up into a tree and remain there, standing close to the trunk or to some branch, stiff, straight and motionless, and looking like a stub of wood. An old partridge may be counted on to do some unexpected thing. It deals in surprises. Its grace and beauty and the readiness with which it adapts itself to changing conditions, as well as the difficult places that it inhabits and the charm of its surround- ings, unite to command the gunner’s admiration. Although where constantly shot at it practices a Although where constantly shot at it practices a variety of such stratagems, yet in regions seldom pene- trated by man and where it has not been pursued, it is absolutely gentle and unafraid, and if startled from the ground flies no farther than to a low branch of a tree where it may sit with outstretched neck and erect crest while half a dozen shots are fired at it from pistol or rifle. If, however, a ball should strike the branch on which it is resting, or if a twig cut from above it drops down and touches the bird, it darts away with the swift flight with which we are all so familiar. If in a park or in private grounds the grouse are left un- disturbed, they may often be seen walking about and feeding, paying little attention to men who may pass near them, recognizing that no danger is to be appre- hended from them. WILLOW PTARMIGAN, Lagopus lagopus. Lagopus lagopus allent. Lagopus lagopus alexandre. In summer the male has the head and neck chest- nut, often becoming darker below. On the neck and chest this is often barred and flecked with black, as it is also on the flanks and generally on the under parts. The belly is more or less slate color. The quills and outer wing coverts are white and the rest of the upper parts irregularly barred with tawny, brown and black. Many of the feathers are tipped with whitish. The female is less deeply colored and is spotted with a paler tawny or yellow. The length is 14 to 17 inches, wing, 7 to 7/4 inches. The Newfoundland form, known as Allen’s ptarmi- gan, is slightly different, and is described as having a few of the secondaries, quills and wing coverts more or less mottled with dusky, and the shafts black. But this difference may be only seasonal. Mr. Austin Hobart Clark, who reported on the birds Mr. Austin Hobart Clark, who reported on the birds collected and observed during the cruise of the ‘Alba- tross in the North Pacific, found in southern Alaska a new form of willow grouse, L. l. alexandre. It is somewhat smaller than the willow grouse of the North, 168 168 WILLOW PTARMIGAN 169 and hasya smaller, weaker bill. In full summer plum- age its upper parts are deep rich chestnut, thickly and finely waved with black, the tip of each feather narrow- ly white. The lower throat and fore neck are chestnut, the breast becoming finely cross-lined with black, which increases toward the belly. The sides and flanks are very dark. The r The range of the willow ptarmigan, often called the willow grouse, is chiefly confined to the Arctic regions in North America, reaching from Alaska, over much of the British Provinces, to the Atlantic, and rarely straggling south into the United States—Minnesota, New York and New England. Twenty-five or thirty years ago it was not uncommon to find specimens of this bird in the hands of taxidermists in New York, the birds having presumably been shipped to that city from southern Canada. In many parts In many parts of the North the birds are exceeding- ly abundant and form no inconsiderable portion of the winter food supply of the Indians, while many are killed about the Hudson’s Bay posts. WILLOW PTARMIGAN, Much of what we know about these birds comes from E. W. Nel- son, who made his observations on the shores of the Bering Sea, and from L. M. Turner, who studied them in Labrador, with his headquarters at Ungava. From these observations we can From these observations we can get a good idea of the life of the willow ptarmigan. In winter these birds seem to be In winter these birds seem to be to some extent migratory, and, moving southward—partly, no doubt, in search of food—are found in considerable numbers as AMERICAN GAME BIRD SHOOTING 170 far south as latitude 50°; yet great numbers remain in the wooded districts of the North as far as latitude 670 Mr. E. W. Nelson, whose studies of the fauna of the Northwest and whose ethnological work among the Eskimos of Alaska are so well known, says that in the northern portions of their respective ranges these grouse are summer residents—frequenting the ex- tensive open country and being most abundant along the barren seacoast region of the Bering Sea and Arctic coast, “but in autumn, the last of August and during September, they unite in great flocks and mi- grate south to the sheltered banks of the Kuskoquim and Yukon rivers and their numerous tributaries. In early spring, as the warmth of the returning sun begins to be felt, they troop back to their breeding grounds once more. “During a large portion of the year these birds form one of the most characteristic accompaniments of the scenery in the northern portion of Alaska. During the winter season these birds extend their range south to Sitka and Kadiak.... “Toward the end of March, as some bare spots commence to show on the tundra, the Eskimos say this will bring the ptarmigan from the shelter of the in- terior valleys, and their observation proves true. At St. Michael these birds commence their love-making according to the character of the season—on some years by the first of April their loud notes of challenge are heard, but the recurrence of cold weather usually WILLOW PTARMIGAN yg! puts a temporary stop to their proceedings. About the fifth or fifteenth of this month the first dark feath- ers commence to appear about the heads and necks of the males. WILLOW PTARMIGAN, During some seasons the males make scarcely any progress in changing their plumage up to the middle of May, when I have frequently seen them with only a trace of dark about the head and neck. In the spring of 1878 the first males were heard calling on the 26th of April, and on April 27th, in 1879, the males were just commencing to moult, show- ing a few dark feathers, but these seasons were un- usually late. In autumn the change frequently com- mences the last of September and by the first of October it is well under way, the winter moult being completed toward the end of this month... . “At the Yukon mouth, on the evening of May 24th, “At the Yukon mouth, on the evening of May 24th, these ptarmigan were heard uttering their hoarse notes all about. As we were sitting by the tent my in- terpreter took my rifle and, going off a short distance, worked a lump of snow to about the size of one of these birds. Fixing a bunch of dark-brown moss on one end of the snow to represent the bird’s head, he set his decoy upon a bare mossy knoll; then retiring a short distance behind the knoll he began imitating the call of the male until a bird came whirring along and taking up the gauntlet lit close by this supposed rival and fell a victim to the ruse. iv At this time the males were continually pursuing each other or holding possession of prominent knolls, frequently rising thence five to ten yards in AMERICAN GAME BIRD SHOOTING 172 the air, with quick wing strokes and descending with stiffened wings, with the tips curved downward. While ascending they uttered a series of notes which may be repeated by the syllables ku-ku-ku-ku, which is changed as the bird descends to a hard rolling kr-r-r-r-r, in a very deep, guttural tone, ending as the bird reaches the ground. Frequently a pair would fly at each other full tilt and a few feathers would be knocked out, the weaker bird quickly taking flight again, while the victor rises as just described and utters his loud note of defiance and victory. On an- other occasion when the birds are more evenly matched they fight fiercely until the ground is strewn with feathers. WILLOW PTARMIGAN, “On May 24th almost all these birds are paired, but some did not complete their nuptials until the first few days in June. This grouse takes but a single mate in northern Alaska, and I am informed by the natives of Unalaska that the same is the case with the rock grouse found on the Aleutian Islands, nor have I ever known of the ptarmigan assembling in numbers about any special meeting place to carry on their love affairs; they scatter about, as previously mentioned, being seen singly here and there on prominent knolls over the flat country. Early in June, rarely so early as the last of May, the first eggs are laid; by June 20th and 25th the downy young are usually out, and when ap- proached the female crouches close to the ground among her brood. When she sees it is impossible to escape notice she rolls and tumbles away as though WILLOW PTARMIGAN 173 mortally injured, and thus tries to lead one from her chicks. The young at the same time try to escape by running away in different directions through the grass. At this season the female and male both moult and assume a plumage which differs considerably The young are fledged and on the wing at varying dates through July and are nearly full grown by the first to the tenth of August.” Mr. R. Mac Mr. R. MacFarlane, long chief factor to the Hud- son’s Bay Company, but now retired and resident in Winnipeg, whose observations on the fauna of many parts of the Far North are so valuable, gives some interesting notes about the willow ptarmigan in one of the regions which he has made famous. He says! “This species is exceedingl “This species is exceedingly abundant in the neigh- borhood of Fort Anderson, on the lower Anderson River, and in the wooded country to the eastward. It is not, however, common in the Barren Grounds, especially from Horton River to Franklin Bay, where it is replaced by L. rupestris. The nest is invariably on the ground, and consists of a few withered leaves placed in a shallow cavity or depression. The female sometimes leaves it only when almost trodden under foot, in fact, several were swooped upon and caught thereon by hand. They usually begin to lay about the end of May or the beginning of June. WILLOW PTARMIGAN, The process of moulting, or the gradual assumption of their sum- mer plumage, commences a week or two earlier. The female lays from seven to ten, twelve, and, occasional- ly, as many as thirteen eggs, which I find was the AMERICAN GAME BIRD SHOOTING 174 greatest number recorded; and we had reason to know that some, at least, of the nests were used by ptarmigan several seasons in succession. When very closely ap- proached as stated, the female would frequently flut- ter off, sometimes spreading her wings and ruffling her feathers, as if to attack or frighten away intrud- ers, and at other times calling out in distressed tones, and acting as if she had been severely wounded. “In one instance, where an Indian collector had found a nest which contained seven eggs, he placed a snare thereon; but on returning to the spot a few hours afterward, he was surprised to find that six of the eggs had disappeared in the interim, and as no egg- shells were left behind they were in all probability re- moved by the parents to a safer place. The male bird is generally not far away from the nest, and his pe- culiar hoarse and prolonged note is frequently heard, the more especially between the hours of 10 p.m. and 2 A.M. Both, however, displayed great courage and devotion in protecting from capture their young, which we often encountered on our return coast trips. “About the end of September, during October, and “About the end of September, during October, and early in November L. lagopus assembles in great flocks, but during the winter it was seldom that more than two or three dozen were ever noticed in single companies. They are, however, most winters very numerous in the neighborhood of Fort Good Hope and other Hudson’s Bay Company posts in the Mackenzie River district; but as spring sets in, they begin to mi- grate northward, and it is very doubtful if many breed WILLOW PTARMIGAN 175 to the south of latitude 68°, at least in the valley of the Anderson.” The change of summer plumage to that of winter begins on the abdomen and gradually extends over the entire body, the head changing last. This change occurs between September toth and the last of October, the young assuming their winter garb a little in ad- vance of the adults. WILLOW PTARMIGAN, In spring the change is reversed, commencing about the head of the birds and thence passing over the rest of the body. After the head has assumed its summer plumage, the change on the rest of the body goes on very slowly, and in many instances is never perfected, the back and abdomen of the ma- jority of summer specimens being never entirely free from white feathers. The wings remain white through- out the year. There is considerable individual varia- tion in the plumage of adult specimens shot at the same season. Mr. Charles Sheldon, who spent the winter of 1908 and 1909 on the shoulders of Mt. McKinley, saw there the willow ptarmigan, the rock ptarmigan and the white-tailed ptarmigan. These birds were all abun- dant, and there was no difficulty at any time in starting out and in the course of a short walk killing with a .22 rifle a sufficient number to last for food during several days. In spring, at the mating season, the willow ptarmi- In spring, at the mating season, the willow ptarmi- gan was extremely noisy, and at that time was seen much in the trees, often sitting on the topmost spray, from which he uttered his crowing call, and often AMERICAN GAME BIRD SHOOTING 176 made short flights. Long before the snow disappeared the birds had commenced to assume the dark plumage of summer, the head and neck changing first. The birds were now very noticeable against the snow. The males fought with much energy. After the young had been brought off the males disappeared. When startled in winter the birds often fly a short When startled in winter the birds often fly a short distance, and then, alighting on the snow, remain crouched there, perfectly motionless, so that they are extremely hard to see. As I have often noticed with the white-tailed ptarmi- As I have often noticed with the white-tailed ptarmi- gan, the willow ptarmigan, when the wind is blowing fiercely and the snow driving, will often alight on a drift and quickly scratching a hole will crouch in it, thus protected from the wind and the drifting snow, which, however, may sometimes quite cover them over. WILLOW PTARMIGAN, In the summer of 1899, while the Harriman Alaska In the summer of 1899, while the Harriman Alaska expedition was in Yakutat Bay, some of the members who were strolling up a ravine back of the sealing vil- lage of the Indians, came upon a female ptarmigan with half a dozen young. The young ones did not at once hide, and one of them was caught in the hand. The mother was anxious and uneasy for her little ones and walked about within three or four feet of us, and when the young one was caught she flew close about the captor. When walking on the ground she clucked like a setting hen, but with a deeper note; and after her young had been released she called to them, warn- ing them to remain hidden, with the same note that a domestic hen uses to warn her chickens that she sees “eySe[W Ul URsTUIe}qd MOTI “eySe[W Ul URsTUIe}qd MOTI WILLOW PTARMIGAN 177, a hawk in the sky. When at last we walked away, leaving the family alone, she also walked off into the alder bushes. The wings and a few breast feathers of this mother bird were white, but the rest of her plumage was black and tawny, much like that of a female spruce grouse. Of this species at Ungava, in Labrador, Mr. L. M. Turner says: “In the spring these birds repair, as the snow melts, to the lower grounds and prepare for the nuptial sea- son. About the toth of April they may be heard croaking or barking on all sides. A male selects a favorable tract of territory for the location of the nest, and endeavors to induce a female to resort to that place. He usually selects the highest portion of the tract, whence he launches into the air, uttering a bark- ing sound of nearly a dozen separate notes, thence sails or flutters in a circle to alight at the place whence he started, or to alight on another high place, from which he repeats the act while flying to his former place. Immediately on alighting he utters a sound similar to the Indian word, chu-xrwan (what is it?), and repeats it several times, and in the course of a few minutes again launches in the air. WILLOW PTARMIGAN, “T cannot speak accurately on the subject, but think “T cannot speak accurately on the subject, but think that seventeen days are required to incubate the eggs. On the 2oth of June I obtained a young bird of this species, which was less than forty-eight hours out of the shell. This was the earliest record. Thousands of these young must perish annually, either from the cold rains or from their parents being killed for food. . . . After the middle of August the birds have ac- quired a good size and are then feeding on berries of various kinds. They are then quite tender, of nearly white flesh, and when properly prepared form a pleas- ant food for the table. The young birds of the year attain their full growth by the first of November.” It thus seems that this ptarmigan is monogamous, a “T cannot speak accurately on the subject, but think that seventeen days are required to incubate the eggs. On the 2oth of June I obtained a young bird of this species, which was less than forty-eight hours out of the shell. This was the earliest record. Thousands of these young must perish annually, either from the cold rains or from their parents being killed for food. . . . After the middle of August the birds have ac- . . . After the middle of August the birds have ac- quired a good size and are then feeding on berries of various kinds. They are then quite tender, of nearly white flesh, and when properly prepared form a pleas- ant food for the table. The young birds of the year attain their full growth by the first of November.” It thus seems that this ptarmigan is monogamous, a It thus seems that this ptarmigan is monogamous, a pair mating and remaining together through the breed- ing season, the male taking part in the care of the young, showing a strong attachment for them and being devoted to the female. The food of this species consists chiefly of the buds The food of this species consists chiefly of the buds and leaves of various willows and birches, as well as berries of different sorts, which during the summer are exceedingly abundant in that country. WILLOW PTARMIGAN, [Early in the morning hundreds of these birds may be heard, continuing until nearly eleven o’clock, when the bird then becomes silent until after three o’clock, when he again goes through the same performance, though with less vigor than in the morning. In the course of a few days a female may be found in the vicinity. The AMERICAN GAME BIRD SHOOTING 178 actions of the male are now redoubled, and woe be to any bird of his kind which attempts to even cross his chosen locality. Battles ensue which for fierceness are seldom equaled by birds of larger size. “In the vicinity of Fort Chimo the nesting of this “In the vicinity of Fort Chimo the nesting of this species begins during the latter part of May. The nest is usually placed in a dry spot among the swamps, or on the hillsides where straggling bushes grow. The nest is merely a depression in the mosses and contains a few blades and stalks of grass, together with a few feathers from the parent bird, which is now in the height of the moulting from the winter to the summer plumage. “The first eggs obtained were two on June 1, 1884, this being the earliest record at Fort Chimo. The number laid for a set varies greatly in different locali- ties. At Fort Chimo seven to nine is the usual num- ber, although in exceptional instances as many as eleven and rarely thirteen may be found. “While I was at St. Michael (Norton Sound, “While I was at St. Michael (Norton Sound, Alaska) I frequently found nests containing as many as fifteen, and several times found seventeen. I was there informed that over twenty eggs had been taken from a single nest. On neither side of the continent did I hear that more than one female deposited eggs in the same nest. I can affirm that a clutch of seven eggs may be taken, and if the nest be not disturbed, the female will deposit nearly the same number again. These may again be taken, and not over three eggs will be deposited, and if disturbed a third time she will WILLOW PTARMIGAN 179 lay no more unless she selects a new location, which, of course, would be difficult to ascertain. WILLOW PTARMIGAN, The eggs, like those of other ptarmigan, are cream-colored as to groundwork and are marked with blotches, spots and cloudings of reddish and dark brown, often entirely covering the ground color. Captain Bendire tells us that all these markings are readily removable from the freshly laid egg. AMERICAN GAME BIRD SHOOTING 180 The form of willow ptarmigan, known as Allen’s ptarmigan, was described from Newfoundland by Dr. Stejneger nearly twenty-five years ago. The differ- ences between it and the bird of the mainland are trifling, being chiefly confined to the color of the shafts of primaries and secondaries, which are black. This is still a common bird of Newfoundland, where it is often called “partridge” by the settlers, and where, in more remote districts, it still offers very good shooting, though nowhere nearly so abundant as formerly. It lives on the barrens, feeding on seeds and berries, and in winter on the buds of alders, willows and birches. It breeds on the ground among the spruces. Perhaps no writer has lived longer in the region in- habited by the willow grouse than Napoleon A. Comeau, who, in his recent work, “Life and Sport on the North Shore of the Gulf of St. Lawrence,” gives some interesting notes on the species. He says: “Of all the above species [of grouse], the willow “Of all the above species [of grouse], the willow ptarmigan is by far the most abundant, and of con- siderable value as an article of food during its years of passage. It is an irregular migrant, in this wise, that it does not come regularly every season, like most other birds, and sometimes we may be three or four years without seeing any. I have gathered consider- able data on this subject, and I find that about every tenth year is one of great abundance. Here are some of the dates: 1863 and 1864, extremely abundant; 1867, disappeared this year; 1871, a few were seen this year, but none between 1867 and 1871; 1872 and 1873, WILLOW PTARMIGAN 181 very numerous again and total disappearance in 1876; 1882, a few observed; 1883, 1884 and 1885, great abundance; 1887, disappeared entirely; 1891, a few seen, gradually increasing each year till 1895, when there were considerable quantities; 1897, none; 1903 and 1904, abundant, and a few seen every winter since to date, 1909. WILLOW PTARMIGAN, “At one time it was supposed that these years of “At one time it was supposed that these years of abundance on the coast were due to heavy sleet in the interior, covering up all the buds and preventing the birds from feeding, and thus forcing them to seek food elsewhere. I have noticed that this will affect them to some slight extent, but the dates given show too much regularity for this to be the true cause. My be- lief is that it is due to the food supply. Having ex- amined thousands of the crops of these birds I found that over ninety per cent. contained the buds of a species of willow, popularly known here as pussy wil- low, Salix arctica? The balance were buds of the birch, poplar and the mountain ash and its berry. I also noticed a few seeds that I could not identify. “After a year or two of great abundance, all the “After a year or two of great abundance, all the willows are destroyed by the breaking of the tips and the buds, and the shrub takes about two years to re- cover, which is generally by fresh sprouts from the roots. As the food fails the birds have to move else- where. It would, therefore, appear from my data that it takes about ten years to go around their circle of migration. I say circle because their line of flight seems to indicate this. They first appear on the AMERICAN GAME BIRD SHOOTING 182 Labrador coast line flying south, and continue so till they reach our large rivers, like the Manicouagan, Bersimis and the Saguenay, seldom going west of this last. These large rivers are followed up in a west and northwesterly direction, the birds scattering inland over a tract that includes the Lake St. John and Lake Mistassini region, then down to the shores of Hudson’s Bay, where Dr. Milne and Mr. Peter McKenzie told me they flew north all along the coast line to Ungava, then south again to the Labrador, and so on. The range of the flight on this side of Hudson’s Bay would cover about ten degrees of latitude and in round fig- ures form a circle around this big peninsula of about two thousand miles. WILLOW PTARMIGAN, As this immense body of ptarmi- gan moves on during a season of abundance, stragglers are left behind, which breed, giving another and lesser batch to migrate the second year, when fewer strag- glers are left, till the third or fourth season, when no more are seen for a time. Their total absence varies from four to six years. They seldom or never breed in the lowlands, always seeming to prefer high and bare mountainous sections. In June, 1893, I saw a pair several times. They probably had their nest with- in a mile of our house (Godbout). The earliest ap- pearance of any large migration was October 29, 1872, but as a rule it begins here from the 15th of November to December. “The regular flight along the seashore lasts about “The regular flight along the seashore lasts about four to six weeks. After that the birds seem to scatter inland and feed. When on the move they fly very WILLOW PTARMIGAN 183 early in the morning, sometimes so early that it is im- possible to distinguish them unless there happens to be some dark background. The morning flight, when abundant, will last an hour or two. The size of the flock varies, in ordinary seasons, from ten to fifteen or twenty. In years of great abundance, flocks of a hun- dred or more are common. On the 14th of December, 1885, I saw at Trinity Bay, six miles east of Pointe des Monts, one flock which contained many thousands. It was a continuous mass of birds over half a mile long and from sixty to a hundred yards wide. I have never seen anything approaching this before, nor have I since. When in large flocks they are, as a rule, shy, especially if the weather is very cold or windy, and will rise long before one gets within ordinary range. On the wing, however, they do not seem to mind any- thing in their way, flying over and around one with- out apparently noticing him. They are very strong on the wing, not any faster than the ruffed grouse, but capable of sustaining much longer flights, occasionally going five or six miles without resting. WILLOW PTARMIGAN, “In small b “In small bunches and in snowy and mild weather they are quite tame, and when feeding in the thick willow bottoms it is hard work to get them to rise out of them. After feeding they congregate around some clump of willow, and scratching a small hollow in the snow, will lie perfectly still for hours, the top of the head just level with the snow, the black eye and beak alone betraying their presence. The popular idea of their diving in the snow to escape pursuit is most AMERICAN GAME BIRD SHOOTING 184 ridiculous. At night they will occasionally burrow in the snow, but only during high winds or very cold weather, the usual way of resting being the small hollow mentioned. Another rather remarkable thing in connection with ptarmigan is the apparent dispro- portion of the sexes. Out of the many thousands that I have killed and examined, only about twenty-five per cent. were males. In winter they prefer low valleys and the borders of rivers and lakes and dense willow patches, but as the season advances they seek the higher ranges, choosing those that face the midday sun. When flying over water, as they often do in crossing bays or large rivers, they keep very near the surface, just about a foot or so above it. Over land the reverse is the case, for sometimes they rise high over the tops of the tallest trees. The most remarkable thing about them, however, is their seasonal change of plumage. In 1885 I had the pleasure of attending the meetings of the American Ornithologists’ Union, in New York. At one of the meetings a very interesting paper was read by Dr. Stejneger on this subject. The doctor exhibited two specimens which came from New- foundland, and which, in his opinion, were a sub- species confined to the island. This distinction was based particularly on the coloration of the primaries. The birds shown had nearly all the tips of the pri- maries black. Since my return I have taken special pains to examine a great number of birds. On those killed prior to 15th November I found the same coloration, more or less, as on the species shown, but WILLOW PTARMIGAN 185 after that date there was a gradual whitening of the primaries, and in many cases only the shafts were white. WILLOW PTARMIGAN, “During the last two migrations, taking the best years, 1895 and 1904, I took some trouble to try and find out approximately how many birds were killed between certain points. During the first year men- tioned, between Mingan and Godbout, 175 miles of coast, 30,000 were killed. In the second (1904), 14,000, but I am sure that during 1885 nearly 60,000 must have been shot or snared. When a flight begins, every man, woman and boy able to handle a gun is out. To avoid accidents, which are very rare indeed, each gun occupies a certain point or station, and shoots at all the birds that pass or light in his vicinity. The ladies keep watch of those that may light near the houses. The bags vary, of course, according to the skill of the shooter and his method of shooting. If he is there for business he will take all the pot shots. He can frequently get five or six in one shot. I have seen fourteen killed in a single shot. A few will only shoot on the wing, but there are many days when the wing shooter comes out ahead. The biggest bag I ever made (it was in 1885), shooting at flying birds, was eighty-two brace in one morning. At Caribou Islands, that winter, nets were tried, but they were not very successful, more being got by shooting. Indians fre- quently snare them by setting their snares around wil- low clumps, where the birds feed. It is a very simple arrangement. A twig is stuck in the snow, a twine AMERICAN GAME BIRD SHOOTING 186 snare is tied to it, a very light support placed under it to hold it in position, and it is ready. In walking around, the bird runs into it, then tries to rise on feel- ing the snare, only to tighten the noose. There is a little fluttering, and it is all over.” ROCK PTARMIGAN. Lagopus rupestris. Lagopus rupestris reinhardi. Lagopus rupestris welch. Lagopus rupestris nelsoni. Lagopus rupestris atkensis. Lagopus rupestris townsend. Lagopus rupestris evermannt. Lagopus rupestris chamberlaint. Lagopus rupestris kelloge. Lagopus rupestris dixont. The bill of the rock ptarmigan is much more slender than that of the willow ptarmigan, and the bird is somewhat smaller, measuring from 13 to 1434 inches. In winter the bird is white, with black tail; but a line running from the eye to the bill is also black. It is said that the female does not always have this black spot covering the lores. In summer the male is grayish brown, the feathers In summer the male is grayish brown, the feathers being crossed with fine black lines, which tend to form irregular zigzag bars. The shoulder feathers, or scapu- lars, are chiefly black, which gives the effect of large black spots on the back. The quills of the wing —primaries and secondaries—and the outer tertiaries 187 187 187 AMERICAN GAME BIRD SHOOTING 188 are white. The lores and top of the head are black, the ends of the feathers on the latter being tipped with brown. Breast regularly barred with blackish and light brown, and the sides the same, but with finer black bars; beneath, white. The summer plumage of the female is brighter, yel- The summer plumage of the female is brighter, yel- lowish or reddish, spotted and barred with black, and having the quills and secondaries always white. The lower plumage is somewhat paler and grayer; the dot- tings and barrings are black, often quite fine; yet the barrings show a tendency to form spots and heavier bars. The head and neck are more yellowish and barred with dusky. The rock ptarmigan, in its various forms, is scat- tered over Arctic America in general, except the ex- treme north, including Greenland and the Aleutian Islands, and is found southeast as far as the Gulf of St. Lawrence, occurring on Anticosti Island. Mr. Nelson reports it a common resident of the mainland of Alaska, where it inhabits the higher ground during the summer, but is driven down by winter to the lower levels. Its habits do not appear to differ greatly from those of the common willow ptarmigan. It breeds in large numbers on the Barren Grounds, from which Mr. MacFarlane reported on its breeding habits, nests and eggs. ROCK PTARMIGAN. It does not appear to be so prolific a bird as the willow ptarmigan, its eggs being fewer in number and apparently running from nine down to four or five. The female sits closely on her eggs, and her color harmonizes so well with that ROCK PTARMIGAN 189 of the surrounding vegetation that usually when dis- covered she is seen only by accident. In Alaska the eggs are laid in May and in the Bar- ren Grounds somewhat later, usually between the mid- dle of June and July. The eggs are quite like those of the willow ptarmigan, but are slightly smaller. The ground color runs from cream color to yellowish, and the spots and blotches vary from a dark brown to a claret red. Sometimes these spots are so numerous as almost to hide the ground color. Reinhardt’s ptarmigan, a sub-species of the rock Reinhardt’s ptarmigan, a sub-species of the rock grouse, is found in Greenland and throughout Labra- dor, and Mr. L. M. Turner, whose notes on the birds of Labrador and Ungava are so familiar to ornitholo- gists, tells us about all that is known of it. He says that it prefers open ground, and rarely enters even the skirts of the wooded tracts. “The mating season begins in May, and during this period the male acts in the strangest manner to secure the affection of its mate. He does not launch high in the air and croak like the willow ptarmigan, but runs around his prospective bride with tail spread, wings dragging like those of the common turkey, or else with head and neck stretched out and breast in contact with the ground, pushing himself in this man- ner by the feet, which are extended behind. The male at this time ruffles every feather of his body, twists his neck in various positions, and the supra-orbital pro- cesses are swollen and erect. He utters a most peculiar sound, something like a growling kurr-kurr, and as AMERICAN GAME BIRD SHOOTING 190 the passion of the display increases, the bird performs the most astonishing antics, such as leaping in the air without effort of wings, rolling over and over, acting withal as if beside himself with ardor. The males engage in most desperate battles; the engagement lasts for hours, or until one is utterly exhausted, the feath- ers of head, neck and breast strewing the ground. ROCK PTARMIGAN. A maneuver is for the pursuing bird to lead the other off a great distance and suddenly fly back to the female, who sits or feeds as unconcerned as it is possible for a bird to do. She acts thoroughly the most heartless coquette, while he is a most passionately devoted lover. He will die rather than forsake her side, and often places himself between the hunter and her, uttering notes of warning for her to escape, while attention is drawn to him, who is the more conspicuous. “When the young are with the parents they rely upon their color to hide themselves among the nearly similar vegetation from which they procure their food. I am certain I have walked directly over young birds which were well able to fly. If the parent birds are first shot, the entire number of young may be secured, as they will not fly until nearly trodden upon, and then only for a few yards, while they may easily be seen. I have found on two occasions an adult female with a brood of thirteen young. All of the flocks were se- cured without trouble. At other times only three or four young would be found with both parents. The young are very tender when first hatched; no amount of most careful attention will induce them to eat, and ROCK PTARMIGAN IQ! after only a few hours’ captivity they die. I never could keep them alive above twelve hours. The change- able weather, sudden squalls of snow or rain, must be the death of scores of these delicate creatures. Their note is a soft piping pe-pe-pe, uttered several times, and has the same sound as that of the young of the bobwhite (Colinus virginianus).” For the first few weeks of their life the young of For the first few weeks of their life the young of this species cannot be distinguished from those of the willow ptarmigan, but when a month old they may be readily told apart by the bill. The food of this species consists of insects, berries, leaves and buds of the birch and willow. Mr. Kumlien, at Cumberland Sound, shot one whose crop was full of moss. Welch’s ptarmigan, described by Mr. ROCK PTARMIGAN. Brewster from Newfoundland, is reported as a highland form of the rock ptarmigan, and confined to the sides and sum- mits of rock hills and mountains in the interior of Newfoundland. The rock ptarmigan is a very local bird, and for the most part spends its life on or near the hills where it was reared. It has been spoken of as a truly alpine species, rarely found below the line of stunted black spruces, except in the depth of winter, when they descend to the lowlands and sometimes min- gle with the willow grouse. The settlers call it moun- tain partridge to distinguish it from the willow grouse, known as partridge. Nelson’s ptarmigan is another form of the rock Nelson’s ptarmigan is another form of the rock grouse, which is confined to the islands of Unalaska, AMERICAN GAME BiRD SHOOTING 192 Akutan and Unimak of the Aleutian group. It spends most of the time on higher ground, but at the breed- ing season comes down to the narrow valleys to rear its young. The nest is usually placed in tall grass in some valley or on the open tundra. Turner’s ptarmigan, another subspecies, is found at Atka, one of the islands of the Aleutian chain. It occurs also on Amchitka Island. The bird is larger than the mainland form, and is quite numerous on these islands. Mr. Turner reports the eggs as from eleven to seventeen in number. The habits of the subspecies are not known to differ from those of the other forms of this ptarmigan. Townsend’s ptarmigan from Kiska Island, Ever- Townsend’s ptarmigan from Kiska Island, Ever- mann’s ptarmigan from Attu Island, Chamberlain’s ptarmigan from Adak Island, Dixon’s ptarmigan from the Sitkan Islands, and Kellog’s ptarmigan from Mon- tague Island, complete the list of closely related forms from the Alaskan Islands, where this species seems to have been very easily influenced by local conditions. Townsend’s ptarmigan from Kiska Island, Ever- mann’s ptarmigan from Attu Island, Chamberlain’s ptarmigan from Adak Island, Dixon’s ptarmigan from the Sitkan Islands, and Kellog’s ptarmigan from Mon- tague Island, complete the list of closely related forms from the Alaskan Islands, where this species seems to have been very easily influenced by local conditions. WHITE-TAILED PTARMIGAN. Lagopus leucurus. Lagopus leucurus peninsularis. Lagopus leucurus. Lagopus leucurus peninsularis. Winter plumage snow-white throughout; the bill and eyes being the only dark spots to be seen. The summer plumage is buff, or, later in the season, clay color, coarsely barred with black on a pale ground. There is little difference between the male and the female. The tail is always white. In the young the tail feathers at first are not white, In the young the tail feathers at first are not white, but are mottled with brownish. They become white with moulting, however. The sub-species, peninsularis, inhabits the alpine The sub-species, peninsularis, inhabits the alpine mountains of central Alaska, northern Yukon, N. W. Mackenzie, south to Cook Inlet, Kenai Peninsula and southern Yukon. In all America, the especial home of the grouse, there is no prettier member of this family than the white-tailed ptarmigan. Like all its kind, it loves the cold and snow, but, unlike the other American mem- bers of the group, it substitutes altitude for latitude and is an inhabitant of the lofty mountains of the West, from central Alaska and northern Yukon south to Washington and New Mexico. Here on the very edge of perpetual snowfields, not far from some brawl- 193 193 AMERICAN GAME BIRD SHOOTING 194 ing ice-fed torrent, and within the sound of the roar of avalanche and the thunder of glacier, the little ptarmi- gan spends his life, seldom disturbed by enemies. In summer he wears a livery of black and clay color, In summer he wears a livery of black and clay color, which harmonizes so well with the rocks among which WHITE-TAILED PTARMIGAN WHITE-TAILED PTARMIGAN his life is passed that it is most difficult to see him, if he wishes to remain unseen and does not move; while in winter, as he journeys over the frozen wastes, his plumage is as white as the snows on which he walks. His must be a contented, care-free life, for he has few enemies to fear. The sneaking coyote and the stealthy wild cat seldom visit his mountain soli- WHITE-TAILED PTARMIGAN 195 tudes. Rarely the eagle’s broad pinions cast their dark shadow over snowfield and rock slope, but the eagle is generally in search of larger game, the tender young of the big horn or of the white goat. WHITE-TAILED PTARMIGAN. The great bears that in early summer prowl over the range, looking for the young grass or digging out mice, or later pick- ing the luscious huckleberries, do not give a thought to the ptarmigan, unless by chance they stumble on her nest, when it suffers the fate of every thing eatable that comes in Bruin’s way. Of all the animals of the mountains, the one that the ptarmigan has most to fear is perhaps the pine marten. He is always traveling about, alow and aloft; equally at home among the trees of the forest and the rocks of the peaks, always hungry, always searching for food; and, while it may be doubted whether he destroys many full-grown ptarmigan, we may feel sure that he compasses the death of many young and pil- lages many a nest. Although the white-tailed ptarmigan is abundant enough in the high mountains which it inhabits, it is scarcely known at all to sportsmen. Only the hardy spirit who climbs above timber line in search of sheep or goats, or that other enthusiast whose highest pleas- ure it is to reach the summit of the loftiest mountain peaks, ever reaches the home of this bird; and as neither of these ever burdens himself with a shotgun, it is almost never killed for sport. In Colorado, where many prospectors and miners carry on their operations far above timber line, the ptarmigan is often killed for AMERICAN GAME BIRD SHOOTING 196 food, but in other parts of the West man interferes but little with the abundance of this species. This ptarmigan is found in considerable numbers above timber line on most of the mountains of western America. It does not occur in the Sierra’ Nevada, nor in the Cascades south of Washington. It is no- where very abundant, though I have seen them, in autumn, in flocks of twenty-five or thirty, and recall an afternoon when, on Mount Jackson, in Montana, two of the party secured twenty-two of the birds. Yet, as a rule, they are nowhere very common, a single brood often seeming to occupy its own range of ter- ritory, which is not encroached on by others. I have not seen them in large flocks as mentioned below by Mr. Trippe. WHITE-TAILED PTARMIGAN. The nest of the white-tailed ptarmigan is built high The nest of the white-tailed ptarmigan is built high up on the mountain-side above timber line, and may be located anywhere among the loose stones and rocks. A little depression is perhaps scratched out near a patch of grass or weeds, and here the mother bird deposits her eight or nine eggs, buff in ground color, sometimes spotted with many small reddish or brown dots, or, again, peculiarly blotched with larger mark- ings of the same color. The nest is usually quite in the open, but the grayish mother bird so closely resem- bles the stones among which she nests as readily to escape observation. Moreover, the confiding nature of the bird gives her such courage that she will remain on the nest, unscared even by the close approach of some great danger. WHITE-TAILED PTARMIGAN 197 Mr. A. W. Anthony wrote to Captain Bendire: “Twice have I escaped stepping upon a sitting ptarmi- gan by only an inch or so, and once I reined in my horse at a time when another step would have crushed out the life of a brood of nine chicks, but an hour or so from the egg. In this case the parent crouched at the horse’s feet and, though in momentary danger of being stepped on, made no attempt to escape until I had dismounted and put out my hand to catch her. She then fluttered to the top of a rock a few feet dis- tant and watched me as I handled the young, constant- ly uttering low, anxious protests. The chicks were still too young to escape; mere little wet bunches of down, that stumbled and fell over one another when they attempted to run. “Miners in whom I have confidence told me that they have lifted sitting ptarmigan from the nest and han- dled the eggs, while the bird stood but a few feet dis- tant, watching her treasures and uttering an occasional squeak like a sitting hen. One which had her nest near the trail between the cabin and the mine was an- noyed in this way so often that she would attempt to regain the nest while the eggs were being handled, and had to be frequently pushed aside. WHITE-TAILED PTARMIGAN. She never failed to peck at the hand and utter her protesting ki-r-r-r-r whenever one attempted to touch her, and made no attempt to fly away.” All the evidence given by people who have found the nests of this bird indicates that the sitting female AMERICAN GAME BIRD SHOOTING 198 is no wilder than the average domestic hen in the same situation. The following very interesting account of this bird The following very interesting account of this bird appears in Dr. Coues’ “Birds of the Northwest,” for which it was prepared by Mr. Trippe; it relates en- tirely to the species in Colorado: ‘The white-tailed ptarmigan is a very: abundant bird on the main range, living entirely above timber line the year round, ex- cept during the severest part of the winter, when it descends into the timber for shelter and food, occasion- ally straggling as low as 10,000 feet. It begins to change color about the middle of March, when a few specks of blackish brown begin to appear in the plum- age of the oldest males, but the change is very slow, and it is late in April before there is much black visible, and the close of May or early in June before the sum- mer plumage is perfect. The ptarmigan builds its nest in the latter part of June and commences hatch- ing toward the close of the month, or early in July. The nest—which is almost always placed on or near the summit of the ridge, or spur, many hundred feet above timber line-—is merely a depression in the ground, lined with a few straws and white feathers from the mother’s breast. The eggs are eight in number, of a light buff brown, thickly sprinkled with spots of dark chocolate brown, somewhat thicker at the larger end. While on her nest the bird is very tame. Once, while walking near the summit of the range, I chanced to look down and saw a ptarmigan in the grass at my very feet ; at the next step I should have trodden upon her. WHITE-TAILED PTARMIGAN. WHITE-TAILED PTARMIGAN 199 Seeing that she did not appear frightened, I sat down gently, stroked her on the back, and, finally, putting both hands beneath her, raised her gently off the nest and set her down on the grass, while she scolded and pecked my hands, like a setting hen, and, on being released, merely flew off a few yards and settled on a rock, from which she watched me till I had gone away. Late in July I came across a brood of young ones, apparently not more than four or five days old. They were striped with broad bands of white and blackish brown, and looked precisely like little game chickens. The mother flew in my face and hit me with her wings, using all the little artifices that the quail and partridge know so well how to employ, to draw me away; while her brood, seven or eight in number, nimbly ran and hid themselves in the dense grass and among the stones. On another excursion above timber line, toward the close of August, I found most of the young ones nearly grown, and strong on the wing; but one brood was of the size of quails, showing that some birds must begin breeding much later than others, or that they: occasionally raise two broods. These little ones were colored much like the older birds, hav- ing blackish-brown bodies and pure white tails. About the first of September the ptarmigan begins to change color again, but, as in the spring, the process is very gradual, white feathers appearing one by one and taking the place of the dark ones. The white on the lower parts enlarges first, then the white areas on the wings, and next, white specks appear on the upper AMERICAN GAME BIRD SHOOTING 200 parts, becoming larger and more numerous as the sea- son wears on; but so gradual is the change that a month after it begins there is not much difference in the plumage perceptible, the general aspect being that of summer. There is much more of the light rufous, however, and the appearance is lighter and grayer, as though bleached. The dark areas predominate, how- ever, throughout October, and, as I have been in- formed by. WHITE-TAILED PTARMIGAN. persons who have killed them throughout the year, it is late in December or January before they become pure white, some few birds showing occasional dark spots even throughout the latter month. “The ptarmigan feeds upon the leaves and stalks of various alpine plants, being particularly fond of those of a species of Cassia, the flowers of which I have frequently taken from its crop. It also lives largely upon insects, and in winter is said to subsist on the buds and leaves of the pines and firs. Its flesh is light- colored, though not as white as thateof the gray grouse, to which it is usually considered inferior for the table. In localities where it is seldom molested, it is very tame, and I have been informed by persons whose word is worthy of belief, that they have frequently killed it with sticks; but when persistently persecuted it soon becomes wild and leaves the range of a shotgun with surprising quickness. After hunting several large flocks for three or four days, they grew so shy that it was difficult to approach within gunshot, although at first they had been comparatively tame. Nimble of foot, the ptarmigan frequently prefers to run away on WHITE-TAILED PTARMIGAN 205. the approach of danger rather than take wing, run- ning over the rocks and leaping from point to point with great agility, stopping every little while to look at the object of alarm. I have sometimes chased them half a mile or more over the rocky, craggy ridges of the main range, without being able to get within gunshot or force them to take wing. The flight of the ptarmi- gan is strong, rapid and, at times, sustained for a considerable distance, though usually they fly but a few hundred yards before alighting again. It re- sembles that of the prairie hen, consisting of rapid flappings of the wings, alternating with the sailing flight of the latter bird. The note is a loud cackle, somewhat like the prairie hen’s, yet quite different, and when uttered by a large flock together, reminds one of the confused murmur and gabble of a flock of shore birds about to take wing. It is a gregarious bird, associating in flocks throughout the year, except in the breeding season. WHITE-TAILED PTARMIGAN. The different broods gather together as soon as they are nearly grown, forming large flocks, sometimes of a hundred or more. The colors of this bird closely resemble those of sur- rounding objects at all seasons of the year. In its summer plumage of speckled black and gray it is very difficult to detect while sitting motionless among the gray and lichen-covered rocks. The ptarmigan is ap- parently well aware of this, and often squats and re- mains quiet while one is walking past, trusting to its resemblance to the surrounding rocks to escape obser- vation. So perfect is this resemblance that sometimes, AMERICAN GAME BIRD SHOOTING 202 on seeing one alight at a certain spot, and withdrawing my eyes from it a moment, I have been unable to find it again, although I knew the exact place where it sat, until a movement on the part of the bird betrayed its position. In summer the white areas of the plum- age are completely hidden while the bird is squatting, although plainly visible while on the wing; in winter the first appearing black specks are concealed beneath the white feathers, and at this period, as I am in- formed, it is almost indistinguishable from the snow. On being pursued, it will dive into the snow and re- appear at a considerable distance.” My experience with this bird has been chiefly in Wyoming, Montana and British Columbia, and usually in the autumn. In the month of November some change in the plumage will be noticed, though this is chiefly in the reduction of the dark-colored areas, rather than in the appearance in them of white feath- ers. Where little disturbed by man, the birds pay but slight regard to the presence of the hunter, and feed along in a close flock without scrutinizing him. If he ventures so near as to alarm them, and this some- times may be within a few feet, they may rise on the wing against the stiff breeze and sail along for a few yards, looking much like a flock of domestic pigeons, to alight after going a very short distance. WHITE-TAILED PTARMIGAN. If the spot where they strike the ground is exposed, and the wind is blowing, the birds crouch flat on the ground, head to the wind, or those that have alighted near stones or rocks projecting from the earth, run and ‘aseun surids “URsTUII peyte}-a ‘aseu surid “URsTUI peyte}-a WHITE-TAILED PTARMIGAN 203 crouch behind them; or, if they alight on a snow bank, they quickly scratch out for themselves a little cavity in the snow, large and deep enough to contain the body, so that the wind blows over them. On the other hand, in Montana, among the high mountains of the Saint Mary’s region, where hunters and mountain climbers are often seen, these birds are sometimes quite wild, rising at a considerable dis- tance with a loud cackle, and flying a quarter of a mile to alight again on some prominent rock, upon which they run about with tail erect and head thrown back, cackling in alarm, and ready at an instant’s no- tice to take to wing again and fly still farther. It is only in this region that I have pursued them with a shotgun, and here they are as quick on the wing and as hard to hit as any of the grouse. Sometimes, when following scattered birds along the rough mountain- side, they would pitch down past me from the rocks on which they had perched, with a flight not less rapid than that of the New England ruffed grouse, as he darts down from the top of some pine tree in which he has hidden himself. Two or three years ago, Joseph Kipp, of Montana, while crossing from the west to the east side of the range, through the Belly River Pass, in July, came upon a brood of half-grown ptarmigan. The mother attacked him vigorously, and was so persistent that he caught her and several of the young, and carried them with him for half a day, when he let them go again, because he had nothing which they could eat. Two or three years ago, Joseph Kipp, of Montana, while crossing from the west to the east side of the range, through the Belly River Pass, in July, came upon a brood of half-grown ptarmigan. WHITE-TAILED PTARMIGAN. The mother attacked him vigorously, and was so persistent that he caught her and several of the young, and carried them with him for half a day, when he let them go again, because he had nothing which they could eat. AMERICAN GAME BIRD SHOOTING 204 It is hardly to be supposed that these birds would do well on food other than that to which they are accus- tomed in their home among the high mountains, and any attempt to domesticate them would be foredoomed to failure. It is not improbable that if some one who resided high up in the mountains should try to rear the young he might succeed, but they could not be taken away from the mountains where they belong. In summer or autumn single birds are often met In summer or autumn single birds are often met with high up on the peaks, presumably the males whose mates are then busy with their nests or young, and these individual birds usually seem wild. They will often stand and look until quite closely approached, and then run swiftly fifteen or twenty yards, and then stopping, stand erect and watch until again approached. While nowhere very abundant, the white-tailed While nowhere very abundant, the white-tailed ptarmigan is yet numerous on all the higher moun- tains which suit the requirements of its life. Half a dozen broods may be found within a range of two miles along the mountain-top, while the number of eggs varies from five or six to fourteen or fifteen. Occasionally, at the approach of winter, a considerable number of the birds will be found together, but I have not seen more than twenty-five or thirty in a flock. Though, in summer, insects, flowers of the heather, Though, in summer, insects, flowers of the heather, berries and seeds undoubtedly constitute a large part of the white-tailed ptarmigan’s food, nevertheless I believe that at all seasons they feed to a considerable extent on the buds and tips of the willow, the largest shrubs which grow near the tops of the mountains. On WHITE-TAILED PTARMIGAN 205 many occasions I have examined their craws and have usually found them stuffed with these willow buds. None of these examinations, however, has been made later than December. -tailed ptarmigan is Of all our grouse the white-tailed ptarmigan is probably the one least exposed to persecution by man. WHITE-TAILED PTARMIGAN. No doubt some are killed by lynxes, martens and weasels, but on the whole its enemies must be few in number. PINNATED GROUSE. Tympanuchus cupido. Tympanuchus americanus. Tympanuchus americanus attwateri. Tympanuchus pallidicinctus. Tympanuchus cupido. Tympanuchus american The familiar prairie chicken or prairie hen of a generation ago was the pinnated grouse, once so abundant in Illinois, Indiana, Kentucky and to the westward. It is remembered by older readers as abun- dant in our markets, where it sold for seventy-five cents per pair. In later years the term prairie chicken has been applied equally to the sharp-tail grouse, a species of more western distribution; and, generally, in the western country, any grouse found in the open are called: “chickens,” The pinnated grouse include four forms, grouped The pinnated grouse include four forms, grouped together under the genus Tympanuchus, a name which refers to the inflatable sac on the neck of all these grouse. All these forms are so similar as hardly to be distinguished by any one save a practiced ornitholo- gist. Birds of this group were formerly abundant on the Atlantic coast, as well as throughout much of the western country until the semi-arid plains were reached. So far as we know, their western boundary, roughly stated, was western Minnesota, eastern Ne- 206 PINNATED GROUSE 207 braska and Kansas, the Indian Territory and western Texas. Birds of this genus are brownish or clay color, cross- banded by dark brown or black. The tail is short and rounded, and the sides of the neck are provided with wh 17044 % =e Su rll || | =~ G5 ( x PINNATED GROUSE 17044 % PINNATED GROUSE conspicuous wing-shaped tufts of straight, stiff black feathers, beneath which is a naked inflatable air sac. In greater detail the color above is pale brownish, In greater detail the color above is pale brownish, barred with dusky and buff; beneath, paler, broadly barred or banded with brown. The quills of the wing are gray or brownish, with buff or whitish on the outer webs. The chin, throat and cheeks are buff, AMERICAN GAME BIRD SHOOTING 208 the cheeks marked with brownish spots. A dark- brown stripe runs from the corner of the mouth under the eye and across the ear, and above this is a stripe of buff. The black tufts of feathers on the side of the neck are stiff and narrow, the longest ones being two and a half inches. The tail feathers are black- ish, tipped with white; the under tail coverts white- tipped. PINNATED GROUSE. In the female the neck tufts are smaller, usually less than two inches in length, and the tail feathers are barred with light brown. In the ordinary prairie hen of the Mississippi Valley, In the ordinary prairie hen of the Mississippi Valley, Tympanuchus americanus, the scapulars are without white dots near the ends, and the neck tufts of the male are composed of more than ten narrow feathers, whose edges are parallel and whose ends are rounded, or sometimes almost square. The feathered young are more or less dotted with patches of white and black and the top of the head is reddish brown. The bird’s length is from eighteen to nineteen inches for the male, and a little less for the female. The Martha’s Vineyard heath hen, T. cupido, is The Martha’s Vineyard heath hen, T. cupido, is slightly smaller than the western prairie hen, has large and noticeable spots of whitish at the end of the scapu- lar feathers. The neck tufts of the male have not more than ten of the narrow feathers, all of which are sharply pointed. The lesser prairie hen, T. pallidicinctus, is also slightly smaller than the Mississippi Valley prairie hen, and is recognizable because each dark bar across the plumage consists of a continuous brown bar en- PINNATED GROUSE 209 closed between narrower black feathers; in other words, the edges of this dark bar are black, and this is true all over the bird. The lesser prairie hen is found in Kan- sas, southwestern Missouri, Oklahoma and _ west central Texas. Along the coast region of Texas and southwestern Along the coast region of Texas and southwestern Louisiana is found a well-marked race of pinnated grouse, which Major Bendire named in honor of Mr. H. P. Attwater, who brought the bird to the attention of ornithologists. It is about the size of the lesser prairie hen, but has the foot feathered only on the upper two-thirds of the tarsus; the tips of the long feathers of the wing-like neck tufts are square. THE HEATH HEN. The bird of the Atlantic coast now known as the heath hen is almost extinct, the only existing colony being on Martha’s Vineyard, an island off the coast of Massachusetts. It was formerly found in Massachusetts, Connecticut, Long Island, New Jersey, Pennsylvania, Maryland and Delaware, but has long been exterminated from most of these regions. Thus, long before any of the other so-called prairie Thus, long before any of the other so-called prairie grouse had been discovered, the pinnated grouse was well known. Indeed, it may very well be that when the Pilgrim Fathers landed at Plymouth Rock, the first flesh meat that they tasted in the new country was the coast form of what in later times was called prairie hen or prairie chicken, then known as heath cocke or heath AMERICAN GAME BIRD SHOOTING 210 henne, later changed in pronunciation to héth’n. The bird was long abundant in Massachusetts, in the open, brushy country around the seacoast, where, no doubt, it fed, as do its descendants to-day, at Martha’s Vine- yard, on acorns, berries, grass and insects. It was well known in New England in the first quarter of the nineteenth century, but disappeared soon after that. The old New England writers speak of the heath The old New England writers speak of the heath cocke as common, so that, according to Wood, “Hee that is a husband and will be stirring betime may kill halfe a dozen in a morning.” Mr. William Brewster, in his interesting and com- Mr. William Brewster, in his interesting and com- plete paper, entitled “The Birds of the Cambridge Region of Massachusetts,” says: “T have been permitted to quote the following in- “T have been permitted to quote the following in- teresting passage from ‘Notes of conversations with Eliza Cabot, written down by her son, J. E. C. (abot),’ and printed for private circulation in 1904: ‘T recol- lect the western prairie grouse in this part of the coun- try. I saw one once in Newton; and once after I was married, your father went down to the cape fishing, and in the woods there I saw a grouse very near me and saw him puff up that orange they have on the side of the neck.’ Eliza Cabot was born on April 17, 1791, and married about 1811. Her granddaughter, Mrs. THE HEATH HEN. Charles Almy, thinks it probable that she saw the grouse in Newton about the beginning of the nine- teenth century, and the one on the ‘cape’ (Cape Cod, no doubt) about 1812. That both birds were heath hens can scarcely be doubted, for there is no evidence PINNATED GROUSE 211 that living western grouse of any kind were intro- duced into Massachusetts at so early a period.” From the evidence given by Mr. Brewster and other From the evidence given by Mr. Brewster and other writers it may be assumed that the heath hen was more or less abundant on the site of Boston at the time that city was founded, and there is no reason why it should not have been numerous in other favorable sit- uations along the New England coast and to the south- ward. Early writings tell us that it was so. It was found along the seaboard south of New Jersey, and the late C. S. Wescott, of Philadelphia, frequently spoke of it as having occurred—according to tradition —in Maryland and Delaware, on the shores of the Chesapeake Bay and on the Peninsula of Maryland and Virginia. Nuttall, as late as 1832, says of the heath hen: “Along the Atlantic coast they are still met with on the grouse plains of New Jersey, on the brushy plains of Long Island, in similar shrubby barrens in Westford, Conn., in the islands of Martha’s Vineyard on the south side of Massachusetts Bay, and formerly, as probably in many other tracts, according to the information which I have received from Lueut.- Governor Winthrop, they were so common on the ancient bushy site of the city of Boston that laboring people or servants stipulated with their employers not to have the heath hen brought to table oftener than a few times in a week!” Linsley, in his list of Connecticut birds, eleven years Linsley, in his list of Connecticut birds, eleven years AMERICAN GAME BIRD SHOOTING 212 later than Nuttall, speaks of the birds as already ex- tinct in that State. Giraud, writing about 1840, says that even then the bird was practically extinct on Long Island, but that it had been abundant thirty years before. Audubon quotes an interesting letter from a Mr. Audubon quotes an interesting letter from a Mr. David Eckley, of Boston, who was in the habit of shooting prairie hens on Martha’s Vineyard. THE HEATH HEN. This letter declares that “Nashawenna is the only other island of the group on which they are found,” and further along adds: “It would be difficult to assign a reason why they are found upon the islands above named, and not upon others, particularly Nashann, which, being large, well wooded, and abounding in feed, seems quite as favorable to the peculiar habits of the birds.” Even at that time, according to this letter, the heath hens on Martha’s Vineyard were scarce, for Mr. Eck- ley says that the result of a few weeks’ residence of a party of three is ten brace of birds. The same gentleman says: “We frequently meet The same gentleman says: “We frequently meet with the remains of such as have been destroyed in vari- ous ways, but more particularly by the domestic cat, which prowls the woods in a wild state, and which often receives a very unwelcome salute for the mis- chief it does. Owls, hawks and skunks also do their part toward the destruction of these valuable but de- fenceless birds. In these ways they are thinned off much more effectually than by the sportsman’s gun. They fre- quent no particular soil, and, like all other hunting, PINNATED GROUSE 213 wherever the food is, there is the likeliest place for the game. In addition to this rule as a guide, we look for their fresh tracks among the sandy barberry hil- locks and along the numerous patches which intersect that remarkable part of the Vineyard called Tisbury Plain. Into this, should the birds fly from the hedges, as they sometimes do, it is almost impossible to start them a second time, as there are no trees or large objects to mark their flight. Being mostly covered with scrub oak of a uniform height, with occasional mossy hollows, it affords them a place of refuge, into which they fly for protection, but from which they soon emerge, when the danger is passed, to their more favourite haunts.” This letter was written in December, 1832. This letter was written in December, 1832. The ornithologists of the first half of the ninete The ornithologists of the first half of the nineteenth century did not differentiate the pinnated grouse of the Mississippi Valley from the eastern bird, and spoke of the pinnated grouse as even then almost extermi- nated from its old range on the Atlantic coast. THE HEATH HEN. PINNATED GROUSE 215 So numerous were they a short time since in the bar- rens of Kentucky, and so contemptible were they as game birds, that few huntsmen would deign to waste powder and shot on them. In fact, they were held in pretty much the same estimation, or, rather, abhor- rence, that the crows are now in Pennsylvania or other of the Middle and Southern States, as they perpetrated quite as much mischief upon the tender buds and fruits of the orchards, as well as the grain in the fields, and were often so destructive to the crops, that it was absolutely necessary for the farmers to employ their young negroes to drive them away by shooting off guns and springing loud rattles all around the plantations from morning till night. As for eating them, such a thing was hardly dreamed of, the negroes themselves preferring the coarsest food to this now much admired bird; while the young sports- man exercised his skill in rifle shooting upon them, in anticipation of more exciting sport among the other prized denizens of the plains and forest. Prairie chick- ens have not only deserted Long Island, Martha’s Vine- yard, Elizabeth Island, New Jersey, and their other haunts to the eastward, but they have also removed even farther west than the barrens of Kentucky. . . .” So numerous were they a short time since in the bar- rens of Kentucky, and so contemptible were they as game birds, that few huntsmen would deign to waste powder and shot on them. In fact, they were held in pretty much the same estimation, or, rather, abhor- rence, that the crows are now in Pennsylvania or other of the Middle and Southern States, as they perpetrated quite as much mischief upon the tender buds and fruits of the orchards, as well as the grain in the fields, and were often so destructive to the crops, that it was absolutely necessary for the farmers to employ their young negroes to drive them away by shooting off guns and springing loud rattles all around the plantations from morning till night. THE HEATH HEN. A con- temporary statement of interest as to the heath hen is that made by Elisha J. Lewis in “The American Sports- man,” published in Pennsylvania, 1857. He says: “The prairie hen was, no doubt, at one time widely “The prairie hen was, no doubt, at one time widely disseminated over our whole country, more particularly in those portions interspersed with dry, open plains sur- rounded by thin shrubbery or scantily covered with trees. Unlike the ruffed grouse, this bird delights in the clear, open prairie grounds, and will desert those districts entirely which in the lapse of time become AMERICAN GAME BIRD SHOOTING 214 covered with forest. These birds are very rare— in fact, may almost be considered extinct in the North- ern and Middle States. Within a few years they were quite abundant on some portions of Long Island. They were also to be found in Burlington County, N. J., and in some few other places. There are, however, still a few to be found on the Jersey plains, and every season we hear of some of our sporting acquaintances exterminating a small pack. We know of ten braces being killed this season (1848), and about the same number last year by the same party; and, as usual, in both instances these scarce and beautiful birds were butchered long before the time sanctioned by the strong —or, rather, the weak—arm of the law. “Thus it is that the destructive hand of the would- “Thus it is that the destructive hand of the would- be respectable poacher, as well as the greedy gun of the pothunter, hastens to seal the fate of the doomed prairie hen in these eastern regions, and we may predict with great certainty that ere long not one will be found, save upon the rich plains of the West; from which also, in course of time, they will be driven and ultimately perish, root and branch, from before the unerring guns of their ruthless destroyers. We un- derstand that there are still a few of these birds to be found in Pennsylvania—we believe in Northampton County—where the pine forests are thin and open and the country about them such as prairie hens delight in. They have always been abundant in the barrens of Kentucky and Tennessee, as also in the balmy plains and fertile prairies of Louisiana, Indiana and Lllinois. THE HEATH HEN. As for eating them, such a thing was hardly dreamed of, the negroes themselves preferring the coarsest food to this now much admired bird; while the young sports- man exercised his skill in rifle shooting upon them, in anticipation of more exciting sport among the other prized denizens of the plains and forest. Prairie chick- ens have not only deserted Long Island, Martha’s Vine- yard, Elizabeth Island, New Jersey, and their other haunts to the eastward, but they have also removed even farther west than the barrens of Kentucky. . . .” Lewis says also in the course of this article that Lewis says also in the course of this article that the pinnated grouse are easily domesticated, and will pair and hatch in captivity—all this from Audubon. The species has been extinct for more than forty The species has been extinct for more than forty years in New Jersey and Pennsylvania. Mr. Witmer Stone says that up to 1868, and probably later, AMERICAN GAME BIRD SHOOTING 216 a few were said to occur on the barren plains which cover portions of Ocean and Burlington counties in New Jersey. This is a part of the pine barren region, an elevated, dry tract covered with dwarf pines, which average not more than a foot and a half in height. Turnbull, in his “Birds of Eastern Pennsylvania,” states that in 1869 a few survived in Monroe and Northampton counties in that State. The heath hen is very similar to the prairie hen, but The heath hen is very similar to the prairie hen, but slightly smaller. The tufts of pointed neck feathers are shorter and, as already said, the bird has slight points of difference in color. Though called a wood- land bird, it is much in the open or in the thick, low scrub oak and pines which cover an area of forty miles square. There are thought to be not more than 150 or 200 of these birds left alive, and they are therefore more nearly extinct than the buffalo. They are already protected by law and should be still more strongly protected by public opinion of the resi- dents of Martha’s Vineyard, who ought to feel proud of this bird and to do everything in their power to preserve it. Not much had been written about the heath hen on Martha’s Vineyard until the year 1885, when Mr. THE HEATH HEN. William Brewster visited the island for the special purpose of studying the bird. He reported the re- sults of this visit in the Awk, and in 1890 repeated the trip and gained additional information, which was printed in Forest and Stream. He said : “Throughout Martha’s Vineyard, the heath hen “Throughout Martha’s Vineyard, the heath hen PINNATED GROUSE * 217 (locally pronounced héth’n, as this grouse is uni- versally called) is well known to almost every one. Even in such seaport towns as Cottage City and Edgarstown, most of the people have at least heard of it, and in the thinly settled interior it is frequently seen in the roads or along the edges of the cover by the farmers, or started in the depths of the woods by the hounds of the rabbit and fox hunters. “Its range extends, practically, over the entire “Its range extends, practically, over the entire wooded portion of the island, but the bird is not found regularly or at all numerously outside an area of about forty square miles. This area comprises most of the elevated central portions of the island, although it also touches the sea at not a few points on the north and south shores. In places it rolls into great rounded hills and long, irregular ridges, over which are scat- tered stretches of second-growth woods, often miles in extent, and composed chiefly of scarlet, black, white and post oaks, from fifteen to forty feet in height. Here and there, where the valleys spread out broad and level, are fields which were cleared by the early set- tlers more than a hundred years ago, and which still retain sufficient fertility to yield very good crops of English hay, corn, potatoes and other vegetables. Again, this undulating surface gives way to wide, level, sandy plains, covered with a growth of bear, chinqua- pin and post-oak scrub, from knee to waist high, so stiff and matted as to be almost impenetrable; or to rocky pastures, dotted with thickets of sweet fern, bay- AMERICAN GAME BIRD SHOOTING 218 berry, huckleberry, dwarf sumac and other low-grow- ing shrubs. “Clear, rapid trout brooks wind their way to the sea through open meadows, or long, narrow swamps, wooded with red maples, black alders, high huckleberry bushes, andromeda and poison dogwood, and overrun with tangled skeins of green briars. THE HEATH HEN. “At all seasons the heath hens live almost exclusively in the oak woods, where the acorns furnish them abun- dant food, although, like our ruffed grouse, they occa- sionally at early morning and just after sunset ven- ture out a little way in the open to pick up scattered grains of corn or to pluck a few clover leaves, of which they are extremely fond. They also wander to some extent over the scrub-oak plains, especially when blue- berries are ripe and abundant. In winter, during long- continued snows, they sometimes approach buildings, to feed upon the grain which the farmers throw out to them. A man living near West Tisbury told me that last winter a flock visited his barn at about the same hour each day. One cold, snowy morning he counted sixteen perched in a row on the top rail of a fence near the barnyard. It is unusual to see so many to- gether now, the number in a covey rarely exceeding six or eight, but in former times packs containing from one to two hundred birds each were occasionally met with late in the autumn. “Only one person of the many whom I questioned “Only one person of the many whom I questioned on the subject had ever seen a heath hen’s nest. It was in oak woods, among sprouts at the base of a large PINNATED GROUSE 219 stump, and contained either twelve or thirteen eggs. The date, he thought, was about June 10. This seemed late, but I have a set of six eggs taken on the Vineyard July 24, 1885, and on July 19, 1890, I met a blueberry picker who only the day before had started a brood of six young, less than half grown. These facts prove that this bird is habitually a late breeder. “The farmers about Tisbury say that in spring the “The farmers about Tisbury say that in spring the male heath hen makes a booming or tooting noise. This, according to their descriptions, must resemble the love notes of the western pinnated grouse. About sunrise, on warm, still mornings in May, several birds may be sometimes heard at once, apparently answer- ing one another. THE HEATH HEN. “During my stay at Martha’s Vineyard, I obtained “During my stay at Martha’s Vineyard, I obtained as many estimates as possible of the number of heath hens which are believed to exist there at the present time. My most trustworthy informants were, credit- ably, averse to what was apparently mere idle guessing ; but when I questioned them, first as to the extent of the region over which the birds ranged, and next as to how many on the average could be found in a square mile within this region, they answered readily enough, and even with some positiveness. As already stated, the total present range of the heath hen covers about forty square miles. The estimates of the average number of birds per mile varied from three to five, giving from 120 to 200 birds for the total number. These estimates, it should be stated, relate to the num- ber of birds believed to have been left over from last AMERICAN GAME BIRD SHOOTING 220 winter. If these breed freely and at all successfully, there should be a total of fully 500, young and old to- gether, at the beginning of the present autumn. When one considers the limited area to which these birds are confined, it is evident that within this area they must be reasonably abundant. I was assured that with the aid of a good dog it was not at all difficult to start twenty-five or thirty in a day, and on one occasion eight were killed by two guns. This, however, can be done only by those familiar with the country and the habits of the birds.” The fact that but a small remnant was left of this once widely distributed species aroused much interest in it, and after a time the Massachusetts authorities began to consider measures for its preservation. Be- fore any steps looking to its preservation had been taken, the numbers of the birds had still further di- minished, and observations made on the island from October, 1906, to May, 1907, at a time when they were collected in large flocks, seemed to justify the conclu- sion that the number of individuals was less than one hundred. The report of the commissioners on fisheries and game for the year ending December 31, 1907, declares that by actual count of the flocks located in various sections of the range, seventy-seven individuals were enumerated. THE HEATH HEN. In May, 1906, a destructive forest fire swept practically the entire breeding grounds, and very few birds were reared that season. The summer of 1907, however, was a favorable one. At least ten *AIOYSIP{ TeINJEN Jo wnasnyy uvollowy ul dnois wong ‘jsou pue 9ssnoIg poajeuulg PINNATED GROUSE 22% broods were successfully reared, and it was believed that the number had more than doubled. The first protective law relating to the heath hen is The first protective law relating to the heath hen is said to have been passed in 1831. This provided for a closed season from March 1 to September 1. In 1837 there was established a closed season of four years, which in the same year was extended for five years more. These Acts, however, permitted any town to suspend the law, so far as that town was concerned, for such a period as might be deemed expedient, and in 1842 the town of Tisbury did suspend the law for a period of ten days on more than one occasion. In fact, the law in behalf of these birds fell into desuetude and no effort was made to enforce it up to about 1905. The inhabitants of Martha’s Vineyard felt a local pride in having there a bird found nowhere else in the world, but this local pride was not strong enough to protect the species. An observer who visited the island in the spring of 1906 made to the commissioners on fisheries and game of Massachusetts a report which gives much _ in- teresting detail as to the habits of the heath hen at the particular time when they are preparing to mate, and is well worth quoting in full. He says: “At 6 p.m. we arrived at the point where we hoped to find traces of the heath hen. Ina cleared field, about thirty rods from the road, we distinctly saw two large birds. On our nearer approach they squatted close and their protective coloration was so effective that although we knew almost exactly the precise location AMERICAN GAME BIRD SHOOTING 222 of the birds, we could not distinguish them. We crawled behind the nearest cover and remained mo- tionless for perhaps ten minutes. At length the long shadows from the descending sun enabled us to dis- tinguish the birds as they crouched with head close to the ground among the very scanty vegetation. THE HEATH HEN. After another interval of motionless activity on our part, one bird quickly arose and began feeding, apparently without suspicion; soon two more birds arose as if by magic from the ground. Then began a most inter- esting series of antics. These birds were joined by five others, coming in singly and on foot from the scrub in various directions. The birds came frequently within forty paces of our hiding place, and in one instance alighted on a small oak tree twenty-three paces from our camera. While not near enough for suc- cessful photographing, we were well situated for using our field-glasses. The birds were all actively feeding in the open field, apparently on grasshoppers and other insects, but nipping red clover leaves very freely. They moved leisurely about. Frequently two birds, some- times as much as a hundred to a hundred and fifty yards apart, ran directly toward each other, dancing and blowing on the way, with the so-called ‘neck wings’ pointed upward in a V form. On facing each other, both squatted and remained motionless from one to five minutes. We could see none of the nodding and pecking motions of the head so commonly indulged in by domestic fowls when fighting; rarely was there sparring with the bill, or striking with the feet or PINNATED GROUSE 223 wings. In twelve or fifteen encounters, only three or four times did they strike thus, and only once did we see ‘feathers fly.’ Most of the energy seems to be spent in posturing and blowing. Generally, one of the combatants backs slowly away, suddenly stopping if the opponent advances too rapidly. In all these fight- ing tactics the similarity of habits with those of the domestic fowl were very marked. From all directions came the peculiar foot, like distant tugboats in a fog, all having whistles of the same pitch. This call may be well imitated by blowing gently into the neck of a two-drachm homeopathic vial. Each call extends over a period of two seconds, and is repeated at frequent intervals. It is prefaced by a run of about one yard, with very rapid, mincing steps. The strides, however, are so short that the bird does not advance rapidly. The tail is spread and the wings dropped after the manner of the strutting turkey-cock. THE HEATH HEN. When the tail is spread, the white under-tail coverts are conspicuous and remind one forcibly of the ‘white flag’ of the deer and antelope, or of our gray rabbit. The head is then depressed and the neck outstretched forward, until it is parallel with the surface of the ground; the neck tufts are elevated to a V shape. The bright, orange- colored air-sacs on each side of the neck, directly be- hind the tufts of feathers, are slowly inflated until they reach apparently the size of a tennis ball, when they appear like two small ripe oranges, one protruding from either side of the neck. The duration of the call appears to closely coincide with the period of infla- AMERICAN GAME BIRD SHOOTING 224 tion, and seems to be emitted as the air enters the sac, rather than when the air is expelled. The collapse of the sac is sudden. The sound is ventriloquial, and it is very difficult to locate the direction or distance whence it comes, unless the bird can be seen. A sec- ond sort of call is much less frequent and closely re- sembles a single syllable of the hoot of the barred owl. “Another characteristic antic was a peculiar combi- “Another characteristic antic was a peculiar combi- nation of a short run, a sudden jump of three to five feet into the air, and a rapid uncoordinated flop and scramble in the air, the bird usually alighting within ten or twenty feet of the starting point, but turn- ing so as to face at least at right angles, or even in the opposite direction from which it started. When in the air it emits a peculiar cacophonous call or cackle, which, when heard at a distance, gives the impression of a hearty burst of laughter. The purpose of these semi-somersault-like maneuvers appeared to be to at- tract the attention of other birds, possibly even as a challenge, for frequently they seemed to precede the somewhat pacific duels described above. The effect of these sounds, together with the tooting calls, in the mists which so often obtain in their habitat before sunrise, is weird in the extreme. At 4:15 A.M., on May 2, these sounds were practically continuous, with- out appreciable interval, apparently from all directions. At 4:45 A.M. six birds could be counted, all in sight at once. THE HEATH HEN. They appeared to resort to a particular, clear space, of about two acres in extent, where the antics just described were carried on. All the birds, except PINNATED GROUSE 225 one, were observed to have the orange-colored air- sacs. These were probably cocks. We saw only one bird which we suspected might be a hen. The other hens were probably nesting, or at least had secured mates, and no longer resorted to the promenading place. As the sun rose high the tooting became less frequent; the birds became more restless, often flying to the neighboring low oaks, resting there until disturbed.” William Hazen Gates, of Williamstown, Mass., worked that spring with the Massachusetts commis- sioners, studying the habits of the heath hen, in order to secure information which might be of use in arti- ficially propagating the species. He says: “On May 31, while wandering across the plains, “On May 31, while wandering across the plains, three heath hens were started, and each taking wing flew nearly out of sight before alighting. As I watched the birds, a call, resembling to a slight degree that of an ordinary barnyard cock calling to the hens, was heard not far distant. The place was noted as nearly as possible, and then cautiously I made my way there. When the place was reached I looked for birds, but could see none. I then sat down and determined to wait, in order to see if any birds could be heard. The ground was covered with leaves, so the least stir would have been heard. I listened and also looked for signs of anything moving, but none appeared. I sat there for fully twenty minutes and, hearing nothing, con- cluded that either there were no birds or else they had gone as IT approached. As I rosea bird flew up within AMERICAN GAME BIRD SHOOTING 226 twenty feet of where I had been watching. The bird had been within sight all the while, but probably had crouched in the leaves and remained invisible. It would have been interesting to note how much longer the bird would have stayed in this position without moving. Another bird was started some fifty feet from this one. “On this same day the foots of one or more heath “On this same day the foots of one or more heath hens were heard between half-past four and five a.m. THE HEATH HEN. The birds are early risers and late bed-goers. Once they were heard to toot at 3:30 A.M., or about an hour before sunrise; and several times their call note was heard as early as this. It is probable, though, that they do not begin to stir quite so early, beginning their breakfasting about sunrise or a little earlier. The mid- dle of the day is generally spent in the shade, or in dust- ing in the sun in the roads. Late in the afternoon, as the air begins to cool, they take to feeding again, and can be seen in the open fields. They will often feed till nearly an hour after sundown. I do not know whether they roost in the low shrubbery or on the ground at night. Mother birds with young, however, stay on the ground, but it is likely that this is done only while the young are too small to roost and need the shelter of the mother. “On June 29 a bevy of heath hens was found. The mother bird took flight, cackling, and flew some fifty feet or so. The young scudded in every direction, and were entirely out of sight by the time I reached the spot. I hunted around through the leaves some, but PINNATED GROUSE 227 fearing that I might accidentally step on one, did not search very carefully, and so did not see any. Two days later, what I think must have been the same bevy was again seen, but about half a mile from the place where they were first seen. This time they were in a more or less cleared space, and six of the young were counted. One or two squatted just where they were, and it looked as if one might go right up to them and pick them up. I did not, however, disturb them. These birds were apparently not over a week old. “On July 2 a mother heath hen and four young were “On July 2 a mother heath hen and four young were seen dusting in a road about 11 A.M. Upon seeing me the mother ran to the bushes and called to the young. As I went by I could hear the mother hen at the side of the road in the bushes. The same day in the after- noon, a mother hen and one young bird were seen. THE HEATH HEN. “On July 7, while walking through the brush near “On July 7, while walking through the brush near the Cromwell cottage, soon after sundown, I heard some peeping ahead. Getting on my hands and knees, I crawled toward the sound. The peeping continued as I approached, so I knew that I had not been per- ceived. Finally, at a distance of some twenty or twenty-five feet, I saw a mother hen with wings spread under the thick foliage of a stunted oak. She was more or less silent, only occasionally uttering a low call, somewhat resembling that of a hen as she calls her chicks at night under her wings. The young, how- ever, peeped quite often as they stole in and out from under the wings of the mother. I think they could not have been much more than a day or two old. AMERICAN GAME BIRD SHOOTING 228 Like the chicks of other fowls, they could not seem to get settled for the night, but would stray in and out. Then as they sought a place of shelter again they would shove one of their fellows out from under the mother’s wing. However, as darkness grew the rest- lessness ceased, and by the time it was too dark to see the group everything was silent. How many there were in the bevy I could not tell, but it seemed that there must have been at least six or eight.” Though the number of these birds is so pitifully small, yet, as already suggested, they pack in the same way as does the western pinnated grouse. The report of the Massachusetts commissioners on fisheries and game for 1907 states that on January 11, 1908, a flock was counted which contained not less than fifty-five nor more than sixty birds. The same report declares that the birds remained in these flocks until late in February and began to utter their calls on the approach of warm weather, and when this takes place the flocks break up and the season for mating begins. The elabo- rate performances earlier described begin about April I, and end about the middle of June, being at their greatest height the last of April and early part of May. The chicks are hatched in June and in July. When about the size of quails they make long flights when alarmed. THE HEATH HEN. Among experiments carried on by the Massachusetts commission was the taking of a set of nine heath hen eggs, which were placed under a bantam hen. Only one of these eggs hatched, and the chick was at once PINNATED GROUSE 229 killed by the hen. The hen was subsequently given some pheasant eggs, and hatched them and reared the chicks with all possible care. That this bird possesses a high interest, not only for the sportsman, but because it is now on the very point of extinction, is obvious. Much has been said and written about its preservation, but the credit for setting on foot a movement which it is hoped may pre- vent the extermination of the species is due to Mr. J. E. Howland. He urged the importance of the situation on the Massachusetts commission, and a permanent guardian was located in the region inhabited by the birds to study their habits and enforce the law. Some- what later, Representative Mayhew introduced a bill into the General Court, placing under the use and control of the commission such lands as may be do- nated, leased, purchased or otherwise placed under temporary or permanent control, as a refuge and breed- ing area for the heath hen. A number of individuals have subscribed money to enable the commission to take advantage of this law, and about sixteen hundred acres have been placed under special protection. The Massa- chusetts legislature has authorized the commissioners to take such unimproved lands upon Martha’s Vine- yard—not exceeding one thousand acres—as they may deem necessary for the purpose of making fire stops for the protection from fire of the feeding and breed- ing grounds of the pinnated grouse, or of otherwise securing the maintenance and increase of such pinnated grouse or of any other species of wild birds upon said AMERICAN GAME BIRD SHOOTING 230 islands. Two thousand dollars was appropriated for work incidental to these purposes and for an inves- tigation and reports upon the best methods and possible cost of protecting and increasing the colonies of birds on the island. This appropriation, and the authority to take the land, ought to protect the heath hen from a danger which has been of late years the most im- portant element in reducing its numbers—the bush and forest fires. The pinnated grouse of the West have suffered from the same cause. THE HEATH HEN. An investigation by the commissioners on fisheries and game of the problems involved in the preservation of the heath hen leads them to believe that one or more extensive areas, whether they be called reserves, sanc- tuaries or refuges, should be acquired by the common- wealth and patrolled and maintained. Such refuges should include the chief breeding and feeding grounds of the birds, and on the land should be sown crops of clover and such cultivated grains as they delight to feed on; that suitable fire stops or breaks should be maintained in order to reduce the danger of bush fires, so destructive to the birds in the past; that every pre- caution should be taken against the contagious diseases which might be transmitted to these wild birds through domestic fowls; that as soon as the number of birds is sufficiently increased, systematic artificial incubation, feeding and breeding should be begun for the purpose of rearing annually an increased number of birds. The commissioners believe that by artificial propagation the number of eggs laid may be increased, while it is PINNATED GROUSE 220 obvious that this means will lessen the loss resulting from the destruction of nests, eggs and young birds by natural enemies, whether mammals or birds. They believe that the expenditure involved would be prac- tically that incidental to ordinary poultry raising, ex- cept that on account of the hardiness and vigor of the grouse it would need little or no winter shelter. The commissioners, in their report for the year 1907, The commissioners, in their report for the year 1907, report contributions from private individuals of not less than $2,420 for the purpose of preserving these birds. Of this sum, the town of Tisbury and the Mid- dlesex Sportsmen’s Association made the largest con- tributions of $200 each, but there are a large number of subscribers, and the list ought to be largely added to, since every sportsman and every naturalist in the land should be ambitious to have some share in the good work of re-establishing this splendid bird. THE PRAIRIE HEN. This is the form of pinnated grouse that has been known to sportsmen. Its range was chiefly in the Mississippi Valley, including Minnesota, Michigan, western Ohio, Kentucky, but perhaps not Tennessee, though it was found in Louisiana, southern Texas, Indian Territory, Kansas, Nebraska and the eastern parts of both Dakotas. It is now found in Manitoba, southeastern Saskatchewan, to eastern Colorado, north- eastern Texas, Arkansas, western Kentucky and In- diana and intermediate regions, where it was at one AMERICAN GAME BIRD SHOOTING 232 time enormously abundant, and, being exceedingly tame at certain seasons of the year, was very readily destroyed. Throughout almost all this region the bird was resi- dent, though in the northern portion it regularly made seasonal changes of location, which, though commonly called migratory, hardly deserve to be so character- ized. On this point Mr. W. W. Cooke, in his report on “Bird Migration in the Mississippi Valley,” says: “The prairie chicken is commonly said to be a resi- “The prairie chicken is commonly said to be a resi- dent bird, and so it is in a larger part of this range, but in Iowa a regular though local migration takes place. This has been mentioned by former writers, and in the spring of 1884 a special study was made of the matter. Many observers unite in testifying to the facts in the case, and, what is still more important, there is not a dissenting voice. One of the observers does not exaggerate when he says: ‘Prairie chickens migrate as regularly as the Canada goose.’ Summing up all the information received, the facts of the case are as follows: In November and December large flocks of prairie chickens come from northern Iowa and southern Minnesota to settle for the winter in northern Missouri and southern Iowa. This migration varies in bulk with the severity of the winter. “During an early cold snap immense flocks come from the northern prairies to southern Iowa, while in mild, open winters the migration is much less pro- nounced. During a cold, wet spring the northward movement in March and April is largely arrested on “During an early cold snap immense flocks come from the northern prairies to southern Iowa, while in mild, open winters the migration is much less pro- nounced. THE PRAIRIE HEN. During a cold, wet spring the northward movement in March and April is largely arrested on PINNATED GROUSE 233 the arrival of the flocks in northern Iowa, but an early spring with fair weather finds them abundant in the southern tiers of counties in Minnesota, and many flocks pass still farther north. The most remarkable feature of this movement is found in the sex of the migrants. It is the females that migrate, leaving the males to brave the winter’s cold. Mr. Miller, of Heron Lake, Minn., fairly states the case when he says: ‘The females in this latitude migrate south in the fall and come back in the spring, about one or two days after the first ducks; and they keep coming in flocks of from ten to thirty for about three days, all flying north. The grouse that stayed all winter are males.’ ” Audubon noticed and spoke of these movements nearly a hundred years ago, for in his account of this species he says: “During the first years of my residence at Hender- son (Kry.), in severe winters, the number of grouse of this species was greatly augmented by large flocks of them that evidently came from Indiana, Illinois and even from the western side of the Mississippi. They retired at the approach of spring.” When John James Audubon first lived in Kentucky, When John James Audubon first lived in Kentucky, the “Barrens”—by which is meant open stretches of land without timber—swarmed with these birds, and they were looked on more or less as a pest. They were credited with committing much mischief among the fruit trees of the orchards in winter, and in the spring they fed on the grain in the new-sown fields. They AMERICAN GAME BIRD SHOOTING 234 were so abundant as to be caught in pens and traps, and any one could kill as many as he wished. Indeed, Audubon speaks of a friend who was fond of practicing rifle shooting, who killed upward of forty in one morn- ing and did not pick them up. Twenty-five years later Audubon speaks of them as Twenty-five years later Audubon speaks of them as at that time not being found in any numbers east of the State of Illinois, and says that there, too, they are decreasing at a rapid rate. THE PRAIRIE HEN. At the approach of spring the large packs, which At the approach of spring the large packs, which have held together during the winter, break up into smaller companies of from twenty to fifty, and before long—in March or April—the mating begins. This has been spoken of by many writers as the booming of the prairie chicken, or the dance of the prairie hen, though this last term is more commonly applied to the spring maneuvers of the sharp-tailed grouse. This mating has been described in a somewhat spectacular manner by Audubon, but recent observers have not seen such fierce encounters as he describes. An excellent account of this mating play was printed in Forest and Stream many years ago by Judge John Dean Caton, an early settler of Illinois, who had been familiar with these birds for almost all his life. He says: “The spring of the year is the season of courtship with them, and it does not last all the year round, as it does with humans, and they do it in rather a loud way, too; and instead of taking the evening, as many people are inclined to do, they choose the early morn- ing. Early in the morning you may see them assemble PINNATED GROUSE 235 in parties, from a dozen to fifty together, on some high, dry knolls, where the grass is short, and their goings on would make you laugh. The cock birds have a loose patch of naked yellow skin on each side of their neck just below the head, and above these on either side, just where the head joins the neck, are a few long black feathers, which ordinarily lie back on the neck, but which, when excited, they can pitch straight for- ward. These yellow naked patches on either side of the neck cover sacs which they can blow up like a bladder whenever they choose. These are their ornaments, which they display to the best advantage before the gentler sex at these love feasts. This they do by blow- ing up these air-sacs till they look like two ripe oranges, on each side of the neck, projecting their long, black ears right forward, ruffling up all the feathers of the body till they stand out straight, and dropping their wings to the ground like a turkey cock. THE PRAIRIE HEN. Now they look just lovely, as the coy, timid maidens seem to say, as ‘they cast side glances at them, full of admiration and love. “Then it is that the proud cock, in order to complete his triumph, will rush forward at his best speed for two or three rods through the midst of the lovesick damsels, pouring out as he goes a booming noise, al- most a hoarse roar, only more subdued, which may be heard for at least two miles in the still morning air. This heavy booming sound is by no means harsh or unpleasant; on the contrary, it is soft and even har- monious. When standing in the open prairie at early AMERICAN GAME BIRD SHOOTING 236 dawn, listening to hundreds of different voices, pitched on different keys, coming from every direction and from various distances, the listener is rather soothed than excited. If this sound is heavier than the deep keynotes of a large organ, it is much softer, though vastly more powerful, and may be heard at a much greater distance. One who has heard such a concert can never after mistake or forget it. “Every few minutes this display is repeated. I have seen not only one, but more than twenty cocks going through this funny operation at once, but then they seem careful not to run against each other, for they have not yet got to the fighting point. After a little while the lady birds begin to show an interest in the proceedings by moving about quickly, a few yards at a time, and then standing still a short time. When these actions are continued by a large number of birds at a time, it presents a funny sight, and you can easily think they are moving to the measure of music. “The party breaks up when the sun is half an hour high, to be repeated the next morning and every morn- ing for a week or two before all make satisfactory matches. It is toward the latter part of the love season that the fighting takes place among the cocks, probably by two who have fallen in love with the same sweet- heart, whose modesty prevents her from selecting be- tween them.” Audubon reports that he tried the experiment of puncturing the inflatable air-sacs on the neck of a male prairie chicken. THE PRAIRIE HEN. He caught one of the birds and passed PINNATED GROUSE 237 the point of a pin through each sac, and found that thereafter it was unable to toot. He performed this same experiment with another bird on one side only, and found that the next morning it uttered the tooting sound with the uninjured air-sac, but could not inflate the one that had been punctured. He states that his efforts to decoy this species by imitating its curious sounds were unsuccessful, ‘‘although the ruffed grouse is easily deceived in this manner.” After the close of the mating operations the locations After the close of the mating operations the locations of the nests are selected. Often they may be in hedges and the margins of clumps of underbrush, in fence cor- ners or along the borders of sloughs, but often, again, in the middle of a field amid the tall grass. The eggs number from eleven to fourteen, and sets of twenty or even twenty-one eggs are not unknown. They vary in color from cream to light olive or pale brown, and are often regularly spotted with fine pin-points of reddish brown. Captain Bendire regards the prairie chicken as one of the most prolific of our game birds. Now, however, comes the season of danger; the eggs Now, however, comes the season of danger; the eggs have been deposited in a slight depression, scratched out among the weeds or grass, and the hen begins to brood. If she has nested early and the season is late, the streams may rise and flood her nest and destroy the eggs or drown the tiny young, if they have already hatched ; or early prairie fires, burning among the dead grass and weeds of the preceding season, may destroy mother and clutch alike; or, later still, the mowing machine may kill the mother or the young, too small AMERICAN GAME BIRD SHOOTING 238 to fly and too inexperienced to force themselves through the thick grass away from the approaching danger. In old times it used to be said that in wet seasons thou- sands and thousands of prairie chickens’ nests were ploughed under when the fields were being prepared for grain. Certain it is that the combination of al! these dangers, together with the insatiate gunner, at one time came very near exterminating the pinnated grouse from the States of Illinois and Indiana. THE PRAIRIE HEN. If the mother bird is fortunate enough to bring off her young, she leads them about much as do other grouse, to the best feeding grounds. She is watchful of danger for them, and at her warning cry the young squat on the ground, which they so closely resemble that it is almost impossible to find one of them. The mother uses every art to lead the intruder away from the brood. The birds grow rapidly, and by the middle of August—the date at which up to within a few years it has been legal to shoot them—are nearly two-thirds grown. They are then very easily killed, and the sport becomes mere butchery. When cold weather ap- proaches, however, they grow stronger of wing, and soon after this pack. Audubon was perhaps the first to announce that Audubon was perhaps the first to announce that the pinnated grouse is easily tamed and easily kept. He declares also that they breed in confinement. A number that he had while at Henderson were turned loose in his garden and orchard, and within a week became so tame as to allow him to approach them. They readily ate corn and vegetables, became so gentle PINNATED GROUSE 239 during the winter as to feed from the hand of his wife, and altogether acted as domestic poultry might act. In spring they went through the operations of mating just as did their wild brethren, and a number of them hatched, but at last the birds proved so destructive in the garden that they were ordered to be killed. nother page, birds sent to Eng- As will be seen on another page, birds sent to Eng- land became quite tame, and many years ago I had a dozen of these grouse in New York, which, when turned out in the spring, so readily accustomed themselves to their surroundings that they followed a man who was spading the garden and scratched and crowded over the freshly turned-up earth in search of insects. They were less wild than so many domestic hens. its ways, the pinnated grouse suggests a In many of its ways, the pinnated grouse suggests a domestic fowl. Though often carrying its tail droop- ing toward the ground, it often carries it upright, as a hen carries her tail. THE PRAIRIE HEN. The mother of a young brood will fight for it, or at least will try to frighten away an intruder. The young chicks constantly talk to each other as they move along, and if one of them discovers an insect and runs after it all those within sight join in the pursuit. pson, in his “Birds of Manitoba,” Mr. E. E. Thompson, in his “Birds of Manitoba,” points out that, while it was only in 1881 or 1882 that the pinnated grouse was found in Manitoba, before 1890 it had become common at many points, such as Winnipeg, Portage la Prairie and other localities. Pre- vious to this, in 1872, Dr. Coues had written: AMERICAN GAME BIRD SHOOTING 240 “T have no reason to believe that it occurs at all in northwestern Minnesota or North Dakota.” In Manitoba they seem more or less to associate with the sharp-tailed grouse, so much so that sometimes birds of both species will be started from before the dog. When winter comes, however, and the sharp-tailed grouse go into the woods, the pinnated grouse stay out on the prairie. LESSER PRAIRIE HEN. The range of the lesser prairie hen has already been SIVED iy" While riding over the prairie through the western part of the Indian Territory in the month of March, I have more than once driven for hours through flocks of these birds, contentedly feeding on the prairie and wholly disregarding the wagon, which passed close to them. On these occasions we must have seen many thousands of the birds, and it is probable that imme- diately after this the packs broke up and the mating season began. The pursuit of this form differs in no respect from that of its slightly larger relative to the North and Fast. The most southern range in Texas of Attwater’s prairie hens is given as just north of Fort Brown, near the coast, and it is very abundant south of San Antonio. 241 PINNATED GROUSE THE PINNATED GROUSE TO-DAY. An inquiry made in 1906 among the game commis- sioners of States where prairie chickens were formerly very abundant brought out a number of replies of great interest. Mr. E. E. Earle, then chief deputy warden of the State of Indiana, wrote to Forest and Stream as follows: “Our supply of pinnated grouse decreased rapidly “Our supply of pinnated grouse decreased rapidly from year to year until 1901. Prior to that time the open season had run from September 1 of any year to February 1 of the succeeding year. Under this law large numbers of these birds were slaughtered every year, they being young, not gun shy, and easily found. “In 1901 a law was passed prohibiting the killing of “In 1901 a law was passed prohibiting the killing of pinnated grouse, or possession of same, at any time between January 1 and November 10, and prohibiting export of such birds. Under the provisions of this act, which was rigorously enforced, pinnated grouse have increased in numbers, and may be found in great droves on our prairies and marshes. “T was in Porter County last March, and one duck “T was in Porter County last March, and one duck hunter told me of having seen in one flock what he estimated to have been one hundred prairie chickens, and such scenes are by no means rare. Wise laws and strict enforcement of same will increase them in any country that is suitable for their habitation.” Illinois, in the youth of men who are now elderly, Illinois, in the youth of men who are now elderly, was the great chicken ground of what used to be called AMERICAN GAME BIRD SHOOTING 242 the West, but the birds were so persecuted that a few years ago it was supposed that this grouse was nearly extinct there. Within the last few years, however, a great change is reported. State Game Commissioner John A. Wheeler wrote: “My deputy game wardens throughout the State re- port prairie chickens, pinnated grouse, rapidly increas- ing. Our deputy wardens in Wayne County report 3,000 birds in that county by actual count. In Sanga- mon County, from personal observation and reports from the deputy wardens, I am convinced that we have nearly that many birds. THE PINNATED GROUSE TO-DAY. From all over the State we are receiving encouraging reports of the increase of prairie chickens.” Almost the northwestern limit of the pinnated grouse’s range is western Minnesota, and of this coun- try Mr. S. F. Fullerton, then the executive agent of Minnesota’s board of game and fish commissioners, reported interestingly. It is obvious that unless the cultivation of the land is such as to provide food for the pinnated grouse they will not do well there, and the character of Minnesota farming, which is largely dairy- ing on small farms, is not such as to encourage occu- pancy by the pinnated grouse. In two letters, Mr. Fullerton said: “The pinnated grouse, or prairie chicken, is disap- “The pinnated grouse, or prairie chicken, is disap- pearing from a large section of our State. It cannot stand civilization, but it is very odd that in new por- tions of the State that have been opened up they are PINNATED GROUSE 243 very plentiful, and we have sections in Minnesota where the pinnated grouse are just as thick as they ever were. “The State game warden of Manitoba states that “The State game warden of Manitoba states that their prairie chickens are not disappearing at all; in fact, they are increasing under rigid protection and stopping the sale. That is only across an imaginary line of Minnesota and Dakota, where these birds are plentiful, but in the southern part of our State, where dairying has taken the place of grain-raising, there are hardly any of the birds left. The sharp-tailed grouse, however, are different, as they are found in the brush country in great numbers where farms are opened up. “A very pleasing thing happened to me last March. “A very pleasing thing happened to me last March. I was up in the northern part of the State, and in a drive of three hours I came across a stretch of land that had been cleared of jack pine. The clearing was several miles in extent, but it was surrounded by jack pine. The snow at the time was over two feet deep. The land last year was cultivated, some corn grown on it and some wheat and other coarse grain. In that clear- ing, the man with me and I counted over two hundred pinnated grouse. THE PINNATED GROUSE TO-DAY. They appeared to have wintered finely and seemed in good condition.” When we reach Nebraska, we approach the limit of When we reach Nebraska, we approach the limit of the pinnated grouse and enter the region of the sharp- tail just as we do in Minnesota. Both species were formerly abundant in Nebraska, but were so overshot by thoughtless gunners and by market shooters that they became rare. Mr. G. L. Carter, then chief warden AMERICAN GAME BIRD SHOOTING 244 of Nebraska’s game and fish commission, wrote fully about birds in Nebraska: “We have both the pinnated and sharp-tailed grouse, “We have both the pinnated and sharp-tailed grouse, and we are so proud of them that it is a pleasure for me to tell you about them. The pinnated grouse are found more abundantly in the central and eastern parts of the State, while the sharp-tailed grouse are found in the northwest portion. “A peculiar thing is that we seldom find a sharp- “A peculiar thing is that we seldom find a sharp- tailed grouse south of the Platte River; when we do, it is late in the season. The breeding and rearing grounds are principally in the northern and western sections, but a few are raised in other parts of the State. “During the winter months, on account of scarcity “During the winter months, on account of scarcity of food in the north part of the State, which is prin- cipally grazing country, the birds are driven farther south, usually along the Platte River valley, some- times going as far south as the northern counties of Kansas. “A few days ago, while going from this city to Omaha, I saw a bunch of perhaps 100 pinnated grouse within eight miles of the Omaha city limits. It is only through our effective game laws, passed during the winter of 1901, that we have these birds to any extent. Prior to that time they were being slaughtered by the market hunters from everywhere and shipped to the market. We were able to convince the Legis- lature of 1901, that there had been shipped out of this State 235,000 of these birds during the year 1goo. We have had this shipping stopped, and, as a result, PINNATED GROUSE 245 ranchmen and farmers throughout the State report birds more plentiful than at any time during the past fifteen years. THE PINNATED GROUSE TO-DAY. “Tf we take care of these birds, we will have them for a great many years to come, as we have bound- less acres of territory which will never be cultivated, and which afford splendid breeding grounds for them.” Missouri is a State where in the past there have been many pinnated grouse, and from this State also we have a good report as to an abundant stock of birds which might easily be increased by proper care. State Game Warden J. H. Rodes’ account of things will be read with interest: “Tn twenty-five, if not more, of the 114 counties in this State, we have prairie chickens remaining in greater or less quantities. Originally, as you know, throughout the prairie districts of this State, these birds abounded in very liberal supply, but having had absolutely no protection up to and prior to the enact- ment of the present game and fish laws, which went into effect on June 15, 1905, they were industriously hunted almost to the point of extermination, and were wholly killed off in many counties where they had formerly been found abundant. “Tt was a common practice—notwithstanding we “Tt was a common practice—notwithstanding we had a statute prohibiting it—to begin shooting these birds after July 1, clear on and through the hunting season, when and wherever they could be found. Now that they are protected during the entire year, except from November 15 to December 15, we hope they will AMERICAN GAME BIRD SHOOTING 246 multiply and re-establish themselves in considerable number. To illustrate: This county, Pettis County, which is little larger, perhaps, than the average size county and fairly densely populated—Sedalia alone, the county seat, having a population of something over 15,000 inhabitants, and many more average-sized towns—has yet remaining in it, I would say, five or six hundred birds. “Of course, it is difficult to estimate even approxi- mately the number of birds remaining, yet it is no un- common sight to see flocks of ten to twenty-five of these birds in the larger pastures and cornfields. A very reliable person told me the other day that he saw in the western part of our county a drove of about forty birds. Doubtless this was an accumulation of several flocks that were feeding together. THE PINNATED GROUSE TO-DAY. “While, as stated, this county is thickly populated, there yet remain many large pastures on which the virgin sod has never been broken, being used as pasture lands, and perhaps some as large as a thousand acres or more. They breed in these pastures and meadows and feed in adjacent oat and corn fields during the sum- mer season. They were seen in unusually large num- bers this winter, which I can but attribute to the fact that special effort was made to protect these birds dur- ing their breeding season last year. “We were fortunate enough to secure one or two “We were fortunate enough to secure one or two early convictions for hunting them out of season, and the gunners took alarm and very few were killed. What is true in this county, is true in many other coun- PINNATED GROUSE 247 ties where like conditions exist. We intend to make these birds the object of special care and supervision in the hope that they may multiply and yet be seen in large quantities in this State. They should be pro- tected during every month of the year for a period of five or six years, but the truth is that very few of the birds can be killed during the thirty days of the open season, as they are then full grown and are very strong, and will not lie before a dog and are very shy.” Kansas is another State where the prairie grouse was formerly abundant, but here the pinnated grouse is always the common bird, and the sharp-tail the ex- ception. Mr. D. W. Travis, State fish and game war- den, gave in brief but very telling form the history of the wholesale destruction and rapid decrease of prairie chickens until about ten years ago, and then of the change of sentiment and an increase of the birds during the year 1905. Mr. Travis says: “Up to the year 1885 pinnated grouse were very plentiful in Kansas and especially so in the western part. From that time they decreased very rapidly, until about 1900. Between those dates a grouse was seldom seen in the eastern half of the State, and but very few in the western half. The decrease was caused by the late burning of the prairies all over the western half of the State. THE PINNATED GROUSE TO-DAY. Settlers were filling this section rap- idly, and it seemed to be the general opinion that all dead grass should be burned, which destroyed all food and insects, starving the birds out and leaving no nest- AMERICAN GAME BIRD SHOOTING 248 ing grounds. Again, meat was scarce and high, and the settlers killed grouse the year round in order to supply their tables, and also killed thousands and sent them to eastern markets. This, with the hundreds of eastern market hunters, caused the almost complete ex- termination of the grouse in Kansas. “About 1900, the people began to realize that the “About 1900, the people began to realize that the grouse were nearly exterminated, and a crusade for their protection started. Stringent laws were passed in 1903, and still more severe ones in 1905, and I am pleased to state that in the fall of 1905 in a number of the central counties the birds have increased to num- bers beyond expectations, and to-day the grouse can be found in many of the eastern counties. With proper protection, pinnated grouse will be abundant in all parts of the State within five years. The day of the game butcher, the pot and market hunter is past.” Texas is now and always has been a State where pinnated grouse were abundant, and it is so large that there are still great numbers of birds there. The growth of the game protective sentiment, which has been so marked within the last year or two, promises to protect these birds. There are other States, as Michigan and Wisconsin, that have a few pinnated grouse, and the bird occurs rarely in the Province of Ontario. Prof. Walter B. Barrows, of the Michigan Agri- Prof. Walter B. Barrows, of the Michigan Agri- cultural College, whose writings on ornithological top- ics are familiar to many of our readers, says: “This bird was formerly fairly common over the “This bird was formerly fairly common over the PINNATED GROUSE 249 southern half of the Lower Peninsula, and up to twenty years ago was abundant in many places in the prairie regions of the three southernmost tiers of counties. Even ten years ago it was not uncommon in this county (Ingham), and undoubtedly a few still exist here. THE PINNATED GROUSE TO-DAY. I am not able to say positively where it is most abun- dant now, but my latest reports, some two and three years ago, indicated that there were still a good many in Calhoun, Kalamazoo and Van Buren counties, and it is fair to presume that they were locally common in the three counties south of those and _ bordering Indiana. “There are vague Michigan accounts of the presence of this species along the Lake Michigan shore as far north as Traverse City, fifty years ago, but I have no reason to believe that the species has ever been common north of the Saginaw Grand Valley in about latitude 43%°. The sharp-tailed grouse may at one time have existed in the upper part of the Lower Peninsula and in parts of the Upper Peninsula, but at present it is not known to occur except in Isle Royale in Lake Superior, where there appears to be a flourishing colony, but as yet I have been unable to obtain specimens so as to determine positively the sub-species.”’ It would seem perfectly practicable to reintroduce this bird in eastern localities where once it was abun- dant, but this of course could be done only in situations where the bird would be sure to be protected; in other words, in large preserves. It is conceivable that the birds might live and do well on some of the large AMERICAN GAME BIRD SHOOTING 250 estates of Long Island, and even on the Shinnecock Hills, but it is hardly to be hoped that they could ever be brought back as a game bird. They would have to be regarded as merely beautiful natural objects. SHARP-TAILED GROUSE. Pediecetes phasianellus. Pediacetes phasianellus columbianus. Pediecetes phasianeilus campestris. Pediecetes phasianellus. Pediacetes phasianellus columbianus. Pediecetes phasianeilus campestris. The sharp-tailed grouse, while about the same size as the pinnated grouse, is a somewhat plumper and stouter bird, and, from the fact that its tail is smaller, does not seem so large. The tail of the sharp-tail is graduated, the middle pair of feathers projecting much beyond the rest; it has no tufts of feathers on the side of the neck. It has a high crest, is brownish or rusty above, varied with spottings and barrings of black and darker brown. Large round white spots mark the wing coverts, and the scapulars are somewhat streaked with white, while the outer webs of the quills of the wing are spotted with white. The lower parts are for the most part white, varied with V-shaped marks of dusky on ‘front and sides of breast. The female is similar, but a little smaller. The differences between the three forms consist chiefly in the shades of colors. The northern sharp-tailed grouse (Pediecetes pha- sianellus—Linn.) is very dark-colored, with the dark markings on the upper parts very heavy, while the white marks on the wings show out strongly against this dark color. The feathering on the legs is dark The northern sharp-tailed grouse (Pediecetes pha- sianellus—Linn.) is very dark-colored, with the dark markings on the upper parts very heavy, while the white marks on the wings show out strongly against this dark color. The feathering on the legs is dark 251 251 251 AMERICAN GAME BIRD SHOOTING 252 brownish gray. In length it varies from about 19 inches for the largest males to about 15 inches for the smallest females, and the wing measures from 8 to 8} inches. Most of the tail feathers are extremely stiff, pointed and white-edged. This northern bird is found in central Alaska, and lf 7, WETEIS Wy eee Dy. oy; Wee atneee eerie: efi SHARPTAILED GROUSE SHARPTAILED GROUSE in the interior of British Provinces, north as far as Great Slave Lake, south as far as Moose Factory, Lake Winnipeg, Temiscaming, the northern shore of Lake Superior and the east side of James’ Bay, in Labrador. It is never seen in the United States. The western sharp-tailed grouse, or Columbian The western sharp-tailed grouse, or Columbian SHARP-TAILED GROUSE 253 sharp-tailed grouse (P. p. SHARP-TAILED GROUSE. columbianus) is very much paler in color, being grayish or clay color and marked with black, but with the black marks less sharp and strong. It is found in central British Columbia and central Alberta, south in the western United States as far as northern California, Nevada and Utah, east to the border of the plains in Colorado. Its range is chiefly west of the Rocky Mountains. It is slightly smaller than the northern form. The more familiar sharp-tail of the Middle West (P. p. campestris) is abundant on the plains from southern Manitoba and southern Alberta, south through the United States to Wyoming and Kansas, east as far as Wisconsin and Illinois, and west to eastern Colo- rado. It is bright rusty in color and its dark mark- ings are much less conspicuous than in the northern form. The sharp-tailed grouse, which of late years has come to be known over much of the West as prairie chicken, is thus—in one or other of its three forms— a bird of wide distribution. It is found from Kansas, on the south, to central Alaska on the north, and from British Columbia, California and Nevada, on the west, to James’ Bay on the east. It occurs, as said, sparsely south of the Great Lakes, but in the United States— except in this locality—its range is chiefly west of the Mississippi River. While during the greater part of the year it seems to be a bird of the prairie, it is yet often found high up in little mountain valleys, and often in a country that is completely wooded. AMERICAN GAME BIRD SHOOTING 254 Like many others of our best game birds, the sharp- tailed grouse has been so unremittingly pursued that it is rapidly becoming more and more scarce, and promises before long, in all regions where it is pursued with dog and gun, to become as rare as its relative, the pinnated grouse. In habits the birds are all closely alike, except that In habits the birds are all closely alike, except that we may assume that the northern form has modified its habits in accordance with its environment. Mr. Roderick MacFarlane found this species breeding in 1884 near Fort Providence. The two last-named forms of the sharp-tailed grouse—which is also called white belly, speckle belly, willow grouse and pin tail—are common all through the northwestern United States. SHARP-TAILED GROUSE. They are birds of the open land, yet at certain seasons of the year resort commonly to willows or brushy ravines, from which sometimes they get up in a thick flock, like a brood of gigantic quail. EK. E. Thompson, writing of the prairie sharp- tailed grouse in Manitoba, describes its prenuptial dancing in the following language: “After the dis- appearance of the snow, and the coming of warmer weather, the chickens meet every morning at gray dawn in companies of from six to twenty on some selected hillock or knoll, and indulge in what is called ‘the dance.’ This performance I have often watched. It presents the most amusing spectacle I have yet wit- nessed in bird life. At first, the birds may be seen standing about in ordinary attitudes, when suddenly one of them lowers its head, spreads out its wings SHARP-TAILED GROUSE 255 nearly horizontally and its tail perpendicularly, distends its air-sacs and erects its feathers, then rushes across the ‘floor,’ taking the shortest of steps, but stamping its feet so hard and rapidly that the sound is like that of a kettle drum; at the same time it utters a sort of bub- ling crow, which seems to come from the air-sacs, beats the air with its wings, and vibrates its tail, so that it produces a low, rustling noise, and thus con- trives at once to make as extraordinary a spectacle of itself and as much noise as possible. “As soon as one commences all join in, rattling, “As soon as one commences all join in, rattling, stamping, drumming, crowing and dancing together furiously ; louder and louder the noise, faster and faster the dance becomes, until at last, as they madly whirl about, the birds are leaping over each other in their excnement, After a/\brief spell) the: enersy! of, the dancers begins to abate, and shortly afterward they cease and stand or move about very quietly, until they are again started by one of their number ‘leading off.’ “The whole performance reminds one so strongly of a Cree dance as to suggest the possibility of its being the prototype of the Indian exercise. The space occupied by the dancers is from 50 to 100 feet across. ... The dancing is indulged in at any time of the morning or evening in May, but it is usually at its height before sunrise. SHARP-TAILED GROUSE. Its erotic character can hardly be questioned, but I cannot fix its place or value in the nuptial ceremonies. The fact that I have several times noticed the birds join for a brief set-to, in the late fall, merely emphasizes its parallelism to the drumming AMERICAN GAME BIRD SHOOTING 250 and strutting of the partridge, as well as the singing of small birds.” I have several times witnessed this dancing in No- vember, just about the time that cold weather sets in, and have seen it carried on for two hours, immediately after sunrise, but have never been so fortunate as to get close enough to the birds to hear their stamping sound like a kettle drum. In the dances I have witnessed, there was heard not only the crowing, of which Mr. Thompson speaks, but also a sharp, high-pitched cackle, each note being separated from the other by a percep- tible interval. Some years ago I sent Major Bendire some notes on this species, which I quote here: “This species is partly migratory, and there is the very greatest dif- ference in the habits of the birds in summer and winter. As soon as the first hard frosts come in the autumn, the birds seem to take to the timber and begin to feed on the buds of the willow and the quaking aspen. At this time they spend a large portion of their time in the trees, and are very wild. In the Shirley Basin, in western Wyoming, a locality where I have never seen any of these birds in summer, they are abundant in winter. At this season they live in quaking aspen thickets, along the mountains, and there I have seen hundreds of them roosting on top of a big barn, which stands just at the edge of a grove of quaking aspen timber. It was always easy in the morning, just after sunrise, to step out of the house, and with a .22 caliber Some years ago I sent Major Bendire some notes on this species, which I quote here: “This species is partly migratory, and there is the very greatest dif- ference in the habits of the birds in summer and winter. SHARP-TAILED GROUSE. As soon as the first hard frosts come in the autumn, the birds seem to take to the timber and begin to feed on the buds of the willow and the quaking aspen. At this time they spend a large portion of their time in the trees, and are very wild. In the Shirley Basin, in western Wyoming, a locality where I have never seen any of these birds in summer, they are abundant in winter. At this season they live in quaking aspen thickets, along the mountains, and there I have seen hundreds of them roosting on top of a big barn, which stands just at the edge of a grove of quaking aspen timber. It was always easy in the morning, just after sunrise, to step out of the house, and with a .22 caliber ‘aye]q S,uoqnpny wo1g "asnoig pareydieys ‘ay S,uoqnpn wo1g "asnoig pareydieys SHARP-TAILED GROUSE 257 rifle shoot off the heads of as many of these birds as were needed for eating for the next two or three days. “T have only one note on these birds which seems “T have only one note on these birds which seems particularly worth mentioning, and of this I spoke in my report to Colonel William Ludlow, on the birds noticed during a reconnaissance to the Black Hills of Dakota in 1874, which was published by the Engineer Bureau of the War Department. The sharp-tailed grouse has a cry which is unlike that of any other grouse with which I am familiar, although something very similar has been observed in the case, I think, of one of the ptarmigans. On the plains of Dakota in 1874, having scattered a brood of sharp-tailed grouse, consisting of a mother and a dozen well-grown young, I sat down to wait for them to get together. The mother had flown to the top of a hill not far off, where she sat on the ground in plain sight, and after a few moments began to call to the young, which immediately answered her from the different points where they had taken refuge. The call of the mother and the young was a guttural, raucous croak, which quite closely re- sembled the croaking of a raven at a little distance. SHARP-TAILED GROUSE. I plainly saw the old bird utter its note, and subse- quently followed up the calls uttered by more than one of the young ones, until I started them, and killed one or two as they flew.” In winter the food of the sharp-tailed grouse consists In winter the food of the sharp-tailed grouse consists largely of rose-berries and the buds of willows, cotton- woods and aspens. In summer and autumn, grass- hoppers, insects and various berries, together with AMERICAN GAME BIRD SHOOTING 258 grass and the leaves of various plants, form their chief food. I have seen them feeding by hundreds in the alfalfa patches of the ranchmen, and have found their crops and throats stuffed with the green leaves, together with a few of the seeds. Almost everything in the nature of berries, insects, seeds and green leaves is devoured by this bird. In the winter and autumn, the sharp-tailed grouse In the winter and autumn, the sharp-tailed grouse inhabiting a prairie country, spend most of their time in the river bottoms, among or close to the willow and cottonwood trees, on the buds of which they feed at this season, and it is not uncommon to see large flocks of them roosting among the branches of these trees in the early morning, apparently too much chilled to notice the approach of man. Mr. E. E. Thompson, in his “Birds of Manitoba,” describes in some detail the habits of the sharp-tailed grouse in winter. He says that it spends the winter nights in the snow, which is always soft and penetrable in the woods, though out on the plains it is beaten into drifts of ice-like hardness. “As the winter wanes, it is not uncommon for a snow storm to be accompanied by sleet. The storm always drives the chickens into the drifts, and afterward levels the holes they formed in entering. The freezing of the sleet then forms a crust, which resists all attempts at escape on the part of the birds, many of which, accord- ing to the account of hunters, are starved and thus perish miserably. I met with a single instance of this myself. SHARP-TAILED GROUSE 259 “Before the winter is over many of the birds, by con- tinuously pulling off frozen browse, have so worn their bills that when closed there is a large opening right through, immediately behind the hook. SHARP-TAILED GROUSE. Early in April the few that have survived the rigors and perils of their winter life spread over the prairie once more and soon scatter to enter on their duties of reproduction.” No one has written about this bird more charmingly than Dr. Elliott Coues, whose article on the prairie form is well worth quoting. He first heard the call of the sharp-tailed grouse in North Dakota, when he was alone in camp, not far from Fort Randall—at the time his home—where he had gone to shoot water fowl. He says: “Awakened before it was light by the sonorous cries of the wild fowl making for the reedy lake where I had encamped, I arose—there was no need to dress—pushed off into the expanse of reeds in a light canoe I had brought with me, and with my gun across my knees sat quietly waiting for light to come. The sense of loneliness was oppressive in the stillness that preceded morning, broken only by the quack or plash of the wild duck, and the distant honking of a train of wild geese winnowing their sinuous way afar. I felt desolate— almost lost—and thought how utterly insignificant man is in comparison with his self-assertion. The grand bluffs of the Missouri, rising past each other intermina- bly, were before me in shadowy outline, that seemed to change and threaten to roll upon me; all around stretched the waste of reeds, secret, treacherous, limit- AMERICAN GAME BIRD SHOOTING 260 less—unmoved, yet whispering to the water about their roots with a strange trickling. But the light came on; the distant hills took shape and settled in firm gray out- line against the sky, and a breath of fresher, purer air, messenger of morning, passed over the lake, dispelling the vapors that hung reluctant, and causing the reeds to sway in graceful salute to the coming sun. A sparrow chirped from her perch with joy; a field lark rose from her bed in the grass, tuning her limpid pipe to a song of gladness; and the wild fowl plashed about right heartily, when the highest hilltop was touched with gold, and another and another, till the scene was il- lumined to the very bosom of the lake. SHARP-TAILED GROUSE. The feathered orchestra sounds never so impressive as when it ushers in the day; never so fine and complete as when familiar voices sing the higher notes to the strange deep bass of the grouse; heard for the first time, as it was on this occasion, the effect is indescribable. No one could say whence the sound proceeded, nor how many birds, if more than one, produced it; the hollow reverberations filled the air, more like the lessening echoes of some great instrument far away, than the voice of a bird at hand. I listened to this grand concert, absorbed in the reflections it stirred within me, no longer alone, but in company I love, till the booming fell less fre- quently upon my ear, and then ceased—it was broad day; the various birds were about their homely avoca- tions, and I must betake myself to practical concerns. “Thus, in no faltering accents of timid expectancy, “Thus, in no faltering accents of timid expectancy, but in the bold tone of assured success, the grouse SHARP-TAILED GROUSE 261 calls upon his intended mate to forget the shyness that will no longer serve their purpose; nor does the invi- tation lack defiance to a rival who may presume to dis- pute his rights. At the rallying cry the birds assemble, in numbers of both sexes, at some favorable spot, and a singular scene ensues as the courtship progresses. There is a regular ‘walk-around,’ as ludicrous, to the disinterested observer, as some of the performances on the comic stage. The birds run about in a circle, some to the right, others to the left, crossing each other’s path, passing and repassing in stilted attitudes, stop- ping to bow and squat in extravagant postures, and resuming their course, till one would think their heads as well as their hearts were lost. But this is simply their way, and they amuse themselves in such fashion till the affair is settled. The cocks have bristled and swelled, strutted and fought, till some have proven their claims to first choice, and others have concluded to take what they can get. Their subsequent history, I am sorry to state, is neither particularly creditable to themselves nor of absorbing interest to us. Leaving them to go about their business in their usual humdrum way, let us look to what now occupies their mates. SHARP-TAILED GROUSE. “A nest will soon be required for her eggs, and the hen has to select suitable premises, though, being an architect of only the humblest order, she has little build- ing work to do; and, moreover, not being fastidious, her choice is made without difficulty. I have found the nests in such various locations that I can hardly determine what her preference is, if, indeed, she have AMERICAN GAME BIRD SHOOTING 262 any. I suppose the site depends much upon circum- stances. She will enter a tract overgrown with the low, scrubby willow bushes, so abundant in our higher latitudes, and settle beneath one of these; she will ram- ble along the edge of a wooded stream and hide in a patch of tall weeds; she will stroll out on the bound- less, bare prairie, and take a tuft of grass at random. But wherever she makes down her bed she is solicitous to conceal it, not only from the rude glances of men, but from the equally cruel eye of her many four-footed enemies. Her method of concealment is most artful— perfected by its witlessness. With admirable instinct, she will avoid a place that offers such chances of con- cealment as to invite curious search; her willow bush is the duplicate of a thousand others at hand; her tuft of grass on the prairie is the counterpart of a million others around; her nest will be found by accident oftener than by design. And when, stooping over a warm nest on the prairie, whence she has just fluttered in dismay, we note how exposed it seems, now that it is found, we wonder how the dozen blades of grass that overarch the eggs, or the rank weed that shadows them, could have hidden the home so effectually that we nearly trod upon the bird before we saw her. She is now but a few yards off, in plain view, amid the scrubby prairie herbage, perhaps squatting, but more likely mov- ing away with a swaying motion of the head at each step. We will not combine murder with the robbery we are about to commit, and let us hope she will be consoled in time. SHARP-TAILED GROUSE. Lifting up the eggs carefully, one by SHARP-TAILED GROUSE 263 one, we find the nest to be merely a few spears of grass, pressed down and somewhat circularly arranged with, in all probability, a few feathers that appear to have rather been mechanically detached from the mother bird than laid down by design. If the place is near our northern border, and early in June, we shall probably find the eggs quite fresh; but by the third week of that month they will be about hatching. At this period, should we, for any sufficient reason, destroy the setting bird, we should find her in sad plight—her plumage, harsh and worn, entirely gone from a large space on her belly; her flesh thin and flabby, and her crop con- taining only a few buds of some weed that grows close by her nest, with some grasshoppers or other insects. “No bird is a more faithful mother than this grouse; no one clings to her eggs more steadfastly, or guards her young with more sedulous care. In proof of how close she will set while incubating, let me mention two instances that came under my observation. One poor bird was actually trodden upon and killed, and some of her eggs smashed. On another occasion, I drove a large four-mule ambulance over a nest; the animals shied as they stepped over it, when the bird fluttered out from between their legs. Stopping instantly, I discovered the nest just between the hinder wheels. © The grouse lies hard and close, never relinquishing hope of escaping observation until the last moment. “The young, as usual among gall moment. “The young, as usual among gallinaceous birds, run about almost as soon as they are hatched, and it is in- ment. “The young, as usual among gallinaceous birds, run about almost as soon as they are hatched, and it is in- . “The young, as usual among gallinaceous birds, run about almost as soon as they are hatched, and it is in- AMERICAN GAME BIRD SHOOTING 264 teresting to witness the watchful solicitude with which they are cherished by the parent when she first leads them from the nest in quest of food, glancing in every direction, in her intense anxiety, lest harm befall them. SHARP-TAILED GROUSE. “Throughout the region of the Red, Pembina and Souris or Mouse rivers, where I observed the birds during the summer, I found them mostly in the under- brush along the streams, which they seemed to seek instinctively as affording the best shelter and protec- tion, as well as plenty of food. Where they were most abundant I frequently observed the ‘scratching holes’ in the bare earth among the bushes, where they resorted to dust themselves, and, most probably, in the instances of ungrown coveys, to roost. Late in the summer and in September, those who cared to shoot the tender young found them to lie well to a dog; in fact, to lie so close that they were flushed with difficulty with- out one. No game birds could be tamer or more readily destroyed. Except when temporarily scattered by molestation, the coveys kept close together, and only occasionally left the covert to stray on the adjoin- ing prairie. They appeared to be feeding chiefly on wild-rose seeds, and those of another kind of plant “Returning to the newly hatched chicks, we will note their characteristics as they progress toward maturity. The down in which they are clothed when hatched is rather dingy yellow, mottled on the crown, back and wings with warm brown and black; it extends to the toes, but leaves a bare strip along the hind edge of the tarsus; the bill and feet are light brown. They are about as large as bantam chickens of the same age, and very pretty little things, indeed. They are very quick in their movements, scrambling to squat and hide on the least alarm, even at this early age. ... “Throughout the region of the Red, Pembina and Souris or Mouse rivers, where I observed the birds during the summer, I found them mostly in the under- brush along the streams, which they seemed to seek instinctively as affording the best shelter and protec- tion, as well as plenty of food. Where they were most abundant I frequently observed the ‘scratching holes’ in the bare earth among the bushes, where they resorted to dust themselves, and, most probably, in the instances of ungrown coveys, to roost. SHARP-TAILED GROUSE. She clucks matronly to bring them to brood under her wings, or to call them together to scramble for a choice morsel of food she has found. Should danger threaten, a different note alarms them; they scatter in every di- rection, running, like little mice, through the grass till each finds a hiding place; meanwhile, she exposes her- self to attract attention, till, satisfied of the safety of the brood, she whirrs away and awaits the time when she may reassemble her family. In the region where I observed the birds in June and July, they almost in- variably betook themselves to the dense, resistant un- derbrush, which extends for some distance out- ward from the wooded streams, seeking safety in this all but impenetrable cover, where it was nearly impossi- ble to catch the young ones, or even to see them, until they began to top the bushes in their early short flights. The wing and tail feathers sprout in a few days, and are quite well grown before feathers appear among the down of the body. The first coveys seen able to rise on wing were noticed early in July; but by the middle of this month most of them fly smartly for short distances, being about as large as quails. Others, how- ever, may be observed through August, little, if any, larger than this, showing a wide range of time of hatching, though scarcely warranting the inference of two broods in a season. SHARP-TAILED GROUSE 265 “Returning to the newly hatched chicks, we will note their characteristics as they progress toward maturity. The down in which they are clothed when hatched is rather dingy yellow, mottled on the crown, back and wings with warm brown and black; it extends to the toes, but leaves a bare strip along the hind edge of the tarsus; the bill and feet are light brown. They are about as large as bantam chickens of the same age, and very pretty little things, indeed. They are very quick in their movements, scrambling to squat and hide on the least alarm, even at this early age. ... SHARP-TAILED GROUSE. Late in the summer and in September, those who cared to shoot the tender young found them to lie well to a dog; in fact, to lie so close that they were flushed with difficulty with- out one. No game birds could be tamer or more readily destroyed. Except when temporarily scattered by molestation, the coveys kept close together, and only occasionally left the covert to stray on the adjoin- ing prairie. They appeared to be feeding chiefly on wild-rose seeds, and those of another kind of plant “Returning to the newly hatched chicks, we will note their characteristics as they progress toward maturity. The down in which they are clothed when hatched is rather dingy yellow, mottled on the crown, back and wings with warm brown and black; it extends to the toes, but leaves a bare strip along the hind edge of the tarsus; the bill and feet are light brown. They are about as large as bantam chickens of the same age, and very pretty little things, indeed. They are very quick in their movements, scrambling to squat and hide on the least alarm, even at this early age. ... “Throughout the region of the Red, Pembina and “Throughout the region of the Red, Pembina and Souris or Mouse rivers, where I observed the birds during the summer, I found them mostly in the under- brush along the streams, which they seemed to seek instinctively as affording the best shelter and protec- tion, as well as plenty of food. Where they were most abundant I frequently observed the ‘scratching holes’ in the bare earth among the bushes, where they resorted to dust themselves, and, most probably, in the instances of ungrown coveys, to roost. Late in the summer and in September, those who cared to shoot the tender young found them to lie well to a dog; in fact, to lie so close that they were flushed with difficulty with- out one. No game birds could be tamer or more readily destroyed. Except when temporarily scattered by molestation, the coveys kept close together, and only occasionally left the covert to stray on the adjoin- ing prairie. They appeared to be feeding chiefly on wild-rose seeds, and those of another kind of plant AMERICAN GAME BIRD SHOOTING 266 equally abundant along the river bottoms. SHARP-TAILED GROUSE. The ma- jority of these birds were ungrown up to September, and scarcely any had at that date begun to assume their new plumage. Up to this time I do not recollect that I ever saw one alight in a tree; and they were still, for the most part, under charge of the parent, as separate families, rather than as the indiscriminate packs in which they afterward associate. With the advance of the month these family associations seemed to break up, the change of plumage was finished, the birds grew strong of wing, and able in all respects to look after themselves. No longer solicitous of shelter, they haunted the innumerable ravines that make down to the streams, and strolled in company far out on the prairie. In this region, at least, they showed little wariness all through the month. I could generally walk up to a covey in fair view on the bare prairie, even to within a few feet, before they would fly, and they seldom went far before realighting. Their appearance when not obscured by the herbage is characteristically peculiar. They seem to stand remarkably high on their legs, and generally carry their short, pointed tail some- what elevated; the singularity is increased when the long neck is outstretched, as it generally is when they are on the lookout. On alighting after being flushed, if not much alarmed, they often stand motionless at full height, but if badly scared, squat closely, and are then difficult to find if not exactly marked down. If without a dog, one may pass and repass among them without finding one, unless he happen to stumble on SHARP-TAILED GROUSE 267 them; and often, going away after such want of suc- cess, one may look back to find the heads of the whole lot raised above the grass, intently regarding his re- treat. It is astonishing how closely they can squat— even laying the head flat upon the ground, and appear- ing scarcely half their natural size. At this season their food appears to be chiefly grasshoppers. I have opened numbers to find their crops crammed with these insects, only varied with a few flowers, weed-tops, succulent leaves, and an occasional beetle or spider. SHARP-TAILED GROUSE. “By the first of October the sharp-tailed grouse have “By the first of October the sharp-tailed grouse have mostly finished the renewal of their plumage, are all full grown and strong of wing; their habits are con- siderably modified. They grow wary and watchful, flushing often at long distances to fly clear out of sight, and running far on the ground. They also be- gin to alight on trees, a habit, however, not confirmed until somewhat later, when, with the advance of cold weather and the failure of former supplies of food, they assume the routine of their winter life. The close coveys of the earlier season are for the most part broken up, and the birds wander often alone in search of food. They haunt the interminable ravines along the Mis- souri, making away from the river bottoms in search of food, but mostly returning at evening to roost in the trees. Early in the morning they may be seen leaving their perches in straggling troops, flying high and swiftly to other feeding grounds; and again in the evening, if one loiter beneath the immense cotton- woods, where, during the day, scarcely a chicken was AMERICAN GAME BIRD SHOOTING 268 to be seen, he will observe their return, till the trees are almost covered, and the air resounds with the hoarse kuk-kuk-k-k-k. Frequently, in very cold and especially in falling weather, the grouse will not leave their perches during the day, but may be seen at any hour roosting quietly in the tops of the tallest cotton- woods. They are decidedly not graceful objects under these circumstances. They look very large, sharply defined among the bare straggling branches against the gray sky, and assume ungainly attitudes, particu- larly when standing erect on their long legs, with out- stretched necks and upturned tails. Their behavior under these circumstances varies in a manner to me inexplicable. Sometimes a group thus scattered among the treetops will permit the closest approach desired, and more than one may be brought down before the rest are off in alarm; not seldom one may fire twice or thrice at the same bird without dislodging it, or kill several without stirring from his tracks. But ordi- narily the chickens’ wits serve them to better purpose than this. SHARP-TAILED GROUSE. As we approach, when just beyond range, the crackling of the underbrush attracts the attention of one of the birds, which before had been squatting ‘like a bump on a log’; he rises on his feet and twists his neck around to have a look. The rest follow his example. A moment more the warning kuk-kuk-k sounds, and the nearest bird leaves his perch—the cry is taken up by the rest, and the whole are off to settle again a few hundred yards away, and tempt renewed pursuit that is likely to end as unsuccessfully. From SHARP-TAILED GROUSE 269 the sportsman’s standpoint, the arrangement is wholly unsatisfactory when the birds behave so; nor when they are tame is it much more attractive; for, unless a supply of meat be the only point, dropping chickens from the trees is no more exciting than robbing a hen-roost. Killed under these circumstances, the food of the grouse is readily ascertained; in the dead of winter it consists chiefly of the berries of the cedar, and buds of the pop- lar or cottonwood and willow, still closely sealed, await- ing the coming of spring. I have taken from one crop a double handful of such food, almost as dry as when swallowed. This diet does not improve the quality of the flesh; a chicken at this season is quite a different thing from one killed earlier in the season. The rating of the grouse as an article of food neces- sarily varies, not only with circumstances, but accord- ing to individual preferences. 1, myself, do not esteem it very highly. A tender young grouse, early in the season, is not to be despised, but all such specially flavored meat is likely to soon become distasteful, espe- cially if, on one or two occasions, a person has been forced upon a surfeit of it. Confined to grouse for a few days, most persons, I should judge, would find relief in mess-pork. “The mode of flight of this species is not peculiar ; it rises with a startling whirr from the ground, till it attains a certain elevation—its straight, steady course, performed with great velocity by alternate sailing and flapping, are points it shares with its relatives. SHARP-TAILED GROUSE. The wing-beats are rapid and energetic, giving it an im- AMERICAN GAME BIRD SHOOTING 270 pulse that enables it to sail long distances, when the wings are held stiffly expanded to their full extent, somewhat decurved, and with the points of the quill feathers separated. The bird’s voice is highly char- acteristic. It is so almost invariably uttered during flight, at particular moments with reference to the de- livery of the wing strokes, that for some time after my first acquaintance with the birds I was in doubt whether the sound were mechanical or vocal; nor was the un- certainty removed until I had heard it from the birds at rest. The ordinary note of alarm is almost invaria- bly sounded just before the bird takes wing, whether from the ground or from a tree, and is usually repeated with each succeeding set of wing-beats, seeming to be jerked out of the bird by its muscular efforts. But we hear it also when, the bird being at rest, it becomes alarmed, yet not sufficiently to fly away; and when a bird is passing at full speed, sufficiently near, we may clearly distinguish the mechanical whirring sound of its wings, as well as, sometimes, the creaking rustle of its tail feathers as it turns its flight. When roosting at ease among the trees, and probably at other times, the grouse have a different set of notes—a sociable cackling or clucking, with which they entertain each other. “In conversation with Captain Hartley, of the Twenty-second Regiment, an accomplished sportsman, well acquainted with the ways of our game birds, I was informed of an interesting point of difference in the habits of this bird and the pinnated grouse. In SHARP-TAILED GROUSE 271 entering a cultivated field the latter goes on foot, and may consequently be readily trailed by a dog, while the sharp-tail flies in, and is only likely to be overhauled by the dog’s winding it, or coming accidentally upon it. The same gentleman has noted the preference of this species for the skirts of woods, brush and broken places generally, in contrast to the entirely open places which the pinnated grouse frequents.” The eggs of the sharp-tail, while often plain cream- The eggs of the sharp-tail, while often plain cream- colored, are at times greenish in color and are some- times marked with small spots of reddish brown and lavender. SAGE GROUSE. Centrocercus urophasianus. SHARP-TAILED GROUSE. They do not appear to lay such large clutches as the pinnated grouse, though, after all, the difference in numbers is not marked. Centrocercus urophasianus. The sage grouse may always be recognized by its great size and by the fact that its tail is longer than the wings, and is graduated, with narrow pointed quills. The upper plumage is gray, variously marked and streaked with black. Some of the tertiary feathers are tipped with white or streaked with white; the chin, cheeks and throat are spotted black and white, but there is usually a distinct white streak on the side of the head running a short way back of the eye. The neck is black in front, while the fore breast is white or grayish, and on either side of the lower neck in the whitish areas are stiff, coarse hair-like feathers. The belly is black, and the lower tail coverts black tipped with white. The male is from 26 to 30 inches long, with a wing of from 12 to 13 inches, and a tail about the same length. The birds weigh from 4 to 8 pounds. The female is much smaller; has the chin and throat white and the black patch on the front of the neck speckled. Half-grown birds are browner than their parents, with a warmer tone of rufous in their plumage, more like the young of the dusky grouse. The length of the female is from 20% to 23 inches. Excepting the wild turkey, the sage hen, as it is often Excepting the wild turkey, the sage hen, as it is often 272 SAGE GROUSE 273 called, is the largest North American gallinaceous bird. Full-grown males sometimes almost equal the female wild turkey in size, and have been reported to weigh about eight pounds. SAGE GROUSE SAGE GROUSE The sage grouse is an inhabitant of the high, dry, artemisia or sage-brush covered plains of the Western States. It is found on the plains and flanks of the Rocky Mountains, in the great central plateau, in the northwest territories, and in parts of southern British AMERICAN GAME BIRD SHOOTING 274. Columbia. Its eastern range covers parts of North Dakota, and extends thence westward through Mon- tana, Wyoming and Utah to Oregon, California and Nevada. The sage grouse is interesting from the fact that it possesses a soft, membranous stomach; in this respect differing from other gallinaceous birds. Its food varies with the seasons, and no doubt in winter the leaves of the so-called sage brush make up the most of this. Centrocercus urophasianus. The sage grouse takes its name from this plant, on The sage grouse takes its name from this plant, on which, at certain seasons, it feeds extensively. Yet it eats other food, including grain. In Forest and Stream of August 29, 1889, Mr. George H. Wyman says: “The sage cock will eat the leaves from a sage bush when it cannot get berries or grain, but it will go farther for a morning’s feed from a wheat field than any bird I know, except the wild geese. I have killed sage fowls with stomachs filled with ripe wheat picked up the same morning in places where none was to be had nearer than eight miles, and in fact with no culti- vation of any kind nearer in any direction. They fly long distances in search of food, but return to roost in the same place at night, generally on some steep hillside, free from shrubs or high grass.” This may have been an error of observation. Very likely there was some unknown source of grain supply nearer at hand. But this, of course, cannot be proved. It is stated by some observers that sage leaves are resorted to only when other food is hard to obtain; but this is a mistake. The leaves and flowers of the sage have been found SAGE GROUSE 275 in the crops of adults and young during the season of greatest food supply. The leaves, blossoms and the pods of other plants, together with many seeds and grasshoppers and crickets, are said by Captain W. L. Carpenter to constitute a large part of this food. Mr. Robert W. Williams is quoted by Captain Bendire as having taken a bird, the crop of which was filled with the blossoms of a species of goldenrod (Solidago). In past years controversies as to the edibility of the In past years controversies as to the edibility of the flesh of the sage cock have often taken place, some writers holding that the flesh is so strongly flavored by the sage that it is unpleasant to the taste, while others maintained with equal earnestness that the flesh was delicate and without unpleasant flavor. Centrocercus urophasianus. Obviously, the flavor of the flesh of any bird depends in great measure on the food which it eats, and birds which stuff themselves with a particular sort of vegetation, whether it be buds of the birch, of the laurel, of the spruce or of the wormwood, if left undrawn for a time may taste of the plant on which they have been feeding. It is a safe plan to dress the sage grouse as soon as may be after it has been killed, but even if this is not done, young birds are not likely to have a strong taste, though old ones may. Like other prairie grouse, the sage cock goes through Like other prairie grouse, the sage cock goes through a courtship which is noteworthy. This is well de- scribed by Captain Bendire in his ‘‘Life Histories of North American Birds,” in which he says: “Early one morning, in the first week of March, “Early one morning, in the first week of March, 1877, I had the long-wished-for opportunity to observe AMERICAN GAME BIRD SHOOTING 270 the actions of a single cock while paying court to sev- eral females near him. ... His large, pale-yellow air-sacs were fully inflated, and not only expanded for- ward, but apparently upward as well, rising at least an inch above his head, which, consequently, was scarcely noticeable, giving the bird an exceedingly peculiar ap- pearance. He looked decidedly top-heavy and ready to topple over at the slightest provocation. The few long, spiny feathers along the edges of the air-sacs stood straight out, and the grayish white of the upper parts showed in strong contrast with the black of the breast. His tail was spread out fan-like, at right angles to the body, and was moved from side to side with a slow, quivering movement. The wings were trailing on the ground. While in this position he moved around with short, stately and hesitating steps, slowly and gingerly, evidently highly satistied with his performance, utter- ing, at the same time, low, grunting, guttural sounds, somewhat similar to the purring of a cat when pleased, only louder. This was kept up for some ten minutes. After having regained his usual attitude it was hard to believe that this was the same bird I had seen but a few minutes before.” Mr. Centrocercus urophasianus. Frank Bond has described at some length a hitherto unnoticed act of the male sage grouse, which he witnessed in Wyoming. The bird, after inflating its air-sacs, bent forward until its breast touched the ground and pushed itself forward along the ground. This action, carried on for days, seems to explain the SAGE GROUSE 277, wearing away of the stiff feathers on the lower neck and upper breast so often noticed in this species. The nest of the sage grou pecies. The nest of the sage grouse is usually placed at the foot of some sage bush, or sometimes at the foot of a bunch of rye grass, whose outer leaves, bending over, may conceal the nest. Commonly it contains from eight to eleven eggs, nearly as large as a hen’s egg, greenish white or brownish in color, more or less heavily spotted with round, but not large, dots of brown and blackish. The period of incubation js given as twenty-two days. There is little There is little or no nest, and the eggs lie in a mere hollow scratched out in the bare ground. The sitting bird harmonizes so wonderfully with the ground on which she rests that one may pass within a foot or two without seeing her. Major Ben Major Bendire quotes Captain William L. Car- penter, U. S. A., who says: “I found a nest at Fort Bridger, Wyoming—where this species is numerous— June 1, with nine fresh eggs. I was standing alongside a sage bush watching butterflies, several times looking down carelessly without seeing anything unusual, when, happening again to glance at the foot of the bush in the very place before observed, I saw the wink- ing of an eye. Looking more intently, a grayish mass was discerned, blending perfectly with the color of the bush, which outlined itself into the form of a sage hen not 2 feet from my foot. She certainly would have been overlooked had not the movement of her eyelids attracted my attention. I stood there fully five minutes AMERICAN GAME BIRD SHOOTING 278 admiring the beautiful bird, which could have been caught in my butterfly net, then walked back and forth and finally passed around the bush to observe it from behind. Not until then did it become frightened and fly away with a loud cackling. Centrocercus urophasianus. The nest was a depression at the foot of a sage bush, lined with dead grass and sage leaves. The spot was marked and visited several times, always passing within a few feet without alarming the bird.” While the mother bird is sitting, the males are scat- tered over the prairie, two or three often being found together, and when alarmed starting off with heavy, lumbering flight to fly half a mile or a mile. When hatched, the young leave the nest and follow When hatched, the young leave the nest and follow the mother. When quite young they are as expert at hiding as are most of the grouse at this age. They are active and hardy, and for the first few weeks of their life bear a certain resemblance to the young turkey, less perhaps in color than in length of neck and the active way in which they move about on their long legs. The mother is devoted, and Captain Bendire quotes Mr. Wm. G. Smith, who caught six young sage chickens one June in Carbon County, Wyoming, as saying: “The female flew at my legs and followed me 200 yards to where my wagon was standing, constantly making hostile demonstrations, while the young kept calling.” The young families roost on the ground, on the sides of shallow ravines or on the prairie above, and *AaAIng yeosojorg “S ‘Q jo uoTsstused Ag "ISNOID a3BS *AaAIng yeosojorg “S ‘Q jo uoTsstused Ag "ISNOID a3BS *AaAIng yeosojorg “S ‘Q jo uoTsstused Ag "ISNOID a3BS SAGE GROUSE 279 the places where they have spent the night remind one much of the roosting place of a brood of quail. When full-feathered and half grown, they are very gentle and unsuspicious, and it used to be common to see a brood of them walk along feeding before a man who was following them up, trying to shoot their heads off with a rifle or a six-shooter. They paid no attention to the shots, but if one was wounded and fluttered about, all flew without delay. At this age they seem very social, constantly calling to each other with a high-pitched, peeping note, and if one wanders off from the flock or is left behind, he becomes very un- easy and runs hard in his efforts to overtake the family. Centrocercus urophasianus. When full grown, and at the approach of cold weather, the birds become more shy and fly more readily. On flying they give a hoarse cackle, somewhat like that of the sharp-tailed grouse, but deeper. The sage grouse always roosts on the ground, and The sage grouse always roosts on the ground, and I have never seen them in what could fairly be called a tree. Captain Bendire reports having seen them on the horizontal limb of the juniper, about two feet from the ground. A number of years ago, in response to an inquiry from him, I wrote Major Bendire as follows: “On a very few occasions I have seen the sage grouse “On a very few occasions I have seen the sage grouse standing on the branches of a sage bush, sometimes 2 or 3 feet from the ground, but I imagine this is quite an unusual position for the bird. This species commonly, I think, goes to water twice a day, flying down to the springs and creek bottoms to drink in the AMERICAN GAME BIRD SHOOTING 280 evening, then feeding away a short distance, but roost- ing near at hand. In the morning they drink again and spend the middle of the day on the upland. The young birds, when feeding together, constantly call to one another with a low, peeping cry, which is audible only for a short distance. This habit I have noticed in several other species of our grouse, notably in the dusky grouse and sharp-tail. “In western Wyoming the sage grouse packs in Sep- “In western Wyoming the sage grouse packs in Sep- tember and October. In October, 1886, when camped just below a high bluff on the border of Bates’ Hole in Wyoming, I saw great numbers of these birds just after sunrise, flying over my camp to the little spring which oozed out of the bluff, 200 yards away. Looking up from the tent at the edge of the bluff above us we could see projecting over it the heads of hundreds of the birds, and as those standing there took flight, others stepped forward to occupy their places. The number of grouse which flew over the camp reminded me of the old-time flights of passenger pigeons that I used to see when I was a boy. Centrocercus urophasianus. Before long the narrow valley where the water was, was a moving mass of gray. I have no means of estimating the number of birds which I saw, but there must have been thousands of them.” Although the sage hen is a large bird, rises slowly, and lumbers off with a good deal of noise, nevertheless, after they have attained their full growth, it is not always easy to hit these birds when in full flight, espe- cially if going with the wind. SAGE GROUSE 281 Except when in flight, the sage grouse is deliberate in its movements, and I have seldom seen a bird on foot that appeared to hurry. More often they walk delib- erately along, with heads stretched high, watching the intruder until the time comes for flight, when they spring from the ground with the cackling cry already mentioned and soon disappear over the next hill. The flight is often very much extended. The only occasion when I recall seeing a sage grouse run was once when a bird that I had started flew several hundred yards and alighted in plain sight on a hillside on the other side of a valley. A marsh hawk, which was hunting near where the grouse alighted, flew to it and several times stooped at it and appeared to reach for it with its feet. The grouse at once started and ran swiftly along the hillside until it reached some high sage brush, the hawk following and now and then making a clumsy dive at it. THE WiLp TURKEY: Meleagris gallopavo. Meleagris gallopavo silvestris. Meleagris gallopavo osceola. Meleagris gallopavo intermedia. Meleagris gallopavo merriamt. America has given to the world its largest game bird and perhaps most important domestic fowl—the turkey. It is purely American, and its ancestry goes back a long way, for it existed here in far-off Tertiary times, por- tions of the skeleton of a turkey having been found in the Miocene deposits of Colorado, and the bones of other species in the post-Pliocene of New Jersey. Of these last, one was about the size of the existing turkey, but taller, while another was much smaller. At this time, the mastodon lived along the Atlantic coast, while the far older turkeys of Colorado had as associates the huge Brontotherium and many other creatures long extinct. When the white men came to these shores they found turkeys in plenty. The flesh constituted a good share of the food of the natives, who wore cloaks or robes made of turkey feathers. Not very long after the discovery of the New World the bird was taken to 282 282 283 THE WILD TURKEY Europe, and there received a variety of names in dif- ferent countries, most of which referred to India, carry- ing out the early idea that America was a part of the Indies. Thus the bird was called by the English “cock of India”; in French, poule d’Inde, contracted to dinde, WILD TURKEY hen of India; in Spanish, gallo or gallina de India, cock or hen of India; in German, Jndianische Henne or Huhn, Indian hen, and also Calecutischer Hahn or WILD TURKEY hen of India; in Spanish, gallo or gallina de India, cock or hen of India; in German, Jndianische Henne or Huhn, Indian hen, and also Calecutischer Hahn or Henne, cock or hen of Calcutta. It was also called by the Spaniards the Moorish hen, referring to a supposed hen of India; in Spanish, gallo or gallina de India, cock or hen of India; in German, Jndianische Henne or Huhn, Indian hen, and also Calecutischer Hahn or Henne, cock or hen of Calcutta. It was also called by the Spaniards the Moorish hen, referring to a supposed AMERICAN GAME BIRD SHOOTING 284 African origin, while in Egypt the Arabic name is Dik runu, fowl of Turkey. THE WiLp TURKEY: Precisely why it should have been called turkey by Precisely why it should have been called turkey by the English it is hard to say, except that as Turkey was a part of the Far East, it may have been supposed to have some relation to India. It has been suggested that the name by which we know the bird is a corrup- tion of a Hebrew word tukki, said to mean peacock, that this term was applied to the turkey, when it was received in Spain, by the Jews, who then monopolized the business of selling poultry, and that from this point of first introduction the name spread with the bird over a part of Europe. This bird, taken to Europe by the Spaniards soon This bird, taken to Europe by the Spaniards soon after the conquest, was the Mexican turkey. The common wild turkey once found over most of The common wild turkey once found over most of eastern North America was for a long time the only form known in the United States, and this was thought to be the progenitor of all the domesticated races of turkeys. In 1856, however, the English naturalist, Gould, described the Mexican turkey as a distinct species, and much later other observers called attention to a turkey from Florida differing slightly from the ordinary wild turkey, and to yet another different one from the Rio Grande. Later still, E. W. Nelson found that the turkey of Arizona presented constant, if slight, differences from the wild turkey of the plains and that of Mexico, and described it as Merriam’s turkey. To the untrained eye the differences between certain THE WILD TURKEY 285 of these subspecies are slight, and the sportsman will do well to try to learn the geographical ranges of the different forms, for in most cases the locality will be to him a better guide in identifying the bird he kills than will any color description. The Mexican turkey is markedly different from the bird of the East, and the two will readily be distinguished. The differences between the various turkeys are found chiefly in the coloring of the rectrices, or tail feathers, and of the tail coverts and the primaries. THE WiLp TURKEY: The original wild turkey—to which the name Melea- The original wild turkey—to which the name Melea- gris gallopavo was given—has been shown to be the bird later described by Gould as the Mexican wild turkey. It is notably different from the eastern form, for its tail, tail coverts and the feathers of the lower rump are tipped with white or whitish, while the eastern and northern turkey has those feathers tipped with deep rusty or even with rich dark chestnut. The ordinary domestic turkey shows the whitish tippings of the feathers of tail, tail coverts and lower rump— characters derived from its ancestor, the turkey of Mexico. The Mexican turkey occupies the wooded mountain slopes bordering the Mexican tablelands on the south and west, ranging north to Chihuahua, but it does not reach the United States. Mr. Nelson has shown where it grades into Merriam’s turkey on the north, while to the south in southeastern Mexico and Central America, it is replaced by a strikingly dis- tinct species, the.brilliantly hued ocellated turkey. The eastern wild turkey, which was long considered The eastern wild turkey, which was long considered AMERICAN GAME BIRD SHOOTING 286 the true Meleagris gallopavo, thus becomes a subspecies of the Mexican turkey and is now known as Meleagris gallopavo silvestris. It and the closely similar Florida race (osceola) have the ends of the upper tail coverts and tail feathers dark chestnut. The common northern wild turkey has the primaries, or stiff quill feathers of the wing white barred with black, while the Florida wild turkey has the primaries black, with small white bars, which are broken and usually do not reach the shafts of the feathers. These differences are constant, otherwise they would not be regarded as subspecific characters. Down in parts of southern Texas and northeastern Mexico is found another subspecies (intermedia) known as the Rio Grande turkey, or Elliott’s turkey. It looks much like the common ‘wild turkey, but the rump feathers have a coppery bronze bar across them close to the ends, and are tipped with dark yellowish. The tail coverts are chestnut, with narrow black cross bars, and are broadly tipped with buff. The tail is mottled pale chestnut and black, has a black bar across it close to the end, and is tipped with yellowish buff. THE WiLp TURKEY: The difference in appearance between Flliott’s turkey and the common turkey is not easily expressed in words, but any one who has an opportunity to compare two specimens of the same age and sex will readily see that they are not exactly alike. Moreover, the female of the Rio Grande turkey has grayish tips to the feath- ers on the upper part of the body, and thus differs strikingly from any other species of turkey. 287 THE WILD TURKEY From the wild turkey of the East, Merriam’s turkey may be distinguished by the whitish tips of the feath- ers of the lower rump, tail coverts and tail. From the Mexican turkey it may be known by its velvety black rump and the greater amount of rusty rufous, succeed- ing white tips on the tail coverts and tail, and by the distinct black and chestnut barring of the middle tail feathers. Merriam’s turkey thus appears to be about mid- way between the eastern wild turkey and the wild tur- key of Mexico, and in fact the eastern wild turkey grades into Merriam’s turkey, as Merriam’s turkey grades into the Mexican turkey, whose white-tipped tail coverts and white-tipped tail, as seen in the do- mestic turkey, are so characteristic. So in North America we have five forms of turkey, the ranges of which are fairly well defined. The Florida wild turkey (Meleagris gallopavo The Florida wild turkey (Meleagris gallopavo osceola), described by Scott about twenty years ago, is confined to Florida, but the precise limits of its range are not yet known. The type specimen came from Tarpon Springs, Fla. The Rio Grande turkey (Meleagris gallopavo inter- The Rio Grande turkey (Meleagris gallopavo inter- media) was described by the late George B. Sennett in 1892. It is a striking bird, found in the lowlands of southern Texas and also in northeastern Mexico, where it ranges from the coast lowlands up to over 3,000 feet in the adjacent mountains. Merriam’s turkey (Meleagris gallopavo merriamt) Merriam’s turkey (Meleagris gallopavo merriamt) was described by E. W. Nelson in 1900. It ranges AMERICAN GAME BIRD SHOOTING 288 from southern Colorado south through Arizona and New Mexico, and grades into the Mexican turkey on the south and into the eastern wild turkey on the east. THE WiLp TURKEY: On all streams flowing east from the Rocky Mountains over the great plains, from the Niobrara, which is near the northern boundary of Nebraska, south nearly to the Rio Grande, turkeys were formerly common, and these were the ordinary bird of the Mississippi Valley. They lived along these various rivers, many of which have their heads in the mountains, and following up these streams to the mountains, there intergrade with the mountain bird. E. W. Nelson has shown where this takes place. In these days, when the common wild turkey is ex- tinct over much of its former range, it is very difficult to define with exactness the former boundaries of that range. We know that it was abundant in southern New England and to the south. Audubon speaks of it rather vaguely as found in southern Vermont, New Hampshire and Maine, and it is certain that it was once very abundant in Massachusetts, where many years ago I saw a skin taken at Mt. Tom about 1848 or 1840. Wm. Brewster, in his admirable volume on the “Birds of the Cambridge Region of Massachusetts,” published in 1906, as No. IV of the Memoirs of the Nuttall Ornithological Club, has gathered much inter- esting information concerning the turkey in eastern Massachusetts and in Maine. He says: “Morton, referring, no doubt, to his experience at “urpesy SAayINT plIM “ SAayINT plIM THE WILD TURKEY 289 Merrymount, now Wollaston, only a few miles south of the Cambridge region, where he lived from 1625 to 1628, and again in 1629 and 30, says: ‘Great flocks (of turkeys) have fallied by our doores;...I hada Salvage who hath taken out his boy in a morning, and they have brought home their loades about noone. I have asked them what number they found in the woods, who have answered, Neent Metawna, which is a tho- sand that day.’ Wood confirms this by stating that ‘sometimes there will be forty, three score, and an hun- dred of a flocke, sometimes more and sometimes lesse; their feeding is Acornes, Hawes and Berries, some of them get a haunt to frequent our English corne: In Winter when the Snow covers the ground they resort to the Seashore to looke for Shrimps and such small Fishes at low tides. THE WiLp TURKEY: Such as love Turkie hunting must follow it in Winter after a new falne Snow, when he may follow them by their tracts; some have killed ten or a dozen in halfe a day; if they can be found towards an evening and watched where they peirch, if one come about ten or eleaven of the clocke he may shoote as often as he will, they will sit unlesse they be slenderly wounded. These Turkie remaine al the yeare long, the price of a good Turkie cocke is foure shillings; and he is well worth it for he may be in weight 40 pound; a Hen two shillings.’ Josslyn mentions seeing, probably at Black Point (now Scarborough), Maine, “‘threescore broods of young Turkies on the side of a Marsh, sun- ning of themselves in a morning betimes, but this was thirty years since [in 1638 or 1639], the English and AMERICAN GAME BIRD SHOOTING 290 the Indian having now [1671] destroyed the breed, so that ’tis very rare to meet with a wild Turkie in the Woods.’ “That the species was formerly found throughout the Cambridge region, there can be no reasonable doubt. Turkey Hill in Arlington may well have de- rived its name from the presence there of this noble bird in early Colonial days. Indeed, Mr. Walter Faxon writes me that an acquaintance of his has seen ‘in a manuscript diary of the ancestor of an Arlington man ...anentry of killing some Wild Turkeys in the region about Turkey Hill.’ At Concord, less than ten miles further inland, the species had not become wholly ex- tinct at the beginning of the past century. The late Steadman Buttrick of that town, a keen lover of field sports and a man of undoubted veracity, who died in 1874, used to delight in narrating how, when a boy, he had made repeated but invariably fruitless expedi- tions in pursuit of the last wild turkey that is known to have lingered in the region about his home. He often saw the bird, a fine old gobbler, but it was so very wary that neither he nor any of the other Concord gunners of that day ever succeeded in getting a fair shot at it. THE WiLp TURKEY: It was in the habit of roosting in some tall pines on Ball’s Hill, whence, when disturbed, it usually flew for refuge into an extensive wooded swamp on the opposite (Bedford) side of Concord River. Mr. Buttrick was born in 1796. As he was presumably at least twelve or fifteen years of age before he began to use a gun effectively, it is probable that his experience THE WILD TURKEY 291 with the wild turkey happened some time between 1808 and 1815.” The turkey was abundant in the southwestern por- tions of the province of Ontario and occurred through much of New York, in Pennsylvania and Ohio, from which last State it has been exterminated in compara- tively recent years. It is said that a few turkeys still linger in Pennsylvania. Michigan, Wisconsin, south- ern Minnesota and Iowa all once had turkeys enough. They were abundant in Nebraska, reaching beyond the northern boundary of the State, for Captain W. L. Carpenter found turkeys on the Niobrara River, and Dr. Coues speaks of good evidence of their occurrence as far north as Yankton on the Missouri—about the same latitude as the mouth of the Niobrara. It is well understood that the turkey was fairly abundant on many streams flowing into the Missouri or its tribu- taries south of the Platte River, and undoubtedly they worked up many of these streams into the mountains. Indians in whom I have confidence have told me of killing turkeys on tributaries of the South Platte in the mountains west of where Denver now stands. From that point south Merriam’s turkey was undoubtedly abundant in the mountains. The turkey found on the plains to the south of the Platte, westward until the mountains are reached, is presumably the eastern form (Meleagris gallopavo silvestris). Captain L. H. North, who as a little boy moved with Captain L. H. North, who as a little boy moved with his family into Nebraska in the year 1856, says of the Captain L. H. North, who as a little boy moved with his family into Nebraska in the year 1856, says of the AMERICAN GAME BIRD SHOOTING 292 streams in eastern central Nebraska forty or fifty years ago: “There were a good many wild turkeys here on the Loup River, the Elkhorn and Shell Creek when we came here. Ed. THE WiLp TURKEY: Chambers tells me they were often seen on the Niobrara River in early days—say in 1877. I do not recall that any turkeys were seen when the Pawnee scouts were out in 1867, guarding the track layers on the plains toward the mountains, but at that time turkeys were found on the Platte River near old Ft. McPherson—not far below the forks of the Platte.” In August, 1909, Forest and Stream printed a letter from me inquiring as to the western range of the wild turkey. This inquiry brought out some extremely in- teresting information which indicates that the former range of the turkey extended regularly to South Dakota. In my letter I asked what the northern and western range of the turkey was, and whether any of Forest and Stream’s correspondents had ever known of its being found in the Black Hills. In response to this Sandy Griswold, of Omaha, Neb., sent to Forest and Stream a letter, from which I quote the essential para- graphs: “The query whether wild turkeys ever got as far West as the Black Hills I am unable to answer; I do know, however, that no longer ago than 1894 they had found their way as far as the foothills this side of the Black Hills in South Dakota. “T was camped on the Lake Creek marshes that fall, “T was camped on the Lake Creek marshes that fall, duck shooting, and on the third of November Alfred THE WILD TURKEY 293 Reshaw, a young halfbreed Sioux, who was one of our camp helpers and guides, killed a twenty-one-pound black and tan turkey in the scraggy pine hills along White River, twenty miles north of our camp and forty- five or fifty miles this side of the Black Hills. He killed the bird flying, from out of a bunch of five which he had jumped from a patch of ground cherries on one of the bluffs. He knew what the birds were, as he and his brother had killed several the previous winter in the same vicinity. “Two days later Alfred, the late George W. Scrib- “Two days later Alfred, the late George W. Scrib- ner, of San Francisco, and I went to White River, where the Sioux had killed his gobbler, and although we hunted assiduously for hours up and down on both sides of the river, we found no turkey. THE WiLp TURKEY: We did find plenty of sign, however, in almost every rose thicket and among the dried ground cherries from which Alfred had flushed his birds. We found fresh tracks and fresh droppings, showing that the birds had been there after the day the Sioux made his kill. “Along the White River in this particular region “Along the White River in this particular region are extensive fastnesses well adapted to the fancy of wild turkeys, low scraggy acorn-bearing oaks, deep arroyos, with numerous springs, thickets of plum, crab and grape, rose fields, ground and choke cherry patches and many vegetable growths on which the birds feed in the fall and summer.” The Reshaws (Richard) are a well-known family of Sioux mixed bloods, descendants of one or more French AMERICAN GAME BIRD SHOOTING 204 Creoles who served the American Fur Company about 1850 or earlier, and who married Sioux women. Through the kindness of Colonel Hugh L. Scott, superintendent of the U. S. Military Academy, I am enabled to give the most northerly definite record of the wild turkey on the Missouri River of which I have any knowledge. Colonel Scott, learning of my interest in this subject, recalled that more than twenty years ago General D. L. Magruder, U. S. A. (retired), had told him of killing wild turkeys near Fort Ran- dall, Dakota, in 1855. He therefore wrote to General Magruder and received from him a letter dated Sep- tember 6, 1909, as follows: “From July, 1855, to October, 1860, I was stationed “From July, 1855, to October, 1860, I was stationed at the different garrisons along that stream [the upper Missouri River] from old Fort Pierre Chouteau to Fort Randall. “From July, 1855, to October, 1860, I was stationed at the different garrisons along that stream [the upper Missouri River] from old Fort Pierre Chouteau to Fort Randall. “On December 17, 1855, I accompanied General Harney upon a hard winter’s march, from Fort Pierre Chouteau to the mouth of the Niobrara River. The march was by land as far as the present site of Fort Randall, where we were compelled by heavy snowdrifts in the ravines to abandon the prairie and take to the ice upon the river, where the march was continued, both going and returning, until our arrival back at Fort Pierre, February 17, 1856. THE WiLp TURKEY: “During the trip, both going and returning, I killed “During the trip, both going and returning, I killed deer, rabbits, grouse and turkeys to supply our mess, finding each of the kinds of game in plenty and quite fat in most of the heavily timbered points along both “During the trip, both going and returning, I killed deer, rabbits, grouse and turkeys to supply our mess, finding each of the kinds of game in plenty and quite fat in most of the heavily timbered points along both THE WILD TURKEY 295 sides of the river. The turkeys were particularly fine- flavored, their food being abundant, consisting mostly of wild grapes, rose apples (the seed pod of the wild rose), cottonwood buds and hackberries, the latter ap- parently their favorite, at least to judge by the quantity contained in their crops. “The last turkey killed by me was at a return camp “The last turkey killed by me was at a return camp about thirty miles above Fort Randall. Beyond that point I have no personal experience, but while stationed at Fort Pierre I was told by the interpreter of the fort that turkeys formerly were quite abundant in the heavy timber about the mouth of the Big Cheyenne River about thirty miles above.” The statement made by the interpreter at Fort Pierre The statement made by the interpreter at Fort Pierre furnishes quite satisfactory evidence that turkeys were once found on the Missouri River as far north as the mouth of the Cheyenne River. Colonel Scott has also called my attention to the diary of Lieut. Rufus Saxton, printed in Vol. I, Pacific R. R. Reports, 1853-4, which says of Cedar Island, on the Missouri River, below Ft. Pierre: “Saw wild turkeys for the first time. They are seldom seen above this point, and have never, I believe, been found beyond the Rocky Mountains.” The reference, of course, is to the northern Rocky Mountains. THE WiLp TURKEY: Alexander Henry, the Younger, states that in 1806 Alexander Henry, the Younger, states that in 1806 the Cheyenne Indians coming up from the south brought with them the tails of turkeys which the Man- dans and Minitari greatly desired for use as fans and for which they traded, and from this we may infer that AMERICAN GAME BIRD SHOOTING 296 there were no turkeys found on the Missouri or its tributaries as far north as the then location of those tribes—Knife River. I suspect that in the Rocky Mountains turkeys sel- dom or never crossed the divide between the north and south forks of the Platte, and that they never got as far west as the Black Hills. No bird is more gentle and unsuspicious than the turkey until it has learned that man is an enemy, and after that no bird is more wary and alert. Mr. Hen- shaw speaks of the lack of suspicion on the part of these birds, and Captain Carpenter tells how—when his com- mand was preparing to establish Fort Niobrara—a wild turkey came to the camp and ventured out of the under- brush to feed on the grain spilled by the animals. When Florida first began to be a resort for northern tourists, turkeys were very abundant and not at all shy or suspicious. They often associated with the domestic turkeys, and one wild gobbler became so tame that he would feed unconcernedly within a few feet of a man. On this point Dr. Ralph, writing about 1890, said to Captain Bendire: “One can hardly believe that the wild turkeys of “One can hardly believe that the wild turkeys of to-day are of the same species as those of fifteen or twenty years ago [that is, 1872 to 1875]. Then they were rather stupid birds which it did not require much skill to shoot, but now I do not know of a game bird or mammal more alert or more difficult to approach. THE WiLp TURKEY: Formerly I have often, as they were sitting in trees on the banks of some stream, passed very near them, “suryynyys ‘AayIny, pli “s ‘AayIny, pli THE WILD TURKEY 297 both in our boats and in steamers, without causing them to fly, and I once, with a party of friends, ran a small steamer within twenty yards of a flock which did not take wing until several shots had been fired at them.” The turkey, while usually resident in a certain sec- tion, is yet said to be prone to wander, and to be by no means as local in its habits as the bobwhite or the ruffed grouse. Sometimes they will remain in a desira- ble location for a long time and then will leave it—for no apparent reason. On the plains the birds used to spend the night roosting in the trees of the bottoms, and after drinking in the morning would wander up on the prairie about the heads of ravines and there feed on grasshoppers and other insects and on sand cherries and tunas, returning in the heat of the day to the shade of the underbrush or even of a cut bank. Turkeys feed chiefly on vegetable matter. In old times the saying, that a good mast year was a good turkey year, passed into a proverb. They eat beech- nuts, chestnuts, various acorns, pecan nuts, persim- mons, the fruit of the cactus, all sorts of wild berries or seeds and grains and other vegetable matter, besides all insects. In the central and southern Rocky Moun- tains the fruit of the pifion forms a large part of their subsistence. As determined by the Biological Survey, the turkey’s food consists of 154 per cent. of animal matter and nearly 84% per cent. of vegetable matter. Of the vegetable matter, buds and leaves constitute nearly 25 per cent., fruit nearly 33, and other seeds AMERICAN GAME BIRD SHOOTING 2098 nearly 20 per cent. In the western country turkeys are great eaters of grasshoppers. They also destroy the tobacco worm and moth when they can get them. The breeding season for the turkey ranges from February to May, according to the latitude which the bird inhabits. At the breeding time, and, indeed, throughout the year until mating in the early spring, the hens and young birds associate together and apart from the gobblers. THE WiLp TURKEY: At mating time the gobbler’s actions are those of the At mating time the gobbler’s actions are those of the domestic turkey. He gobbles loudly, struts and spreads his tail, drags his wings on the ground and puffs him- self out until he has made the proper impression on the hen. Often several birds are going through this performance about a single hen, and fights between the males are common, and, it is said, sometimes with fatal results. The nest is a mere hollow scratched in the ground, lined or not lined with straws, grass and a feather or two. The eggs vary in number from eight to four- teen. Captain Bendire reports a case where there were twenty-six eggs in a nest, but two hens were at the nest, one sitting on the eggs and one standing close by them. It is likely, therefore, that occasionally two hen turkeys share a nest, as two quail sometimes do. Like many ground-nesting birds, the turkey is ex- Like many ground-nesting birds, the turkey is ex- ceedingly hard to see when on her nest, and of the turkey, as of other birds, various instances of this have been related. Captain B. F. Goss, writing May, 1882, in southern Texas, says: THE WILD) TURKEY 299 “We were encamped quite near the nest; one morn- ing I noticed a hen turkey stealing through the bushes and suspected she was going to her nest. We watched her carefully for three mornings, and having pretty nearly located the nest, commenced a close search, and examined, as we thought, every inch of ground. I was about giving up, when looking down almost at my feet, I saw the bird sitting on the nest. She at once ran; she had allowed me to pass several times within a foot of her without moving, and seemed to know at once when she was seen. THE WiLp TURKEY: I have often noticed this trait in birds of this genus; as long as unseen you can tramp all around them, but they seem to know at once when they are seen and lose no time in getting away.” As long as the hiding bird, which sees and hears its pursuer, is convinced that he is still ignorant of its position, it feels safe, but the moment it recognizes by the expression of the man’s eye that its hiding place has been detected, it is off without delay. The eggs of the wild turkey are not at all unlike The eggs of the wild turkey are not at all unlike those of the domestic bird. Usually they are cream- colored, dotted with finer or larger spots of reddish brown, chocolate and sometimes lavender. Captain Bendire states that the spots are more often very small and fine than large. The young follow the mother as soon as hatched, but The young follow the mother as soon as hatched, but Audubon says, “As the hatching generally takes place in the afternoon they frequently return to the nest to spend the first night there.” AMERICAN GAME BIRD SHOOTING 300 The young are believed to be very tender and subject to many dangers from dampness. Some writers de- clare that the mother leads them on high ground for the first week or two of their life in order that they may escape the dangers of dew or rain from the grass. Audubon says: “To prevent the disastrous effect of rainy weather the mother, like the skilful physician, plucks the buds of the spicewood bush and gives them to her young”! The little birds are able to fly at about two or three weeks old, and soon after that leave the ground and roost on the low branch of a tree sheltered under their mother’s wings. When danger threatens, the mother turkey, like many other gallinaceous birds, calls to her young, which at once crouch and hide and cannot then be seen. It is said that if the male turkey finds a nest of eggs It is said that if the male turkey finds a nest of eggs upon which the hen is sitting he will destroy them, and that if he comes upon a brood of newly hatched young he will kill them. THE WiLp TURKEY: It is certain that during the autumn and winter the young birds and the females associate together, while the old males keep by themselves and do not begin to seek the society of their mates until the approach of spring. In the Rocky Mountains the nests are built at an al- In the Rocky Mountains the nests are built at an al- titude of from 3,000 to 5,000 feet, but as the weather grows warmer and the snow disappears, the old hen leads the young up to the higher mountains, so that they finally summer at from eight to ten thousand feet. In the late autumn, when the weather grows cold and snows come on the mountain ranges, the birds move THE WILD TURKEY 301 down again to sheltered cafions or timbered river val- leys, where they spend the winter. In the southern States turkeys have always been In the southern States turkeys have always been abundant and their stronghold is still there—parts of Virginia, the Carolinas, Georgia, Alabama, Mississippi, Louisiana, Arkansas, Missouri and Texas. Merriam’s turkey is said to be almost extinct in Colorado, but is still abundant in Arizona and New Mexico. That there should be occasional outlying colonies of a few birds in Iowa and Nebraska, such as Mr. Griswold is told of, seems very surprising, but such colonies cannot last long unless protected by the owners of the land on which they live. The turkey, grandest of game birds, has been ex- terminated over much of its former range. Great in size, and valuable for food, he is an object of pursuit wherever found. So, throughout the farming country of the North and West the turkey is gone and gone for- ever. As the country is settled up, is his complete ex- termination to follow? Domesticated, he will always survive, but should we not strive to retain the old wild turkey of the eastern States in his untamed wild state, self-dependent, one of the typical and interesting in- habitants of our primitive forests and our far-stretch- ing southern plains? PART. 3 UPLAND SHOOTING sash hours Cassia Pertes.. AHOEN SCO BALTIMORE hours Cassia Pertes.. From Bull. 21, Biological Survey, Dept. of Agriculture, By Permission. WOODCOCK SHOOTING. Many years ago I contributed to the Century Maga- gine an article on this species, and from it, by the kind permission of the Century Company, I extract a few paragraphs on some details of the woodcock shooting of those days. Birds were plenty then, as we may hope that some day they will be again, if wise measures are taken for their protection. The woodcock is not often seen and is quite con- The woodcock is not often seen and is quite con- tented to be overlooked. He has no brilliant song to catch the ear, no gaudy plumes to attract the eye, nor does he perform graceful evolutions high in the air in the broad glare of day. He is truly a modest fowl, and except at night, or during the twilight of morn- ing or evening, he does not willingly venture into sit- uations where he can be viewed by the casual wan- derer through field or wood. One who desires to make his acquaintance must penetrate into the depths of the most tangled swamps to find him at home. Even here, during the day, he is usually half asleep. Not so drowsy, however, as to be unaware of the approach © 305 305 305 AMERICAN GAME BIRD SHOOTING 306 of an intruder. The soft rustling of the leaves, the occasional snapping of a dry twig, and the sound of the heavy footfall rouse him from his doze or his day dream. He moves sideways beneath the spread- ing leaves of a tuft of skunk cabbage, and with head turned on one side, and great eyes spread to their wid- est, watches for the approaching form. Once in a while something may cause him to take the alarm and dart away before it is within sight; but usually he lies close, and when he rises it is near at hand. He springs from the ground uttering a shrill, twittering whistle and twists about in his upward flight to dodge the branches which spread in a network above him, until he has topped the undergrowth, and then darts off in a straight line for fifty or a hundred yards, to plunge once more into his beloved cover. The dog is a most important auxiliary in woodcock The dog is a most important auxiliary in woodcock shooting. WOODCOCK SHOOTING. A very few sportsmen employ cocker or field spaniels, which are trained to range close to the gun, and to give tongue as soon as they strike the scent, thus warning the shooter of the proximity of the bird and preparing him for its possible rising. But most men use the setter or pointer. A good wood- cock dog should work close; that 1s, within sight of the gun. Often where the undergrowth is very thick, it becomes necessary to attach a bell to the dog’s col- lar, so that if it pass out of sight for a few moments its whereabouts may still be known by the sound. Late in November you will still find a few birds, and at this time they will be lusty and strong of wing, ‘spilg oures) BulysIUeA OMY ,, SJOUSIYy “MV Iq wor “a *SUIYINOIN YIOIPOOM ‘spilg oures) BulysIUeA OMY ,, SJOUSIYy “MV Iq wor “a *SUIYINOIN YIOIPOOM WOODCOCK SHOOTING 307 and will test your skill. Cross the meadows then and go down into the swamp, working along near the edge, where the underbrush is not too thick, and the soil under the leaves, as you can see in the cattle tracks, is rich and black. Just beyond you, on the left, a steep hillside rises sharply from the edge of the swamp, its surface overgrown with low cedars, sumacs and bay- berry bushes. The old dog comes out of the swamp and turns toward the slope, and as he crosses before you glances back inquiringly. He knows the hillside, and understands as well as you do that a cock is usu- ally to be found on that warm southern exposure at this time of the year. No need to wave the hand or use any elaborate signal to tell him to work up among the cedars and through the brush. A little sidewise movement of the head, and he is breasting the steep ascent and rustling among the twigs and the crisp leaves, while you walk along a cowpath at the foot of the slope. If there be a bird there it will be sure to fly toward the swamp, and must therefore cross in front of you. WOODCOCK SHOOTING. For a few moments you hear the dog as he works above you; then the sound ceases, and as you pause to listen for it there comes to the ear that shrill whistle, so like the midsummer twitter of the kingbird, that warns you to “mark cock.” You see a brown flash among the green cedars, and the bird darts out to plunge into the swamp; but as he sees you he turns sharply and flies down the path, straight away. You have plenty of time; bring up your gun deliberately, cover the bird, and when it is AMERICAN GAME BIRD SHOOTING 308 about thirty yards distant fire, and it is yours. At the report of the gun your dog appears on the bank above, pauses a moment until you have slipped an- other cartridge into the gun, and then dashes off. to- ward where the bird lies. A word steadies him as he approaches it, and after quartering once or twice the scent reaches his nostrils. He feels for it, then pauses, and at command steps forward, gently takes the bird in his mouth and trots slowly toward you, expressing as much pride and satisfaction in his face and in his slowly wagging tail as if he had captured the prize without any assistance of yours. On again, along the border of the swamp, sometimes stooping low to pass beneath the tangled underbrush, or forc- ing your way through the thick alders, making the dead stems crack and fly, or passing through a spot free from low shrubs, where the tall, gray trunks of the hardwood trees stand apart, and the footfall is scarcely heard on the damp, dead leaves. For some time the dog works quietly ahead of you, manifesting none of the signs which would lead you to suspect that birds were near; but as you approach a little arm of the swamp which runs up a narrow ravine, the merry action of the setter’s tail warns you to be pre- pared for a point. Yes, there, where the wind has swept aside the leaves, exposing the black mud _be- neath, you see in it dozens of little round holes, which tell you that the long bill has been at work here. Sud- denly he stops, and stands quite still, except that the tip of his tail moves a little from side to side. WOODCOCK SHOOTING. As WOODCOCK SHOOTING 309 you step up to him he moves again, very slowly and cautiously, and then suddenly stops and remains mo- tionless. It is a pretty picture, and one that the sports- man never tires of watching and admiring. The dog’s fore foot is raised in the act of stepping, his tail is straight and rigid, head a little above the line of the back and slightly turned to one side, ears a little pricked. Walk up beside him and look at his face, and you will see, what his attitude already indicates, that he is laboring under strong excitement. His nose is perhaps within a few inches of the bird and the scent is strong. You will see his eyes roll as he looks over the ground before him. His forehead is knotted into a frown, which shows how thoroughly in earnest he Tse The bird is likely to fly up from beneath the dog’s nose, so close to you that you cannot shoot without run- ning the risk of either missing altogether or else blow- ing him to fragments, and will then, perhaps, dart be- hind a thick cedar or twist into some alders through which you can hardly see to shoot. The alder runs, so numerous through the New Eng- The alder runs, so numerous through the New Eng- land States, are most satisfactory places to work for woodcock. These are usually the channels of little brooks a few feet below the general level of the open meadows through which they pass. The ground is too damp to be successfully cultivated and the farmer gives it up to the black alder, which attains a height of from fifteen to twenty feet. Beneath these in the wet, springy soil, the skunk cabbage (Symplocarpus), AMERICAN GAME BIRD SHOOTING 310 a variety of ferns, and many other moisture-loving plants, grow in wild luxuriance. These runs, or swales, are often so narrow that the best way to hunt them, if two are shooting together, is for each to take a side and let the dog work between them. The birds, when started, will either show themselves above the alders, or, what is more likely, will break out on one side or the other, and fly forward along the edge of the bushes, giving a perfectly open shot and one which not even a tyro ought to miss. WOODCOCK SHOOTING. In working out such places the bell should be put on the dog, for it is often so dark beneath the thick growth that it is difficult to see him. Should he come to a point, and the bird decline to rise, a heavy stick or stone thrown into the bushes just in front of him will often flush it. One of the most instructive articles on woodcock One of the most instructive articles on woodcock shooting ever written is from the pen of Mr. B. Wa- ters and was published in Forest and Stream in the year 1903. Iam glad to be able to reproduce it—with a few minor changes—in these pages. Of all the kinds of shooting of field and forest the Of all the kinds of shooting of field and forest the sport of woodcock shooting holds the warmest place in the hearts of its devotees. The woodcock shooter is an enthusiast of enthusiasts. He may take a keen pleasure in bringing other game birds to bag, but when woodcock shooting is under consideration comparison ceases. And indeed this sport possesses many fas- cinating features peculiar to itself. First of all, it can at best be indulged in only in very small quantities. There is but little of it when compared with the abun- WOODCOCK SHOOTING 311 dance of other kinds of shooting, for the woodcock is comparatively a rare bird and its season is a short one; therefore the keen edge of enjoyment of woodcock shooting is never dulled by surfeit. The habitat of the bird is distinctly different from the home of all other game birds, and on the earth’s surface only tiny spots here and there meet the wants of its nature, and many vast tracts of fertile country have no wood- cock ground at all. It, too, is a bird of mystery, of whose coming and It, too, is a bird of mystery, of whose coming and going no one knows. It is nocturnal in its habits, and its haunts have been such secluded and unused spots —rarely invaded by man—that it is seldom seen. WOODCOCK SHOOTING. The residents of sections wherein is the home of the wood- cock may never see one from year’s end to year’s end, and, indeed, may go through life with no more knowledge of them than that derived from hearsay ; or, seeing one, may still remain in ignorance of its identity. While the quail, the partridge, the snipe, and other game birds are not unfamiliar to country residents and are readily identified by them, the wood- cock and its doings are shrouded in mystery. The large woodpecker in some sections is called woodcock by the country folk, while in other sections any plover which has a long bill is often called by the same name. So little is the bird known, that sometimes when killed it is called snipe, and sometimes the snipe is called woodcock, by those who have not given the bird spe- cial study or attention. Its life being so entirely with- out the sight of man and in general so little being AMERICAN GAME BIRD SHOOTING 312 known of it, it is not strange that the little accurate knowledge is obscured by the air of much mystery, and that those who seek the bird find a fascination in it greater than that of any other form of game bird shooting. The bird itself is of peculiar form and of rare richness in its colorings, and its flesh is esteemed a morsel of choice excellence fit for the palate of the most fastidious epicure. Thus it affords great sport in its capture and is pleasing to the eye and to the palate. The scarcity of the bird, its beauty, and the delicate flavor of its flesh, all serve to enhance its value, and its mystic life adds a charm to its pursuit which is distinct from all others. Its home is generally in densely shaded nooks in Its home is generally in densely shaded nooks in out-of-the-way places where man rarely enters and where the soil is soft and moist, for in such places is its food obtained. The alder runs and slopes in the birches and nooks in the woods where springs or rivu- lets or excessive moisture makes the ground soft, are its favorite haunts, and sometimes in certain sections it finds spots in the cornfields which are desirable feed- ing grounds, though haunts and feeding grounds are never plentiful. WOODCOCK SHOOTING. In Mississippi it is occasionally found in open sedge fields. Many places which to the eye have every appearance of being a suitable home for it, still have no birds in them. The difficulties of woodcock shooting have been The difficulties of woodcock shooting have been greatly exaggerated, particularly as concerns the ex- traordinary skill required by the shooter, and the still WOODCOCK SHOOTING eiiie, more extraordinary labor, and consequent fatigue, im- posed on the dogs, the latter being an indispensable factor in the sport if any success worth considering is sought. While intrinsically the sport possesses all the requirements of the highest degree of wing shooting, the writers on it have deemed it fitting that it be dressed in a glamor of romance, presumably that a lit- tle knowledge might be presented in an elaboration of high colors which touched on the sky, the sunshine as it glinted through the alders, the beautiful color of the foliage, the balmy zephyrs laden with nature’s per- fumes, ad infinitum, all of which are present in all other kinds of shooting, or, indeed, present if there is no shooting at all. The shooting of woodcock is diffi- cult, it is true, but not so extraordinary in its diff- culty as to be distinctly special, and far from being so difficult as most shooters make it from injudicious selection of guns, loads, etc. Woodcock shooting is close shooting, the closest of Woodcock shooting is close shooting, the closest of any kind of shooting recognized as legitimate sport with the shotgun. While the woodcock is called a game bird, it is gentle and mild in its habits, with none of the pugnacity or extraordinary vitality pos- sessed by members of the grouse family. The small- est of shot is sufficiently heavy to kill it, and the cylin- der-bore gun is amply close enough for the ranges which one must accept in shooting it. The choke bore of any kind is out of place in such extremely short ranges, and unfit to use on a bird so easily killed; though, strange to say, the use of it is not uncommon, AMERICAN GAME BIRD SHOOTING 314 owing, no doubt, to the fact that many men, owning but one gun, must use it for all kinds of shooting, and in other instances to the further fact of thoughtless- ness concerning the proper requirements of the sport. WOODCOCK SHOOTING. Short barrels, too, are desirable, the difference in the handling of a 30-inch barrel and a 24-inch barrel in cover being far away in favor of the latter. Woodcock shooting is largely a matter of snap Woodcock shooting is largely a matter of snap shooting; therefore, a wider range at a much shorter distance is a requisite if one is cultivating success in- stead of nursing a fad in respect to the use of choke- bore guns for all kinds of shooting, whether the guns be fitting or otherwise. In the shooting of quail or chickens or ruffed grouse—to a lesser degree with the latter—a certain degree of deliberation and quick aim can be practiced, but in woodcock shooting the oppor- tunities for deliberation are the rare exception; hence the need of adopting an. open gun to meet the require- ments of quicker work and short ranges. Light loads and smaller shot can be used successfully, some noted shooters using dust shot exclusively. With a short cylinder-bore gun—a true cylinder bore, not the modi- fied choke bores, which are often called cylinder—such a pattern can be secured at 15 or 20 yards as will insure fair success to the average shot and the best of success to the good one. It might be said that such a gun and load are too murderous, and indeed they would be in the hands of a man who could shoot with any degree of precision if he could exercise de- liberation; but as in the greater number of instances WOODCOCK SHOOTING 315 the shooter has but an instant in which to act, the results are far from being so fatal as one might fancy them to be. Often there is but a momentary glimpse of a dusky shadow flitting through or across a small vista in the dense growth, and the shooter must fire then or not at all, unless he is pleased at a purpose- less tumult, that being the sum total when he shoots and trusts to luck for the execution of his purposes. WOODCOCK SHOOTING. As in all other shooting, experience enables the As in all other shooting, experience enables the sportsman to recognize the promising nooks for wood- cock and the signs which denote its presence, these being the holes made by it in boring in the ground for its food, and other indications well known to the shooter, and which can only be recognized by experi- ence. As to the labor and fatigue imposed on the dog As to the labor and fatigue imposed on the dog while seeking for woodcock, they are largely an ex- aggeration. Wilson, in his work on the birds of North America, specifically mentions the fatiguing efforts which the dog encounters in woodcock shooting, and mentions that relays of dogs are necessary. As a matter of fact, the work of the dog in woodcock shoot- ing is the easiest of all kinds of shooting. He must range close to the shooter—at most not beyond a gun- shot—if he is to serve the best purpose in that kind of shooting and it is not at all essential or desirable that he work at high speed. It is essential, however, that he be intelligent, and know thoroughly the best manner of working to the gun and assisting the shooter to get his shots in a manner to insure success. He AMERICAN GAME BIRD SHOOTING 316 should work diligently, but not hurriedly, and it is hardly necessary to add that the work should be done as silently as possible, though this also is true of all other kinds of shooting. The dog should be a good retriever, otherwise a The dog should be a good retriever, otherwise a large percentage of the birds will be lost, for many times it is as difficult to find the bird after it is killed as it is before. Dogs which run riot in this shooting can soon tire themselves out, particularly in summer shooting, when dogs are out of condition, and the weather warm, but the consequent fatigue from such overexertion and unfit condition cannot be justly at- tributed to the difficulty of the sport. It is rather hard work for the shooter, particularly him of the North, where the quest must be made afoot, and where the footing is difficult and insecure, though after all it is but little more difficult than any other shooting in which the shooter walks. WOODCOCK SHOOTING. As the dog often comes to a point in thick cover As the dog often comes to a point in thick cover out of sight of the shooter, even though the point may be but a few steps away from him, a bell attached to the dog’s collar has been found of great assistance in determining his whereabouts, and its silence indicates when he stops on point, a matter very essential in con- ducting the sport. Not every dog is a good wood- cock dog, even though he may be excellent on quail, snipe, chickens, etc. Some dogs appear to dislike the work intensely, others refusing to recognize the bird at all. A few take to it very kindly, and work to the gun from observation to a useful degree far above WOODCOCK SHOOTING 317 what could be established by the most careful train- ing. The spaniel is but little used in the United States for woodcock shooting, or any other kind of shooting, for that matter, though there is no doubt but they could be made eminently useful in field sport. In Louisiana, and other sections of the South, where the woodcock seeks a clime more genial than that of a northern winter, the conditions of shooting change almost entirely. In sections at certain times, gener- ally in the last of December and the fore part of Janu- ary, they may be found in great numbers, and a bag of twenty, thirty or forty in a day is not then consid- ered remarkable. They frequent the switch-cane bot- toms, or woods in the timbered prairie, in which the heavy fall rains have softened the ground, and where abundance of food can be found. Their stay in the South is very short, for they start North immediately on the lessening of the winter cold—after a stay of not more than a few weeks—their coming and going then being quite as silent and secret as in the North. They are then killed in great numbers, both day and night, by market shooters, and shipped to the home and dis- tant markets. They have their choice feeding grounds even in that land of abundance, and skill, diligent ef- fort and knowledge of habitat are quite as essential to success in the southern winter shooting as in the less bountiful shooting of the North in summer and fall. WOODCOCK SHOOTING. So scarce has the woodcock been for the last dozen years that some young gunners have never seen one, and know them only from books. Happily, for the So scarce has the woodcock been for the last dozen years that some young gunners have never seen one, and know them only from books. Happily, for the 318 AMERICAN GAME BIRD SHOOTING past two or three years they seem to have been rapidly increasing, a result, no doubt, of their protection in summer over most of the country. It is interesting to recall the days of woodcock shoot- It is interesting to recall the days of woodcock shoot- ing a generation ago and to compare their results with those of recent times. For the past few years many a gunner who devoted a week or ten days to faithful tramping and shooting in eastern covers has thought himself fortunate if in that time he killed three or four woodcock. An item published in Forest and Stream in 1874 speaks of three gunners who went out early in the summer season and in one day killed sixty- four woodcock. Twenty, twenty-five and thirty a day were ordinary records for a single gun, and in No- vember, 1876, we recall an account by a friend of a day’s shooting which yielded him twenty-six woodcock besides a less number of partridges and quail. SNIPE SHOOTING The snipe is notorious as an uncertain bird, and snipe shooting as a sport that can never be depended on. In old times one used to walk mile after mile in the hope that some of the birds might be started from some favorite bit of feeding ground, but too often only dis- appointment and weary leg muscles rewarded the en- thusiastic tramper. On the other hand, snipe were occasionally found in great abundance, and could hardly be driven away. Recollections of occurrences of both kinds are laid away in the memories of all of the older gunners. Near Vincennes, Ind., many years ago, there was a famous feeding ground for snipe to which, during mi- gration, the birds resorted in great numbers. Thither I went one season with a companion to whom the grounds were well known, and there I saw more snipe han at that time I supposed existed. We were driven to the edge of the marsh, and there, as the team drew up and the wagon cramped to let us jump out, a dozen snipe rose almost under the horses’ hoofs, flew fifteen or twenty yards and alighted. Disregarding the advice of my friend, I had brought Disregarding the advice of my friend, I had brought a dog with me, the best on partridges that I had ever seen, but he proved absolutely useless here. The birds 319 319 AMERICAN GAME BIRD SHOOTING 320 were so many, the scents so numerous and confused, that the poor old dog entirely lost his head and gal- loped about aimlessly until called in and made to fol- low at heel. The birds got up, a dozen at a time, flew a few yards ahead and alighted, and then a number of others got up and did the same thing. The report of the gun put a considerable number on the wing, yet at first did not seem to make the birds wild. Mean- time I could hear my companion, who had gone off by himself, shooting very regularly, and felt that his greater experience was now standing him in good stead, and that he must be acquitting himself very much bet- ter than I was. For the multitude of the birds, and the way in which they were rising on every side, con- fused and unnerved me almost as much as it did my dog. SNIPE SHOOTING Although that day I killed more snipe than ever before or since, yet I do not look upon it as one of the shooting days especially worth remembering. The birds were too many, and I was not in condition to take advantage of my opportunities. I have had more satisfaction from a single ruffed grouse, neatly killed as he darted away through the thick underbrush of the swamp, than I did in that heavy bag of snipe, where I loaded and fired all the afternoon, until my ammu- nition was exhausted. Some such feeling as this, I believe, animates most sportsmen when they reach grounds where birds are so numerous that all the uncertainty of shooting is taken away. Few of us, I think, care greatly to catch SNIPE SHOOTING 321 brook trout—strong and gamey though they may be— out of a preserved pond. Something of this sort Mr. Waters expresses in Something of this sort Mr. Waters expresses in the admirable article on snipe shooting which follows this. He has tramped the marshes where snipe were scarce, and again where, as in Louisiana, they were enormously abundant, and he, better than most gun- ners, knows the oddities and eccentricities of this re- markable bird. Mr. Waters’ article, with slight changes, says: According to the writings of ornithologists, According to the writings of ornithologists, the breeding grounds of the snipe begin on their south- ern boundary, at about 42 degrees of latitude, a paral- lel through the northern part of Nebraska, Iowa, etc. The grounds extend thence north to the Arctic Circle. The snipe migrate leisurely southward as the winter season approaches, tarrying on the available feeding grounds, ultimately going as far south as the West Indies and northern South America. It is a bird of the wet lands, and as said of the woodcock, the available area affording its food sup- ply is small as compared with the earth’s surface. Relatively, the places which are soft enough to be bored with its sensitive bill, which contain food to its liking, and enough to supply its needs, are exceedingly lim- ited in number and area. SNIPE SHOOTING Soft and wet land may also be gravelly or sandy or Soft and wet land may also be gravelly or sandy or clayey, and therefore unfit to sustain the animal and vegetable life on which the snipe subsists; or from its refractory nature it may be impervious to the deli- AMERICAN GAME BIRD SHOOTING 322 cate weapon with which nature has provided the snipe for the capturing of its food. It therefore is appar- ent that of all the wet lands there are only certain parts which contain snipe food. Of the places which afford snipe food some are good Of the places which afford snipe food some are good throughout the whole season, as, for instance, the sloughs and marshes and parts of river valleys of the prairie country wherein it makes its summer home. Other places are but temporarily available, as lands made soft and wet by heavy rains. Such places may serve it well for many weeks, as in Louisiana and Texas in the fall and winter months, during the rainy season, which in those States is largely the equivalent of winter. Again, the snipe may seek its food in places which are quite wet, as in some of the large wet marshes, and again, in some other sections, it may make its haunts on upland so firm that the hunter may walk thereon pleasantly and dry-shod. While the woodcock, its long-billed confrére, is a While the woodcock, its long-billed confrére, is a bird of the covert, the snipe is a bird of the open. On these birds nature lays a more severe restriction con- cerning a late stay in the North than she does on any other game bird, for a snipe or woodcock attempting to gain a subsistence in a frozen country is in a pa- thetic situation indeed. Its food is said to be larve, tender roots of plants, and worms, which it secures by boring, and also such insects and other edible food as it can secure on top of the ground. To the local sportsman the snipe’s habits in the SNIPE SHOOTING 323 shooting season—which is mostly the migratory sea- son—seem erratic and unknowable, if its unstable char- acteristics may be called habits at all. It is in one place to-day, and to-morrow in another. To-day there may be an abundance, to-morrow a dearth. SNIPE SHOOTING Or it may go contrary to its erratic reputation and remain a num- ber of days about the same grounds. Still, the shooter is largely in ignorance of what the snipe will do next. The weather and food conditions may be the same, so far as observation can determine them, and yet the birds come and go in their own whimsical way, regardless of conditions. Some subtle, mysterious impulse seems to impel the birds of a certain locality either to come or go, though not in the manner of birds which flock. Snipe fly mostly in ones or twos or threes, some- times more, but always in small numbers. Being inde- pendent in flight, it is difficult to uaderstand how the common impulse to seek other grounds is at the same time felt and acted on by all the snipe of a certain neighborhood, or at least by most of them. There are many exceptions, as a matter of course; as, for instance, in a section where there are snipe in abun- dance on a certain day, a part only may leave at the same time. Indeed, a few snipe may be found on cer- tain grounds throughout the whole season. Yet, how- ever much the exception may affect the rule, the greater part of the birds are erratic and lawless most of the time. No doubt that which seems whimsical and mysteri- No doubt that which seems whimsical and mysteri- AMERICAN GAME BIRD SHOOTING 324 ous in the life of the snipe is really in harmony with the needs of its nature. As it is nocturnal in habit, it is difficult to study, and it is specially difficult for the resident of one locality to observe its general habits with any degree of precision. Seeing it in but one small corner of its habitat, the local sportsman can gain, at best, but a fragmentary knowledge of its needs and its habits. Being swift of wing and enduring of flight, the snipe undoubtedly feeds over vast areas of ground many miles apart, twenty or thirty miles of flight being of no more effort to it when in search of food than twenty or thirty rods are to the prairie chicken. SNIPE SHOOTING When snipe invade feeding grounds in vast numbers, as is frequently the case, the grounds are soon thoroughly bored, and all the food within reach is consumed; thus it may be a necessity for them to seek food else- where till the exhausted grounds have time to replen- ish themselves. Many writers lay great stress on the difficulties of Many writers lay great stress on the difficulties of snipe shooting. They treat it as a bird of phenomenal swiftness and erratic flight, and the shooting of it as requiring something extraordinary in the matter of skill. As a matter of fact, snipe shooting, at certain times, is the easiest of shooting. On warm days, when the birds are fat and lazy, flying slowly and tamely, with pendulous bills, as is often the case in the fall, in the South, no bird a-wing is more easily killed. They are then disinclined to fly. They indolently lie to the dog’s points till the shooter walks them up, ‘adiug surjurod 19339S 5 oe ‘adiug surjurod 19339S SNIPE SHOOTING 325 The books teach that the snipe rises with a zigzag flight against the wind, darting to right and left with such rapid flashes of speed that the best of skilful sportsmen are puzzled, and consequently make many a miss. The snipe, it is true, goes against the wind when there is a wind, and zigzags a few times to rise up- ward before taking a straight course. Many writers on snipe shooting lay it down as correct that the shooter, to take advantage of this peculiarity in ris- ing, should walk down wind, or advance to the dog’s point down wind, so that when the snipe is flushed it will fly toward him. All such teachings savor of the novice, or of a skill which needs nursing. All the difficulties are greatly exaggerated, zigzag, swift flight and all. The zigzag of the snipe a-wing is in the beginning of its flight, and nothing is easier than to wait a moment till it straightens out on a straight flight. Then the killing is a matter of shooting on the wing, similar to other wing shooting. SNIPE SHOOTING As to walking down wind to secure a better shot, the sportsman need not concern himself about it in the least, excepting perhaps on such days as are cold, and days when the birds are very wild and rise at the extreme range of the gun. As with pigeon shooting, the really good shot does not let his birds get far if they rise within range. Whether they zigzag or not, he snaps them as soon as they are on the wing; or being well on the wing, he permits them to get into steady flight, and then delivers his fire. There is on AMERICAN GAME BIRD SHOOTING 326 the part of the experienced shot no particular attempt to reach the bird from a weak quarter. He takes the shooting as it comes. On windy days, or when the weather is cold, the On windy days, or when the weather is cold, the snipe may be very wild and rise at extreme ranges. Shooting then is quite as much a test of the gun as it is a test of the shooter’s skill. Few writers, how- ever, pay any heed to the distinction, and consider it all, be the rise far or near, as a matter of skill alone. At best, walking down wind on snipe is an uncertain advantage, for they can fly down or across wind with a swiftness and ease which dispose very quickly of any trifling advantage of a few yards taken up wind for the start. The habits of snipe, as oftenest described, are their habits when they are lean and wild, or wild from a change from warm to cold or from calm to windy weather. But to teach that such is their regular man- ner of flight would be on a par with teaching that quail live in the tree-tops because they sometimes take refuge there. Even when lean and wild, on a calm day the snipe does not strain the skill of a good shot. But on a windy day it is a different proposition. The wild, lean snipe can dart very swiftly across or down wind, and if to this be added rise at long range, the shooting becomes really difficult, though then, as mentioned be- fore, it is also a test of the gun. SNIPE SHOOTING When wildest, the snipe is exceedingly restless and When wildest, the snipe is exceedingly restless and moves fitfully from place to place. It then takes alarm SNIPE SHOOTING 327 quickly, flying high out of range, with its bill extended straight ahead. It can pitch to the ground from its highest flight, darting downward with stiffened wings and alighting with the greatest ease. In the course of migration the birds stop in favorite In the course of migration the birds stop in favorite places where food is abundant, and oftentimes there remain till the weather becomes unpleasant. As a rule, they arrive in the South in a lean condition. When lean they are also wilder, regardless of weather conditions. Shooting then, if limited to times when they are wild, is shooting in its most difficult phases. But as mentioned before, such difficulties of snipe shooting are not the average of snipe shooting. Snipe shooting as to possible quantity varies widely, Snipe shooting as to possible quantity varies widely, one locality with another. One locality may contain but a few snipe to reward the shooter’s efforts, while in other nearby localities they may fairly swarm, as in Louisiana and Texas in the fall and spring months, when the birds are migrating. In those States they generally remain several weeks to enjoy the food abun- dance. Some scattered ones in the South may be found all through the winter. The heavy rains of fall and spring, frequently a downpour of days in the far South, soften the fat alluvial prairie lands, thereby fitting hundreds of square miles for the snipe’s habitat. In particularly favorable sections of the prairie, cotton, corn and sugar fields, they may at times be found in thousands. A dog in such shooting is an encumbrance, except to act as a retriever. There is no woodcraft AMERICAN GAME BIRD SHOOTING 328 necessary in such shooting. The sportsman walks along till the birds are walked up. So rapidly will he sometimes flush them that at every step it is fire and load, and fire and load again. At such times the gun becomes too hot to hold, and the shooter must perforce stop till it is cool enough to handle. SNIPE SHOOTING Enormous bags of snipe have been made, particu- Enormous bags of snipe have been made, particu- larly in Louisiana and Texas, where the greater part of the flight of North America congregates for a few weeks in the period of snipe migration. One of the greatest, and I believe that it is referred to now as the greatest bag, was made many years ago by Mr. Pringle, a wealthy sugar planter, of Louisiana, who had great fame as a sportsman of rare skill. He bagged 400 and some odd snipe in one day. This is a large bag, indeed. It is but one of thousands of large bags in that section, so common as to excite no special comment. I have told of these matters to shooters in the North whose ‘success was measured by a dozen snipe, more or less, as the result of a day’s shooting. Such large bags being outside of their personal experience, they have been pleased to consider it an idle tale. They seemed to think that their narrow experience in shoot- ing a few birds over a few acres of ground each year was the measure of the world over. In regard to the big bag made by Mr. Pringle, it In regard to the big bag made by Mr. Pringle, it may be added, by way of explanation, that he had ne- groes to assist him, some to carry the spare guns, oth- ers to carry the ammunition and to retrieve the dead SNIPE SHOOTING 329 birds. I have been told by men who have hunted with him that he is a most indefatigable walker, and pos- sesses extraordinary quickness and accuracy in the use of the shotgun, snapping the birds almost on the instant that they take wing. In that land of game abundance, at that day, it was not considered unsportsmanlike to kill all that the sportsman pleased to kill, for however great the bags, there was no apparent diminution in the numbers of the birds. If the sportsmen killed many, their neigh- bors derived the benefit of it. The killing, too, was at irregular intervals, differing from the steady drain made on the bird supply day after day by those who shoot for market. SNIPE SHOOTING This circumstance of the record bag was a happen- ing of many years ago, when the sentiment concerning game preservation was different everywhere, North and South, from what it is to-day. As to snipe shooting, and the way of it, the proper As to snipe shooting, and the way of it, the proper manner to shoot them is to go forth and shoot them. In other words, the set manner of doing this thing and that thing, as taught by some writers, is all very well if one can do no better. There is no rule whereby snipe shooting can be There is no rule whereby snipe shooting can be made soft and easy, and there is no sportsman with proper ambition who will care to have his skill less than the best test that the bird can offer. If the sports- man’s skill is equal to the test, practice will improve it. In any event, there is at least the pleasure of try- ing to cope with the conditions. The proper skill is AMERICAN GAME BIRD SHOOTING 339 that which takes the shooting as it comes, instead of picking out the easy shots, or easy combinations to secure them. The best snipe gun is moderately choked, or an im- proved cylinder bore. As in all open shooting, good work may be done with a full choke, since the shooter can pick his distance to shoot his birds. However, it is not every man who can wait on his bird, or who can estimate distances at a glance; therefore, it is bet- ter to have a scatter gun which will be available for instant use when the bird rises. A 12-bore is most commonly used, and as for the size of shot, No. 8s or Qs or 10s are good, the latter being quite large enough when the birds are fat and lazy. As a bird to shoot over dogs, the snipe is inferior. Sometimes it is in such abundance that a dog is un- necessary. At other times it is so wild that it will not lie to the dog at all. If fat and tame, it may lie too well. Again, it will frequent marshes so wet, cold and rank with marsh grasses that it is impossible for a dog to work satisfactorily, however good his intentions and ability may be. SNIPE SHOOTING Very few dogs have a natural fondness for work Very few dogs have a natural fondness for work on snipe. It is acquired in most instances. Some dogs, good on upland game birds, thoroughly detest the snipe and refuse to recognize it. On the other hand, some dogs like snipe as a bird to work on, though such are exceptional. Considered strictly as a bird of the open, the snipe affords excellent open shooting. Yet there is never SNIPE SHOOTING 331 the weird uncertainty about it that some writers have discovered. No doubt an easy explanation of the diffi- cult shooting is found in the manner in which the shooter handles his gun rather than in the manner of the snipe’s flight. When snipe are in great abundance, and can be When snipe are in great abundance, and can be bagged with little effort by walking them up, the sportsman soon tires of the sport. It is too easy then to be considered sport. On the other hand, when they are scarce, wild, and will not lie to a dog, it is too difficult and uncertain. The dog is eliminated then as a factor, and the shooting is largely a matter of taking chances. When the happy medium is found, the birds being neither too wild nor too tame, it 1s excellent sport indeed, though in Louisiana I noted that where there was an abundance of both snipe and quail the sportsman quickly tired of snipe shooting and gave quail the preference. QUAIL SHOOTING. The quails of America cover a greater territory than any other of our game birds, and are more faithfully pursued than any. They are found, interruptedly, from points in Canada far down into Central America, and beyond. Over the eastern half of the continent, and far better known than any other species, bobwhite stands alone. Moreover, by one means or another, he has extended his range, first to the Rocky Moun- tains and then beyond that to the Pacific coast, while the Texas form, and some Mexican species, drift far to the southward. Bobwhite has been called the game bird of America, Bobwhite has been called the game bird of America, and from early days has furnished sport to north- erner and to southerner alike. It was never abundant in Canada, though the gunners of southwestern Ontario used to get a few; but forty or fifty years ago it was very abundant in central New York, from which coun- try, we believe, it has long been exterminated. Many years ago the late Prof. O. C. Marsh, of Yale Col- lege, used to relate with gusto stories of the good quail shooting that he had had near Lockport, N. Y., in the days before the Civil War. Hard winters and overshooting have long ago al- Hard winters and overshooting have long ago al- most exterminated the quail in Massachusetts, Con- necticut, New York, and portions of Pennsylvania, yet 332 332 332 332 QUAIL SHOOTING ga it cannot be doubted that self-control on the part of gunners would result in the re-establishing of these birds in goodly numbers through many sections of their former range. In a certain Connecticut town, where the quail had been practically exterminated, the gunners, for several years, have had an understand- ing that no one of them should disturb these birds. The result of this protection has been that in the spring of 1910 quail might be heard calling from every side in certain sections of this town. It is said by the gunners there that there will be great quail shooting this fall. This may mean that the gunners will again kill off all the quail, and that again for six or seven years they will have no shooting. QUAIL SHOOTING. it cannot be doubted that self-control on the part of gunners would result in the re-establishing of these birds in goodly numbers through many sections of their former range. In a certain Connecticut town, where the quail had been practically exterminated, the In the West are the beautiful plumed and helmeted quails of the mountain, of the valley and of the desert —and those other birds of curious plumage and soft, thick crest, Mearns’ quail and the scaled quail, also known, respectively, as “fool quail” and ‘“‘cotton-head.” Except for Mearns’ quail, these western forms do Except for Mearns’ quail, these western forms do not, we are usually told, offer good shooting. They run like deer before the dogs, rise at long distances, make long flights, and as soon as they reach the ground start running again. Mearns’ quail is said to be an exception to this rule, and lies close and hard. So difficult are valley quail to shoot over points, that many gunners of southern California do not attempt to use a dog to find them, while others use dogs only for retrieving, or to flush the birds while running. Few people in the United States have had greater AMERICAN GAME BIRD SHOOTING 334 experience in quail shooting than Mr. B. Waters, who has written one of the very best articles on this sub- ject that has ever been penned, which in substance reads as follows: BOBWHITE SHOOTING. From the personal point of view, each one gener- ally has his own preference in respect to the bird which he prefers to shoot to secure the greatest pleasure, and this preference in turn determines the shooter’s opin- ion that such bird is therefore the best of all birds for the purpose of sport. Thus, one prefers ducks, and not considering that his own personal idiosyncrasies, or greater success, or habit and long association, or what not, may have much to do with his preference, he solemnly affirms that duck shooting is the best of all shooting. And so with him whose choice of sport is the shooting of some other bird—that bird is sure to be exalted above all others. But from the standpoint of the greatest good to the greatest number, quail shooting, for many reasons, is the best of all shooting. It is a kind which affords such mixed shooting—open and cover, slow and swift —that shots can be found to meet the skill and fancy of all, be the former little or great and the latter fastidious. There is much of the open quail shooting which is not so difficult as to dishearten him of moderate skill, while, on the other hand, shooting in cover tests the QUAIL SHOOTING 335 skill of the most expert sportsman. And again, taken all in all, whether in open or cover, the quail shooter of good average skill can compass a good showing in results, and thus enjoy the pleasure which comes from reasonable success. In this connection it may not be amiss to maintain In this connection it may not be amiss to maintain that a certain degree of success is essential to the shooter’s pleasure. Many writers deprecate the con- sideration of the bag, treating it as an irrelevant, gross incident, so dominated by the beauties of nature and the ethics of shooting, in the abstract, that it should be mentioned in hushed tones or viewed with eyes askance. The beautiful and the useful of sport should go hand in hand. Each is a part of the great whole, and as such should be equal factors of sportsmanship. To the sentimental, which ennobles and adorns the useful of life, there must be added the material and the practical. To the shooter there must be a reward for his efforts. BOBWHITE SHOOTING. It has often been said that it is not all of shooting to shoot, nor all of fishing to fish, for- getting the converse, that, all of shooting or fishing being absent, there is no shooting nor fishing at all. As to quail shooting in respect to quantity, there As to quail shooting in respect to quantity, there is more of it than there is of any other kind of shoot- ing, hence each shooter can better satisfy his longings for sport if it be measured by the possibilities of the bag or the number of opportunities offered. And there is also more of it when measured by the matter of time, for it extends through a season of about five months, taking it as it is in the North and South. AMERICAN GAME BIRD SHOOTING 336 Thus, the man whose business cares leave him but a few days for shooting, and these at no definite time, has more possibilities of sport on quail than on any other bird. But the very abundance of the quail seems to have But the very abundance of the quail seems to have checked the proper appreciation of it. Not that it is treated with neglect, but there seems to be a lack of the enthusiasm and lavish use of the superlative, so often to be noted when writers are discoursing on the ruffed grouse or the woodcock. Nevertheless, it is not uncommon to have a keen relish for what is rare, even if it be not of the best, while the good may be so common as to escape notice. The quail is more uniformly and widely distributed The quail is more uniformly and widely distributed throughout the United States than is any other game bird. Its habitat generally comprises both open and cover, though whole districts are exceptions, as will be touched on later; thus, besides giving the sports- man a mixed style of shooting, is added the charm of constant variety, and testing of the sportsman’s skill in woodcraft. It differs from the ruffed grouse, whose home is in the woods, a much smaller section relatively. BOBWHITE SHOOTING. All works on natural history, so far as I know, teach All works on natural history, so far as I know, teach that the quail’s home comprises conjointly both open and cover; and while this is true in a general way, there are important exceptions to it, so much so that a work devoted to the habits and habitat of the quail as they are in one locality might be distinctly erroneous if applied to the quail of some other locality. In this respect it differs from the prairie chicken and the ruffed QUAIL SHOOTING 337 grouse—for of the one it may be said, without quali- fication, that it is a bird of the prairie; of the other, that it is a bird of the woods. Such sayings of them will be found to be true wherever those birds may be found. The quail thrives wherever it can obtain a food sup- ply, in open or in cover. It readily adjusts its habits to the dominating circumstances of food and cover, whether it be in prairie or woods, or a country com- prising both open and cover. In the country north of the Ohio and east of the In the country north of the Ohio and east of the Mississippi River, it frequents the open fields largely, preferring such as have a good food supply, with hedges or old walls and fences fringed with brush, or nearby woods and thickets to which it can run or fly for shelter or safety. In such sections it rarely goes far into the woods, preferring to skirt around the outer edges of them, merely for protection and shelter. The hawks are its deadly enemies, and it needs ever to be alert in avoiding them. The quail oftenest roosts in the open fields, where there is at least a few inches growth of grass, stubble or weeds for concealment, and it uses the same place many times if not constantly disturbed. This is indi- cated by the grass or other vegetation being beaten down in the roost, a small circular opening, about two feet in diameter, and the pile of droppings in the center of it. BOBWHITE SHOOTING. The birds huddle on the ground, bunched up close The birds huddle on the ground, bunched up close in a circular form, with their heads outside; thus all AMERICAN GAME BIRD SHOOTING 338 face toward the outer circumference of the circle, which cannot be approached without coming to the view of some bird. This arrangement is said to prove ad- mirable for the safety of the whole. In theory it seems a wise arrangement; in practice it works very badly, since they often fly reluctantly when they have comfortably adjusted themselves for a night’s rest. The pointer or setter may also draw very close to them then, generally doing it with greater precision than when they are more scattered about, the evening hours being more favorable for strong scent and accurate pursuit. Were not dogs trained to such stanchness as is required in shooting, they could easily, at such juncture, spring in and capture, as indeed some par- tially trained dogs will do under the circumstances, In the States of greatest bird abundance, as in Ar- kansas, Mississippi, etc., and where there are many ragweed fields, very destructive shooting often takes place near the twilight hours, when the birds have set- tled themselves for their slumbers. When the dog points the bevy the shooter places himself at the proper distance from the roost to obtain the best scatter of the shot. Then the huddled birds, being flushed, swarm up loosely together for three or four feet, when the shooter takes a snap shot at them, and often does nearly as much damage as if he had potted them on the ground. It is hardly necessary to add that this practice is disapproved by all true sportsmen. In Minnesota, Dakota, Nebraska, Kansas, and other In Minnesota, Dakota, Nebraska, Kansas, and other prairie States, the quail readily adapts itself to the QUAIL SHOOTING 339 local peculiarities of the different sections, utilizing such slight advantages as may offer, as hedges, fences, the cover with which most streams are fringed, or high weeds. The quail sadly needs cover for its protection, its destroyers being both of air and earth—hawks, foxes, cats, dogs—and the eggs, too, fall a prey to the rapa- cious appetites of some of its enemies. BOBWHITE SHOOTING. In the South the cur dogs of the negroes—every family owning one or more, all kept in a kind of half-famished condition— prowl through the fields seeking for food; they are the very worst of egg destroyers. Were the quail not so hardy and prolific, its fate would be swift, and extermination certain. The negroes’ dogs seem to be almost omnivorous. In the fall they may be seen making daily visits to some persimmon tree, under which they eat the fallen fruit with apparent relish. Those which have some claim to hound blood are not averse to making a meal in the corn field, on corn when it is in the milky stage. With such rapacious enemies to contend against, the destruction of the quail must be great, but in addi- tion to all that, many are trapped and netted, methods which destroy whole bevies at a time. But to return to the matter of the quail’s habitat: But to return to the matter of the quail’s habitat: in certain parts of the South, as in the oak woods in sections of Alabama, Texas, Arkansas, or in the pine woods of Louisiana, Mississippi, etc., the quail may live wholly in the woods, food, always a first consid- eration, being there secured in abundance. AMERICAN GAME BIRD SHOOTING 340 In cover, some of the shooting is easy and some of it very difficult, though hardly ranking in difficulty with ruffed grouse shooting. The quail is neither so wary nor so wild as the ruffed grouse. Shooting in some parts of the pine woods is almost as easy shoot- ing as shooting in the open, the ground being bare except for its covering of dry pine needles. The smooth trunks of the pine trees, standing several yards apart, and free from limbs for thirty or forty feet. offer no serious obstacle to the shooting. In other sections of the pine woods, where the growth of the trees is more stunted, and the limbs grow from near the ground up, the difficulty of the shooting is second to none, and in some sections is almost prohibitive. BOBWHITE SHOOTING. Again, there are sections wherein the quails live on the open prairie, as in parts of Arkansas, and, the shooting being strictly open, it much resembles chicken shooting, excepting the difference in the size and speed of the two birds, the quail being much the quicker to get away at the start. The quail makes its flight in the open prairie, alighting near any little bit of shrub- bery, be it no more than a bush or two of sumach, which, by the way, grows here and there on the prairie in Arkansas. In the woods, when pursued, it fre- quently takes to the tree-tops for safety, where it is hidden indeed. On warm days, or when there has been a long spell of pleasant weather, it is far less wild than when the weather has been stormy, or when there has been a sudden change from warm to cold. Such changes add to the difficulty of the shooting. On point. On point QUAIL SHOOTING 341 In the North, the quail makes its home where some buckwheat or other grain field is available for a food supply. It so arranges its haunts that it has some cover within easy flight, in the densest part of which it seeks safety when flushed, not refusing the heavily timbered swamps if too much persecuted by the shooter. In such places it has an excellent chance of escape from pursuit, or may foil its pursuer by simply run- ning away; or, if pressed to take flight, it has many chances for safety owing to the difficulty of shooting accurately in the dense cover. New England shooting is the most difficult of all New England shooting is the most difficult of all quail shooting, excepting, perhaps, shooting in the dense pines and cat briers of the South. Then, to have any satisfactory success, the scattered birds must be diligently followed and sought in the thickets, be they ever so dense. In this respect it differs from shooting in the sections of more abundance, where such close attention to the scattered birds is unneces- sary, either for sport or the interest of the bag. In the South, where there is an abundance of birds, comparatively, the sportsman rarely tarries with a bevy which gives him any special difficulty. It is much easier, and more satisfactory, to go on and seek more birds. BOBWHITE SHOOTING. For this reason, even under favorable oppor- tunities, the scattered birds are never, as a rule, hunted till the last one is flushed, and flushed again, when it is possible, as in the North. In New England, buckwheat fields are the quails’ In New England, buckwheat fields are the quails’ choicest resorts for food, and any adjacent brush, or AMERICAN GAME BIRD SHOOTING 342 long grass of swamp or upland, or the skirts of woods, afford the shelter and protection that they need or seek. No doubt the birds become wilder in the North than in the South, for, first of all, the inclement weather of the North tends to make them so, and there is a much more relentless pursuit of them by the shooter. The birds being scarce, after the bevy is scat- tered the search continues while there is a hope of finding a single remaining one; and if success with them has been unsatisfactory, the shooter may return later to catch them, when they are whistling to each other in the attempt to come together as a bevy. In the broad plantations of Mississippi, Alabama, In the broad plantations of Mississippi, Alabama, Tennessee, Arkansas, etc., a dog of reasonably wide range is necessary, much wider than would be either desirable or useful in New England, or similar sec- tions in respect to cover; for there is much of the country in the South, open and cover, which every- where affords a bountiful food supply, and, therefore, the birds are to be found in the most unexpected places. The cover and cultivated fields of the South do not aid the hunter’s judgment as to quail haunts to the degree that they do in New England shooting. In the latter place there are comparatively few areas in which the birds can get both food and cover together, or even food alone, and the sportsman soon learns to distinguish the favorable places. In the South, in cover and open, there is food in abundance everywhere. There are vast fields, some of which are overgrown with sedge grass, others with weeds, with fields of QUAIL SHOOTING 343 cotton and corn interspersed, any part of which is a fit habitat for the birds; thus, the dog working out such ground in the South can, as a rule, beat out all parts of it with probable success. BOBWHITE SHOOTING. In the more open grounds of the South the dog can be seen at long dis- tances, so that a wide range is not detrimental in itself, providing that the dog is really working to the gun, and not self-hunting or semi-hunting. As to the manner in which the dog should hunt, no As to the manner in which the dog should hunt, no hard and fast rule can be laid down which would apply to all sections. Whatever may be the quail’s abiding place, it learns Whatever may be the quail’s abiding place, it learns to make the most of its surroundings in promoting its own safety and interests. It learns whether it is better to fly or run in evading its pursuer, and the best strat- egy to attain that end. If good cover is conveniently near, it may trust to its wings at once for safety, and to its legs and wings if followed into the cover. If the country is open, or with narrow and insuffi- If the country is open, or with narrow and insuffi- cient cover, as in parts of Louisiana, and other sec- tions where the ground is thoroughly cultivated, it takes a great deal to its legs and cunning devices. In working on such birds the dog must learn to govern his work by the circumstances of it. He might be an excellent performer on quail in the North, and a poor one in the South, or he might be a good one in Mis- sissippi and a poor one in Louisiana, though the pre- sumption is that if he were good in one section he would become so in any other section after the neces- sary experience. AMERICAN GAME BIRD SHOOTING 344 Many of the plantations of Louisiana are drained by open ditches running parallel at reasonably equal distances from each other, though the distances may vary greatly one field with another and may be 50 or 200 yards, more or less, apart, while other ditches of like arrangement intersect them at right angles; thus a plantation may be cut with more or less regu- larity into small squares surrounded by ditches. Some plantations may be irregularly ditched, while others with a far greater watershed may not be ditched at all. BOBWHITE SHOOTING. The heavy rains round out the banks of the ditches and their bottoms, and a fringe of weeds and brush, thick and thin in places, strings along the banks and makes a fairly good shelter for the quail. On these land squares are grown cotton, or corn, or nothing, as the case may be, though, if not cultivated, there is always certain to be a good crop of weeds, affording plenty of quail food. When flushed in such places the quail may fly a few yards to the first ditch, or may cross over two or three ditches before finding a place to its liking. As mentioned elsewhere, the state of the weather may greatly affect the quail’s habits. The bevy having gone to the ditches for safety, the dog, to be useful, must have great superiority in roading if he pursues satisfactorily. When in the ditch the birds run swiftly along the bottom. It is almost impossible, at first, to induce the green dog to go into the ditch, or, being in, it is impossible to make him remain there, though it may not be over a foot deep, and dry. The green dog ‘sourd aq} ur 3urjo0o4S ‘sour aq} ur 3urjo0o4S QUAIL SHOOTING 345 will promptly cross out from one side to the other, missing the scent, and accomplishing nothing useful. He does not know what is required of him. But once he catches the idea, he soon improves on it, following carefully along the bottom of the ditch and pointing the scattered birds here and there, every few yards apart, in ones and twos, the shooter having a good opportunity from his position on the outside to kill as the birds fly out. The shooting along ditches is not so easy as one The shooting along ditches is not so easy as one might imagine. Sometimes the birds run swiftly sev- eral hundred yards or more in the ditch, and may then run out and across to other ditches, giving a trail which may try the most experienced dogs to follow. BOBWHITE SHOOTING. If the birds happen to be near a cotton or corn field, where the ground is bare, and there are no ditches for concealment, they may run so fast and far that the dog may never approach near enough to them to secure a point, and the shooter who is inexperienced in this work will be likely to think that his dog is surely deceiving him. When near the woods, or switch-cane, the birds often take shelter therein, and when in the latter cover it is well to abandon further pursuit of them. In the sugar country, where there may be corn fields In the sugar country, where there may be corn fields here and there among the broad levels of the sugar- cane, the character of the shooting again changes. Many birds will be found in and around the corn fields, and then it is very pretty shooting. It may not be amiss to mention, for the benefit of It may not be amiss to mention, for the benefit of AMERICAN GAME BIRD SHOOTING 346 those who shoot in the sugar country for the first time, that they should keep their dogs out of sugar-cane fields as much as possible. The cane, in harvesting, is cut diagonally across with a knife, thus leaving a stump with an edge which will cut a dog’s foot almost as a knife would. It is a common matter for a dog to split his toes or heel on cane stumps, with the result- ant crippled condition, and no more work from him for a time. In Mississippi, in the midwinter season, the birds stay mostly in the woods. Good shooting may be had in the South from the middle of November to the first of March. Many of the Southern States have a longer legal open season, but the dense cover and warm weather make a natural limitation to the sport. The weather is mild, the birds are strong, and the sport is at its best, in the winter months of the South. Quail shooting, in the main, is close shooting, as to the ranges at which the birds are killed. Most birds drop within twenty-five yards, some much nearer than that. A gun weighing from 6% to 7% pounds is of ample weight. The 12-bore is most commonly used, though the 16 and 2o-bores are excellent, and are preferred by many sportsmen. BOBWHITE SHOOTING. Some shooters use guns of 28 caliber, and are enthusiastic over their work. As a matter of course, the smaller bores may be much lighter than the 12-bore. The 16 and 20-bores being smaller, their killing The 16 and 20-bores being smaller, their killing circle is less, though they shoot with good force in QUAIL SHOOTING 347 comparison with the 12-bore. Closer holding is re- quired to shoot them well. Whichever bore is used, it should not be closely choked. There is no need of a choke bore in quail shooting. It is an extremely difficult matter to induce the It is an extremely difficult matter to induce the average shooter to use an improved cylinder-bore gun. The recommendation to use an open bore seems to be construed as reflecting on his ability to shoot a close gun, instead of being accepted as sound information concerning the gun fit for that particular kind of game. It requires time to effect a reformation concerning the use of the choke bores in quail shooting. The sports- man should go forth equipped for his sport according to his needs, and not to the whimsicalities of senseless fashion. He should not take a full-choked gun in cover, nor a cylinder-bore gun to shoot ducks. There should be intelligent adjustment of means to ends. In- dustry and skill and woodcraft should not be balked by visionary theories and inappropriate weapons. The foregoing is written of the quail as it refers to man’s pleasure afield with dog and gun. Naturally it is not fearful of man, and rather prefers to dwell near his haunts, not from an affection for him, but from the fact that near cultivated sections there is always more food to be found than in the uncultivated. The matter of providing food for itself and its young is quite as constant and insistent in the life of the quail as it is in the life of man. It often nests in or near the cultivated field. Its It often nests in or near the cultivated field. Its cheery, ventriloquial whistle, reiterating its favorite AMERICAN GAME BIRD SHOOTING 348 utterance, bobwhite, may be heard about the farms, particularly in the morning hours, and bobwhite has come into use to designate the bird itself. BOBWHITE SHOOTING. Sometimes the call is uttered with a short introductory note, and these, with a few alarm calls, or calls of inquiry when the birds have become scattered, seem to be about all the vocal means of communication which these birds have. In many parts of the South, where the quail are in greater abundance, their sweet notes may be heard in many directions, at many distances, in the early morn- ing and evening hours. These calls work great harm to it, for by them the shooter learns the whereabouts of all the birds in his neighborhood, and locates the haunts of every bevy. , In the fall, the rallying cry often serves to inform the sportsman as to the best course for the morning’s hunt. In the South the quail is called partridge. In the fall, when the shooting opens, the quail soon In the fall, when the shooting opens, the quail soon learns of the gunning dangers, and its habits thereupon change quickly to conform to a life of greater safety, though it will run many risks to be near an abundance of food. However, when danger is impending, it avoids the open much more than when danger is not, and is more alert, quicker to suspect mankind, and quicker to take alarm. When spring returns, the quail seem to lose their When spring returns, the quail seem to lose their fears of man, and they breed with little reference to concealment from him. Their confidence is unimpaired QUAIL SHOOTING 349 till fall approaches, when there is a repetition of all the fears and troubles and dangers of preceding years. He who can average three kills out of six shots, He who can average three kills out of six shots, cover and open, is an excellent marksman. The shooter may make a run of ten or twenty straight kills, but soon there are sure to come misses if he does not pick his shots. In winter the shooting is much more diffi- cult than in the fall. Of course, the man who never misses is of the parlor, not of the field. The home of the greater number of the American quails of the extreme southwest or on the Pacific coast is in a country of great aridity. BOBWHITE SHOOTING. Moreover, much of the vegetation of that country consists of thorny and spiny plants, of which many are cactus, yucca or mesquite. It is evident that over much of this country the use of dogs in shooting these birds would be diffi- cult, if not impracticable. For much of the year the ground is so dry that no scent would lie, and a dog, unless trained for that particular work, would be at a great disadvantage. Moreover, in the swift traveling performed by hunting dogs, as they are trained in the South for field trial work, a dog, in a very short time, would inevitably become crippled by the spines of the cactus, which he could not help constantly running over or into. If to these difficulties we add the fact that the quails seldom or never lie to a dog, but run as hard as they can at the first intimation of danger, endeavoring to get into the thick brush, through which they can thread their way faster than any animal can AMERICAN GAME BIRD SHOOTING 350 follow them, it will be seen that many of these quail cannot be pursued for sport, as sport is commonly un- derstood; that is to say, the shooting of a game bird over a pointing dog. This a priori conclusion is con- firmed by statements made by sportsmen who have lived for many years in Arizona, one of whom, Mr. Herbert Brown, not only a sportsman, but a field natur- alist of great ability, tells me that he has never heard of a dog being used on Gambel’s partridge in all the time he has lived in the southwest. Yet Allen Kelly says that in the irrigated districts of the Imperial Valley, Cal., Gambel’s quail lies well where there is cover, but on the bare ground runs like a deer. I have seen the valley quail in southern California I have seen the valley quail in southern California and the mountain quail in the Sierras, but have never yet seen either hunted with dogs. That the mountain quail can be shot over dogs is hardly to be doubted, but the case is different with the valley quail living in the lowlands of dry California. On the other hand, in Vancouver Island the introduced valley quail sometimes lies to a dog among the thick undergrowth, much as the eastern quail would lie. BOBWHITE SHOOTING. When startled they get up in a thick coveys of fifteen or twenty birds and scatter and sometimes lie well. VALLEY QUAIL. Of the California valley quail in the vicinity of Pasadena, N. P. Leach says on this point: “Up here on the mesas and among the sage brush and grease- QUAIL SHOOTING 351 wood in the foothills we use dogs when hunting quail, chiefly for flushing and retrieving, but down on the plains, amid the cactus and low brush, some hunters use both setters and pointers. There, when you flush a covey of quail and shoot in among them or over them, they will very often scatter and then hide under the cactus and lie to point. It is a cruel place to work a dog. The first day that I took my dog down there, he filled his shoulders and breast full of cactus spines. We pulled out most of them, but even then he was in no condition to work for some days. “Over one-half of the quail hunters here do not “Over one-half of the quail hunters here do not use a dog, and ninety per cent. of the other half use spaniels for retrieving. The other small minority use either setters or pointers. “When my spaniel flushes a covey, some of them “When my spaniel flushes a covey, some of them fly my way, so that I am very sure of a double shot, and sometimes an extra double at some of the laggards. Then, as the dog can outrun the birds, he keeps them moving, which often gives me shots at fast-flying birds. I have him so trained that I can control him with my whistle; that is, bring him into heel, where he will remain until ‘I tell him to go.” It is hardly to be doubted that if either the valley, It is hardly to be doubted that if either the valley, the mountain, or, in fact, Gambel’s quail, were to be introduced in any section where the climate was mild enough, they would, if properly protected, do well and increase. The beautiful valley quail readily adapts itself to confinement, becomes not at all shy, and has been frequently known to build its nest and lay its AMERICAN GAME BIRD SHOOTING 352 eges. We do not know that any have been reared, but no doubt this might readily enough be done, were proper care exercised. GAMBEL’S QUAIL Dr. Coues, in an article in the Jbis many years ago, said of the shooting of this bird in its home: “Compared with the eastern quail (O. virginianus), “Compared with the eastern quail (O. virginianus), from the sportsman’s standpoint, Gambel’s plumed quail is more difficult to kill. Not that it rises with more startling suddenness, or flies faster, for I noticed no material differences in these respects; but when a bevy is flushed, and one, or at most two, birds secured, it is exceedingly difficult, and usually only by chance, that other shots are obtained; for except under certain cir- cumstances, they lie very badly, and when they drop after being for the first time started it is, usually, not to squat and remain hidden, but to run as fast and far as possible; so that, if found at all, it will be dozens of yards from where they were marked down. This pro- pensity to run, which is also a great obstacle to their being flushed within proper distance, is exceedingly troublesome both to the sportsman and his dog; so much so, that the best trained dogs can often be of lit- tle or no service. It is true that this habit of running affords many shots on the ground, and often places the whole bevy directly under fire, but no true sports- man would thus ingloriously fill his bag by potting a bevy of such noble game birds. Like all their tribe, QUAIL SHOOTING 353 their flight is exceedingly rapid and vigorous, but it is always even and direct, so that it only requires a very quick hand and eye, and the usual intuitive calcu- lation for cross shots, to kill them readily. Notwith- standing all I have heard to the contrary, I consider them far from being tough birds, and No. 8 shot is abundantly large enough for them. The fault in most cases, I presume, is with the shooter rather than the shot. I may add that many of the places in which bevies are found would compare unfavorably with the worst woodcock brake of the Eastern States as regards facilities for obtaining a fair shot. I have had a bevy flushed all around me and hardly caught a glimpse of a feather. But these, and all other difficulties, should only increase one’s ardor and confer additional value on the lovely birds when obtained.’ MOUNTAIN QUAIL. In the California Mountains, near various hill towns, the mountain quail are often abundant, and late in October, or early in November, after rain, or snows turning to rain, we have occasionally started coveys which, flying for no great distance, would pitch down and lie so close that they could be walked up by a gunner and a few good shots had. In such situations, and at a time when the ground was moist enough to make the scent lie well, I should expect to have good shooting with a careful dog. In the dry West, however, conditions for shooting In the dry West, however, conditions for shooting AMERICAN GAME BIRD SHOOTING 354 differ so widely from those which prevail in the more humid East that it will probably take a long time for gunners and dogs to work out a system, at once agreeable and effective, for shooting these game birds. Dr. Elliot declares that many are shot over dogs, but Dr. Elliot declares that many are shot over dogs, but that far more are trapped and netted. The situation is still more difficult when birds of The situation is still more difficult when birds of the extremely dry country of Arizona and New Mexico are in question, though, as will be seen further on, Mearns’ quail appears in quite a different category. SCALED QUAIL. Of his attempts to shoot the scaled, or blue quail, Dr. Coues says: “This species is a bird of noticeably terrestrial hab- “This species is a bird of noticeably terrestrial hab- its, rarely taking to trees or bushes unless hard pressed in one of those extremities into which some people are fond of forcing any birds large enough to be worth a charge of shot, and wary enough to make it exciting sport to penetrate their poor bodies with it. It generally trusts to its legs rather than its wings, though these are not at all deficient in size or strength. On level ground it glides along with marvelous celerity, and makes good progress over the most rocky and difficult places. As a consequence, it is rather diffi- cult to shoot fairly, though it may be ‘potted’ in great style by one so disposed; and it will probably require several generations in training before it can be taught to lie well to a dog. I am inclined to think, indeed, QUAIL SHOOTING 355 that the lying of quail, an essential feature for the chase in its perfection, is almost as much a result of education, as the ‘pointing’ that the intelligent brute who helps us kill them has learned. In a primitive and strictly natural condition, quail, as a general rule, rather use their legs to escape pursuit than squat and attempt to hide. That the reverse is the case with the Virginia quail, I am perfectly aware, but this proves nothing to the contrary, and I am inclined to think its crouch- ing, till almost trodden upon, to be an acquired trick. This would surely be a poor way of escape from any of its natural enemies—any carnivorous bird or mam- mal; yet they find it to succeed so well against their chief persecutor, that he has had to call in the aid of a sharper-sighted, sharper-nosed brute than himself, else he might stumble over stubble-fields all day with- out seeing a bird, except by accident. I presume that Virginia quail in the days of Captain Smith and Poca- hontas were very much in the social status of the Arizonian to-day; and these certainly trust to their legs and wings rather than to the artifice of thrusting their heads in tufts of grass and then fancying they are safe.” Dr. D. G. SCALED QUAIL. Elliott, a sportsman of long experience, has this to say about the habits of the blue quail when pursued : “This bird runs with great speed and seems to be “This bird runs with great speed and seems to be able to keep it up for a long distance, and flies with much reluctance, alighting almost immediately and be- ginning to run at once. When compelled to take wing, AMERICAN GAME BIRD SHOOTING 356 it rises with the usual whir-r-r, and proceeds on a slightly curved line, rather straight ahead, and if, on alighting, it should stop for a moment, it is almost at once under the cover of some cactus or other low bush, which affords a place of concealment; and from which it can watch its pursuer, before starting to run again. “A dog is practically useless for hunting the “A dog is practically useless for hunting the scaled partridge, for if he is well broken and attempts to point a covey, the birds will run several hundred yards, while he is standing, and then will add several hundred more while he is trailing them, and the poor animal becomes bewildered and disgusted and is apt to run also. I know nothing so trying to the patience of a sportsman as the tactics of this species, unless it be the similar habits of other crested quail. Asa rule, this species was not very much hunted in the localities I met with it, and it always seemed to me rather singular that they should be so wary, for that is an attribute that wild creatures usually acquire, after having made the acquaintance of man and learned that his presence always brought wounds and death, and that safety to themselves was only to be obtained by leaving his vicinity as soon as possible. But these birds seem instinctively to have ascertained this fact before they ever saw a human being, and decamp at once whenever a man appears.” QUAIL SHOOTING 357 MEARNS’ QUAIL. With Mearns’ quail the case is quite different. This bird, while not at all shy, possesses markedly swift flight, and lies exceedingly close. Dr. L. C. Frick, who has had much experience in Dr. L. C. Frick, who has had much experience in shooting them in New Mexico, considers them a better game bird than bobwhite. In shooting them he uses pointer dogs, which have proved very efficient. Dr. Frick believes that if this bird could be introduced into the Mississippi Valley and the country east of it, it would at once become enormously popular as a game bird. He mentions as a great point in favor of this species, that when the birds rise the sexes can always be distinguished, and that therefore cocks may be se- lected for killing and the hens preserved. He believes that the males greatly exceed the females in number, and that therefore it is practicable to do a good deal of shooting without lessening the productive power of the birds in a particular district. The eggs are given by Captain Bendire as eight or ten in number, but, on the other hand, Mr. Nelson, who has been much in the range of these birds, says: “T have never seen the Massena partridge in coveys larger than would be attributed to a pair of adults with a small brood of young. Frequently a pair raise but three or four, and I do not remember having ever seen more than six or seven of these birds in a covey.” Mearns’ quail is odd in appearance and odd in habits, but one of its chief peculiarities seems to be that in AMERICAN GAME BIRD SHOOTING 358 a country where all the quail take to their heels and run like race-horses, it alone, when it pitches down, lies hard, and will let the dogs get up close to it before flushing. We do not understand that.the birds are particularly hard to hit. Though they fly swiftly, the shooting is usually open, and only reasonable quickness is required to hit the bird. RUFFED GROUSE SHOOTING. Dwelling in many sorts of country, sometimes, as in the East, where it is constantly pursued, cunning to the last degree, and practicing every stratagem; or again, in the wild regions, dreading only its natural enemies, and thus fearless and bold, and trusting wholly to strength of wing, the ruffed grouse is killed in many ways and under varying conditions. Where it is ignorant of firearms or of the danger from man, it permits half a dozen shots to be fired at it at close range, or sometimes may even be noosed from its perch on a limb by a bit of string tied to the end of a pole. A graphic account of ruffed grouse shooting in a A graphic account of ruffed grouse shooting in a region where they had been little disturbed, yet were not wholly tame, was contributed to Forest and Stream many years ago by that charming writer and good sportsman, Mr. T. S. Van Dyke. In those days ruffed grouse were abundant in the region referred to, as, in fact, they have been up to comparatively recent times in that country, as well as in certain sections of Michi- gan, where, within only a few years, we have heard of from twenty-five to thirty birds being killed in a day by two guns. We give the substance of Mr. Van Dyke’s account as follows: 359 359 AMERICAN GAME BIRD SHOOTING 360 Who that has heard the somber shades of the dense pine forest throb beneath the strokes of his hoarse re- sounding wing, or in the autumn woods has seen him flash for an instant amid the hues of crimson and gold, or pierce like a shaft of light the dark green of the cat-brier swamp, can ever forget the ruffed grouse? What sportsman can forget the feelings with which he has heard his drum-beat echo from the dark moun- tain side, or through the bursting woods of spring, or in those soft, still autumn days when the leaves are falling through the mellow haze of Indian summer, or, as sometimes heard, in the noon of night, in the depths of the forest primeval? RUFFED GROUSE SHOOTING. Few pictures hang more bright in the inner chamber of the sportsman’s soul than the broad fanlike tail spread along his path as he treads the trail of the deer, or its dark bands shining on the carpet of checkered leaves or sweeping over the mossy carpet of wintergreen or vanishing in the heavy green of the laurel brake. Not even the majestic woodcock, with his solemn Not even the majestic woodcock, with his solemn dignity; not bobwhite, with his sweet, graceful ways and artless beauty; not the brilliant but erratic little genius of the boggy meadow; not the noble turkey, with his beamy bronze and bearded breast, can raise such tender memories as this grouse. For all these must be sought, and often sought in vain, in their native haunts. But the ruffed grouse is a more famil- iar spirit, and many a time plays across the sports- man’s path when wandering over the sapling-clad slope where the autumn woodcock lies in the full bloom "yjNour Ur pIIg YIM Buyurod 19}39g "yjNour Ur pIIg YIM Buyurod 19}39g SHOOTING THE WOODS GROUSE 361 of life and fatness, or when following bobwhite through the hazel thicket, or when roaming from pond to pond in search of ducks among the vine-clad arbors of the river bottom. And often the hunter of the deer sees him strut before him as he sits resting on a fallen log, and often, when on the trail of the deer in winter, sees him shake the snow from his lightning wings, as, bursting from its cover, the bird goes whizzing away amid the snow-draped trees. Few of those who most love this noble bird have Few of those who most love this noble bird have ever seen him in the simplicity of youth, before he has left his mother’s side and gone forth to roam alone the spangled shades of the rugged mountain side or the somber shrubbery of the tangled glen. For his hearthstone is too often in the dense mass of sum- mer’s wealth, and few are the eyes that can follow him into the deep, dark brake or into the shaggy cov- ering of the mountain’s breast, until autumn’s frosts have tattered their gay banners and trailed their green glory in the dust. RUFFED GROUSE SHOOTING. For certainty of finding this grouse at home in his For certainty of finding this grouse at home in his early days, with comfort in hunting him, few places have ever equaled the bluff regions of the upper Mis- sissippi. Here this grouse lived and loved, and stayed until long after the autumn leaves were scattered on a thousand winds; and even after the deep snows of winter fell upon his early playground many a one re- mained instead of seeking the covers of the bottom lands. Here he may be found while the trees stand in the full green of summer and before any hue of AMERICAN GAME BIRD SHOOTING 362 death has touched their shining heads. Years ago these bluffs were studded from base to crest with large oak trees, scattered more or less along the slopes, and more abundant and dense of foliage around the sides and heads of the numerous ravines. Where they hap- pened to fall, the rich verdure of the white birch gen- erally filled their places, and in the bottoms of the ra- vines and along the base of the slopes the crab-apple and wild plum and scrub oak formed abundant cover. Everywhere along the hills the ground below the trees was densely carpeted with green, upon which the sun- light flittered in a thousand shades through the open- ings in the leaves above. And yet the walking was always good, and the view generally free in all direc- tions beneath. It was one of the fairest days in 1867 that, with a friend and two dogs, I first roamed these pleasant shades and found my old friend in a new kind of home. From nearly the foot of the bluffs, where the outer guard of soft maple and white-oak saplings be- gan to encroach on the black oak of the hills, to very near the top, where the birch was flying its bright green flag from its snowy staff, the dogs were soon racing to and fro, while we were strolling along be- hind them, half way up the hillside. We soon came to a shallow ravine where the ferns and the prairie grass that covered the ground were taller and greener, and the shade of the black oak and maple was deeper and cooler than on the rest of the hillside. RUFFED GROUSE SHOOTING. The elder dog, named Jack, had hunted such ground before and SHOOTING THE WOODS GROUSE 363 seemed to know all about such places, and at once started up the leeward side of the ravine with slow and cautious trot, while the younger, named Frank, seemed to have an intuition that the other dog knew more than he did, and slowed down his pace to about the same. And soon Jack’s trot subsided to a walk as his nose caught the faint breeze that played over the shady side of the hollow, and his tail slackened its lashing motion and settled down to a slow wavy swing. Quietly he moved along, with nose upraised just above the deep green of the ferns and prairie grass and the bright golden hue of the lady-slippers and the carmine of the wild peas, raising it from time to time still higher, with inquiring sniff, and swinging steadily off to the leeward so as to keep the breeze fairly in his nose all the time. And soon the old dog’s tail began to straighten and the joints of his legs to stiffen, and he turned his head slowly from side to side, and snuffed the air more cautiously as he moved, more and more slowly, along. And all the time Frank coming up the other slope, some hundred yards away, with eyes fixed intently upon Jack, imitating all his movements, even more strongly than if he had smelt something himself, instead of taking Jack’s word for it. Suddenly Jack stops, and as suddenly Frank does the same, and at the same instant a line of mingled white, black and gray, with roaring wings enveloping the whole in a haze of brown, bursts from the rank ferns some ten yards ahead of the dog and darts like an arrow through the green arcade. AMERICAN GAME BIRD SHOOTING 364 Bang-whang go two barrels of the guns almost to- gether, a feather parts from the long, outspread fan behind the booming wings, and in a second more the brown streak fades among the distant trees. RUFFED GROUSE SHOOTING. B-b-b-b-b goes another from almost the same place, almost before the first one is out of sight, and bang goes one barrel of each gun exactly together, and a cloud of feathers floats from the downward whirling bird, while with boisterous b-b-b-b-b seven or eight more birds rise, curling, flashing, darting and whizzing from the ferns in all directions. But Jack seems to have no anxiety about the birds But Jack seems to have no anxiety about the birds that have fallen, and after going cautiously a few steps forward, stops again, with slowly waving tail. Care- fully he moves along, sniffing daintily at the air on high, and swinging off occasionally to one side so as to catch the full breeze, then, as he advances a few paces beyond where the other birds had risen, his limbs and tail gradually stiffen, until he again becomes quite rigid, with Frank, on the other side of the ravine, imi- tating all his motions almost as accurately as if the two were connected by an electric wire. As we come up to him he suddenly relaxes, moves As we come up to him he suddenly relaxes, moves off a few yards to one side again, and then, with nose high upraised and body sunk low in the grass, he crawls forward a few feet, in shape more like an alli- gator than a dog, and then comes again to a standstill. As we advance a little in front of the dog three grouse burst roaring from the ferns some twenty feet ahead of us and dart away in different directions. One SHOOTING THE WOODS GROUSE 265 whirls downward out of a cloud of feathers; another changes his course at the report of another gun, and mounts skyward through the tree-tops; the third, dash- ing the sunshine from his glistening wings, scuds away through an open place, with the guns belching flame and smoke vainly at the place he had just left; while the one that had mounted above the trees, poising for a second aloft, closes his wings and descends with a heavy thump to the earth. The fallen birds retrieved, we went on to find the scattered birds. Some three hundred yards we wan- dered along, and suddenly Frank began to dawdle in his pace. RUFFED GROUSE SHOOTING. With gently oscillating tail, he sniffed in- quisitively at the breeze that swept up the hillside from the long ravine below. To our senses it was laden with the fragrance of ferns and wild buckwheat and wild peas and white clover, with wild rose and mint; but the dog smelt something more, for he suddenly stopped with the quickness of thought, and at the same instant a grouse broke, with uproarious wing, from the deep green cover, some fifteen feet from his nose. Two charges of shot shivered the blended white and green of the birch behind which he disappeared, the air throbbed no more beneath the beat of his hoarse wings, and a faint nebula of fine feathers drifted into sight on one side of the tree. Up and down the hill again both dogs were soon Up and down the hill again both dogs were soon beating the ground. In about five minutes Jack, com- ing down the hill on a gentle canter, dropped into the grass as suddenly as if shot, and lay there with only AMERICAN GAME BIRD SHOOTING 366 the tip of his nose visible above the ferns. As we came to him a bird rose like a rocket, only a yard from the dog, and whizzed upward as if bound for the stars. My triend’s first barrel decimated the banded feathers of its broad, outspread tail, and he caught it with his second barrel as it was speeding its bobtailed career high among the branches of the old oak trees. As it fell, another bustled, with riotous hubbub, almost from the same spot from which the last one rose, and wheel- ing, with its breast, mottled with black and white, in full view, cleft the breeze so fast that the shot from my gun was held back by the resistance of the air waves. At least that was my theory then, and it ought to suffice at this lapse of time. Some ten minutes passed away, and we found Frank Some ten minutes passed away, and we found Frank anchored apparently to a stump in a little ravine far up the hillside, with Jack indorsing his draft on our confidence with his most statuesque attitude, about thirty yards behind him. RUFFED GROUSE SHOOTING. The birch was waving in the breeze above him, and the ferns were swaying gently below his nose, the raspberries and blackberries were still bright on the bushes in the ravine, and the young oaks were as green as in the spring, but other signs of life there were none. We threw stones in ahead of the dog, but nothing moved. We tried to urge the dog to flush them, but he would not budge. At the risk of losing a shot I went in, for the ravine was deep and steep-sided. A few feet ahead of the dog I slipped and fell, and in a twinkling the sky above me seemed alive with roaring wings and meteors SHOOTING THE WOODS GROUSE 307 of white and black and brown mixed in a whirl that made the air tremble even more than the thunder of my companion’s gun, which was spouting flame and smoke above my head. When I recovered myself I found that four birds had made all the uproar, and that my friend had pacified two of them. The grouse were so scattered that it was better to The grouse were so scattered that it was better to search for a new flock than to try to find the single birds that had flown far up and down the hillsides. So we moved along several hundred yards until we came to a broad-bottomed ravine. Along the hills near its head the oaks stood larger and closer than before, the ferns were brighter, longer and greener, the birches were taller, and maples and aspens were jostling them aside. A soft fragrance of wild honey and thyme haunted the dark, cool shades, and everything hinted strongly of the favorite home of the ruffed grouse. Old Jack at once took the hint, and with gingerly Old Jack at once took the hint, and with gingerly tread went marching up the bottom of the ravine, with nose aloft and slowly undulating tail. Though he had yet smelt nothing, the spirit of the place whispered grouse so strongly that his fancy kept him on a half point from the start, just as many a good old dog’s imagination makes him change his pace the instant he enters a dark, damp swamp, where everything breathes the magic word, woodcock. RUFFED GROUSE SHOOTING. And even Frank seemed enthralled by the cool, green, silent shades, and threaded the birchen bowers and the beds of fern with more than usual care. But Jack went far up the hill several times, and came AMERICAN GAME BIRD SHOOTING 368 trudging back, looking somewhat dispirited; and Frank, after making his way as often up and down through the ferns, seemed as badly muddled; yet both seemed to think there must be game there. We passed around the head of the ravine, over ground that seemed especially made for grouse to spend the day in, but they seemed to have that provoking trait that game often exhibits, of ignoring the fine places you pick out for it and preferring to make its.own selection. Further down the ravine, below where the scrub oaks and ma- ples and aspens broke into the heavier black oak that robed most of the hills, and where the bottom widened out into a little valley, lay a long thicket of crab-apple and wild plum, edged with black haw and hazel where it broke into the oak and maple of the hills. Knowing that the birds ranged low as well as high, along these hills, we went to it. The dogs soon disappeared within the dense green shrubbery, and naught was heard of them in a minute or more but the light rustle of their feet. And not another minute seemed to pass away before that, too, ceased. Leaving my friend on the outside, where he would Leaving my friend on the outside, where he would be able to get a shot at anything that came out, I went into the thicket. There stood Jack, bent: like a bow, with tail and jowl nearly parallel, as he had evidently thrown himself with a sudden whirl, upon striking the scent from one side. And a few yards behind him, half hidden in the deep green, stood Frank, with the solemnity of a tombstone on a winter night. As I stopped behind Jack there was a bewildering burst of SHOOTING THE WOODS GROUSE 369 uproarious wings, and a dozen or more birds went darkliing through the green, some wheeling out of the top, some scudding straight away, some darting low toward the edges. RUFFED GROUSE SHOOTING. Quick as a flash I dropped on one knee and sent a charge through the leaves where one’s fanlike tail was vanishing on a sharp curve as I raised the gun. But by the time the shot reached there it was gone, and by the time I discovered it was gone the rest were all gone. But dimly through an opening 1 could see my friend on the hillside, with half a dozen grouse swiftly driving toward him. One went past him like an arrow feathered with white and brown, and was gone before he could raise his gun. Another, whirling into sight above the brush, with its full white breast, broadly mottled with black, brightly flashing in the sun, just a trifle too late for me to shoot at, went spinning by him with unruffled feathers at the report of his gun. And then five or six more went roaring on past, and above and behind him, while he, in con- fusion, shifting his gun from one side to the other, and hardly knowing what to shoot at, let them all go by, and stood as if looking for more to come. Few American sportsmen have had so much expe- Few American sportsmen have had so much expe- rience in upland shooting as Mr. B. Waters, whose excursions have covered the game fields of almost the whole United States east of the Rocky Mountains. Famous as a handler of hunting dogs, and equally fa- mous as a crack shot, both at the traps and in the field, his views on the shooting of any game bird will receive respectful attention, for he is past master of the art. AMERICAN GAME BIRD SHOOTING 370 This is what he has to say about ruffed grouse shooting : From the time when the mind of man runneth not to the contrary in matters of shooting for sport, the ruffed grouse, by common consent, has been classed with the most difficult of game birds which the sports- man endeavors to bring to bag under the approved conditions of sportsmanship, if indeed it be not the most difficult of all. RUFFED GROUSE SHOOTING. For it taxes the sportsman’s nerve, patience, skill, woodcraft and endurance as no other bird can tax them; and all these requirements are necessarily supplemented by a gun of good killing powers, one selected with special reference to cover shooting; and last, but not least, a dog of more than ordinary intelligence and good intent and good train- ing, if the sport is to have any successful results and pleasing finish in its action. If any element of the sportsman’s ruffed grouse craft be missing success is marred accordingly. The ruffed grouse, in every art and article, is a bird The ruffed grouse, in every art and article, is a bird to fill the sportsman’s ideal. Its habitat is in nature’s most picturesque setting; the bird is beautiful in its delicate tracings and markings, and rich and varied in its colorings; racy of form and faultless in sym- metry; wild, dashing, daring, alert, and infinitely re- sourceful in its crafty devices when pursued; exclu- sive in its habits, and, withal, a bird of rare excellence for the table, its flesh being of a delicate texture and pleasing flavor—so palatable, indeed, that it is by many epicures more highly prized than is the flesh of any SHOOTING THE WOODS GROUSE age other game bird. With those who may vaunt the ex- cellence of the woodcock, the snipe, the prairie chicken, the duck, the turkey, etc., it also holds a high place in their esteem; and the exceptional man, whose fancy for one particular kind of bird prejudices him against all others, will not speak unkindly of it. And yet, delicious as it is when properly prepared for the table, it can easily be spoiled by ill cooking, and of bad cooks there is no end. The art of cooking it properly is as rare as is the skill of killing it properly. RUFFED GROUSE SHOOTING. If it be cooked too much, or if it be cooked improperly, it loses much of its rich delicacy of flavor and texture, and becomes dry and unpalatable; and in that unfortunate condi- tion it probably was when that eminent authority, Wil- son, partook of it, and thereafter, in his “American Ornithology,” wrote of it: “At these inclement sea- sons, however, they are generally lean and dry, and, indeed, at all times their flesh is far inferior to that of the quail or of the pinnated grouse.” Yet, as tastes are not all alike, the superlative will probably be placed | according to the individual fancy in matters of food, as in all other matters, and it is well that it is so. If all fancied alike, all would be monotony. Nevertheless, a man who cannot have a culinary spell cast over him by a skilfully cooked ruffed grouse, it having been kept a proper length of time after killing—not too long— has no music in his soul, and may not even be fit for treason and spoils. The ruffed grouse chooses rough and timbered sec- The ruffed grouse chooses rough and timbered sec- tions, for it is strictly a bird of the woods and thick- AMERICAN GAME BIRD SHOOTING 372 ets, preferring the roughest parts of a hilly or moun- tainous country, and of these it many times selects the densest recesses, or the timber of seamy and rocky hillsides, or where ledges, fallen tree-trunks and tree- tops in the woods, secluded from man, guard against intrusion, and even the timbered swamps are not ob- noxious to it. For man it has the most uncompromising aversion. It selects its home in the places least frequented by him, though once the home is determined on it holds to it with dauntless persistency, let the gunner disturb it as often as he may. In choosing its habitat it prefers that it be near a In choosing its habitat it prefers that it be near a supply of good water and an abundance of good food, for it is a good feeder. Whortleberries, blackberries, beechnuts, acorns, chestnuts, partridge berries and buds are readily accepted as food in their proper season. The bud of the laurel is said to render the flesh poisonous for food purposes, though the belief seems to rest more on tradition than on any direct evidence. RUFFED GROUSE SHOOTING. Unlike the quail, which prefers to make its home near Unlike the quail, which prefers to make its home near the homes of man, and the prairie chicken, which sticks closely to the grain field, the ruffed grouse is ever in- tent on choosing its home and haunts distinctly apart from those of man. In the East it is called “par- tridge’”’; in sections of Pennsylvania and the South, “pheasant.” In the breeding season, when it has been free from In the breeding season, when it has been free from pursuit and harassing alarms, it sometimes strays a short distance from cover into the adjacent fields, SHOOTING THE WOODS GROUSE 373. where grow palatable huckleberries and blackberries, though rarely venturing further than a short flight, and often but a few yards from cover. Though always a wary bird, and ever avoiding man, Though always a wary bird, and ever avoiding man, it is not so wild and quick to take wing before the frost and unsettled weather of fall set in as it is afterward; yet if the gunner disturb it once or twice, the full wildness of its nature, and its constant alertness to avoid man, are fully and permanently aroused. Then man and the places he frequents are shunned as much as possible. Indeed, it is not a social bird with its own kind. After the young birds have matured they separate, and in the fall the gunner will find them in ones and twos, and at rare times in threes. Given to the sportsman the conditions of an open field, and therein a ruffed grouse on the wing, within range, then the difficulties of killing it are but little, if any, greater than those which obtain in the killing of a prairie chicken on the open prairie; though whether in open or cover, the ruffed grouse is always swift and decisive in its flight. But in the open, whether it be on field or prairie, there is an even light and an unobstructed view. Then, for safety, the bird can rely only on its swiftness of wing, all too slow when pitted against the sportsman who can, under those circum- stances, with studied quickness and deliberation, com- mand a large circle around him. RUFFED GROUSE SHOOTING. Thus the ruffed grouse is at a fatal disadvantage when shot at in the open field, as is also every other bird pursued under the same conditions; but these conditions are rare in- AMERICAN GAME BIRD SHOOTING 374 deed in ruffed grouse shooting, for, as mentioned be- fore, it ventures into the open only on such infrequent eccasions as it is tempted to search therein for food, and then only in places seldom invaded by man, where it fancies there is freedom from pursuit. To all fixed habits there seems to be an exception for a short pe- riod in the fall, when it is subject to a crazy wayward- ness. While in the open field it is strong and swift of wing, in cover it is at its best. It will, on occasion, dash through the densest thickets with apparent ease, with no diminution of its swiftest speed, seemingly having a charmed manner of flying through tree-tops and thickets as if they were but phantom trees of the woodland, or shadows offering no obstruction to its onward flight. And in its favorite haunts it is a master of self- And in its favorite haunts it is a master of self- defense. It can utilize thickets, trees, old fences, ledges, stone walls, swift wings and endless cunning to evade its pursuer. Be the position of the shooter what it may in reference to this bird in cover, it, when flushed, takes instant advantage of the nearest thicket or the trunk of a tree or old fence, keeping one or the other between itself and the gunner in its line of flight, thus in a great measure blocking all opportunity to shoot, or at least hampering the shooter greatly, and oftentimes causing a miss. The bird, in most instances, times its rise so as. to The bird, in most instances, times its rise so as. to have the advantage of some nearby object as a shield to its flight. On occasion it will display a courage SHOOTING THE WOODS GROUSE 375 bordering on audacity, permitting the shooter to pass close by, and flushing after he is some yards further onward. This wile is oftenest practiced after it has been flushed, marked down and pursued. Both man and dog are apt to pass it then, though they may fol- low in the exact line of flight. RUFFED GROUSE SHOOTING. The shooter may hear the irritating roar of the bird’s wings behind him, on ground but a moment before passed over, or catch a shadowy glimpse as it dashes away from some tree- top. Owing to its short flights and its proneness to take Owing to its short flights and its proneness to take a straight, or nearly straight line, the persistent shooter may be able to mark and flush the bird again and again. It sometimes, in repeated flights, returns to near the place where it was first found, and it always takes the flights so that ground and cover are to its advantage in avoiding danger. Once in a while a foolish bird will be found, which will do the very thing it ought not to do, commonly paying for the lapse with its life; so that if there is anything in the theory of heredity, the ruffed grouse should be uniformly of high capabilities, the fool birds being killed promptly, and never breeding. By far the greater part of the shooting is at close By far the greater part of the shooting is at close range, as it needs must be in thicket or woods, where the longest views are short, and obstructed by trees or ledges or the undergrowth or the hilly nature of the ground, where in the early season the view may not be greater than a few yards or feet, if the leaves have not fallen. AMERICAN GAME BIRD SHOOTING 376 It then is not an infrequent occurrence that the shooter will hear the startling whir of wings close by him, and yet be unable to shoot or to mark the bird’s course, from inability to see the bird at all. The light of the woods, broken and broken again, as it is through the irregular openings in the tree-tops, and branches and leaves interposing, with here and there shafts of clear light, and masses of shadows interspersed every- where, tends to interfere with quick and clear vision, and adds to the difficulty of accurate shooting, thus differing widely from shooting in the open. The successful ruffed grouse shooter must be ever The successful ruffed grouse shooter must be ever promptly ready to shoot, and further, must be quick of eye and action. RUFFED GROUSE SHOOTING. He must instantly decide on the manner of making the shot, taking advantage of all the few opportunities offered, and avoiding the ob- structions which interpose. No studied effort at aim- ing is possible. Cover shooting of all kinds requires quick action, but ruffed grouse shooting requires the quickest. Of all snap shooting, ruffed grouse shoot- ing is the snappiest, and the successful shooter of that bird must exce! in that kind of shooting, since in most cases he will have but a brief instant’s glimpse of the bird in the unfavorable mixed lights of shadows and cover. For this shooting the gun should be light, short of For this shooting the gun should be light, short of barrel—26 to 28 inches—and a cylinder bore, for a full choked barrel is entirely out of place in such cover shooting, equally unsatisfactory when it does or does ‘aspoyH ‘yO Aq paydeis0qjoug “HOBI}SeWIOp UI paival Y909 asnoIg payny ‘yO Aq paydeis0qjoug “HO UI paival Y909 asnoIg payny SHOOTING THE WOODS GROUSE 377 not kill, it being a miss in the first instance, and often a badly mutilated bird in the second. The average shooter will find that he has success far below his opportunities, even when equipped with the gun most fitting for the work. In this shooting there is no waiting for opportunities to fit the gun. The successful hunter must take the shots that are The successful hunter must take the shots that are offered, and as they are offered; it matters not how difficult they appear or how brief the opportunities may be. He may catch but a momentary glimpse of the bird as it crosses some diminutive opening, or he may see it for an instant in a maze of leaves and branches, or he may get a partial glimpse of it and some dis- turbed leaves in the course of its flight, yet these are the opportunities which are the most numerous, and which must be relied on for the bulk of the shooting; in short, that is ruffed grouse shooting. RUFFED GROUSE SHOOTING. If the shooter be too indolent or apathetic to be ever ready to shoot, or if he is too slow to take advantage of the opportunities, his success will be but meager so far as material results are concerned, though he may be greatly encouraged by the belief that his last ill success was due to faults in the bird, and that if he can have another opportunity he will acquit himself nicely. The opportunity comes, and failure again evokes more excuses. Once in rare whiles the shooter will have a good opportunity, catching the bird in some corner so favorable that the advantage is with the shooter; but such instances are rare indeed, and by themselves would make but little sport. 378 AMERICAN GAME BIRD SHOOTING To be ready for the opportunities the sportsman must be quiet, and never relax his vigilance, and his gun must be so held that it can instantly be brought into position to shoot. The nerves of the shooter must be constantly at a high tension in readiness for the rise of the bird and the instantaneous shot. Every faculty must be at a high key. The very moment that the shooter relaxes his attention will be the moment that the bird will rise, and before the sportsman can get ready the opportunity is gone. It will be seen that the man who dawdles with his gun, who is slow in the handling of it, or who is noisy, cannot hope for any satisfactory success in shooting the bird of game birds, the ruffed grouse. On the other hand, he can be keyed up to too high a pitch, overready when the bird rises. A nervous flurry does nearly as much to disar- range the desired results as does the more indolent dawdling. There are those, however, who can never overcome the nervous start at the roar of the bird’s wings, though they may be perfectly undisturbed in any other bird shooting. And the skill of the shooter, be it ever so high in degree, must be supplemented by the work of a quiet, well trained, industrious, intelligent dog, for the shooter is much better off without any dog at all than with one that is riotous or one that ranges too far or that is heedless of his work. Loud orders to the dog have no place in ruffed grouse shooting. RUFFED GROUSE SHOOTING. The sports- man himself cannot observe too great a silence. The human voice alarms and puts the birds to flight. SHOOTING THE WOODS GROUSE 379 The work required of the dog in this shooting is dis- tinctly different from that required in any other kind of bird shooting, except, perhaps, woodcock shooting, which in a way it resembles, though a higher degree of dog intelligence and obedience are required, as the ruffed grouse is far more cunning and wary than the woodcock. The “partridge dog” should not work far from the gun in cover, and he should be silent and diligent in his quest. Many experienced shooters highly com- mend the use of a small bell tied to the dog’s collar, its low tinkling constantly indicating the dog’s where- abouts in the thick cover; and generally, when the bell stops, it indicates that the dog is on point, thus in a way keeping the shooter posted by ear as to his dog’s doings and whereabouts. The rattle-headed, highly nervous dog, or the one The rattle-headed, highly nervous dog, or the one which gallops swiftly and merrily about, is distinctly out of place in this kind of shooting. The esthetic shooter, whose dog must carry a high head and a tail lashing his sides merrily as he gallops and bounds about in the ecstasy of his enjoyment, as the dogs many times do in the idealists’ tales of great work afield, would better take his fiery dog into the open, where he can better disport himself unhampered, and where his pretty ways may be admired without any unpleas- ant interposition of the ruffed grouse. Such manner of the dog’s seeking is incompatible with ruffed grouse shooting, for the shooting should be the dominant fea- ture, not the joyousness of the dog. AMERICAN GAME BIRD SHOOTING 380 A dog of fair gait and persistent industry can easily beat out the necessary range, and the one which makes his quest patiently and soberly and quietly, working with judgment and honesty to the gun, will bring the shooter satisfactory success in the results, to say noth- ing of the incomparable comfort and pleasure in shoot: ing over him. RUFFED GROUSE SHOOTING. Nine out of every ten dogs which are running with high head and merry actions are running because they are in high spirits and for their own pleasure, with no thought of the birds or of work to the gun. When they come on birds this is often a matter of chance, and their point work is marked by deplorable errors. This kind of dog leads his partial master to believe that when he wears off the wildness and wire edge he will steady down to a useful grade of work; but often when such dog has worn off his exuberance he has worn off all there is in him of field performance, and he either loafs, or does his work in the same slov- enly manner, though, loafing, he does less of it. In shooting for sport, the shooter takes his birds on the wing. Of course, in shooting for market, the market shooter has no thought of sport or its prac- tices. His one object is to kill the bird and bring it to bag. The manner of it is of the least importance. His theory and practice are founded on commercial principles; therefore, he shoots his birds as he can, whether they be on the limb of a tree, the ground, or flying. Some hunters have dogs trained to seek for the Some hunters have dogs trained to seek for the SHOOTING THE WOODS GROUSE 381 birds, and, finding them, they flush and follow them. When flushed by the dog, the bird generally takes to the trees, and the dog, barking, so engages their atten- tion that they fall an easy prey to the hunter, who often bags every bird in the covey under such circum- stances. Often, when flushed by the dog, they fly to the tree-tops immediately overhead, where, in fancied security, they watch the dog. The shooter then drops them one by one, taking the lowest birds first. The falling of the lowest ones does not disturb those above, though if a top bird is dropped the others fly away forthwith. As to the number a shooter can kill in a day, so much depends on the shooter’s skill, the bird supply, and the local shooting conditions, that these things alone determine it. RUFFED GROUSE SHOOTING. In some sections of New England, two or three In some sections of New England, two or three birds at the end of a day of diligent effort is consid- ered a highly successful result, and it is not an infre- quent occurrence in that section that a diligent day may have no birds at all at its ending. In certain sections of New York, Wisconsin, Minne- sota, Dakota, etc., and in the mountain sections where the ruffed grouse abound, such a bag would be consid- ered an absurdity, if held forth as an index to good shooting, industry and superior results. While in North Dakota I have heard of one bag While in North Dakota I have heard of one bag of eighty birds, made to one gun in a day, something extraordinary. They were shot at the air holes along the banks of the Red River, after it had frozen over, AMERICAN GAME BIRD SHOOTING 382 the birds coming to those places for water. This was not recounted to me as a matter of sport, nor is it so set forth here, but will give an idea of the numbers of the ruffed grouse in the sections where it is in the greatest abundance. But the sportsman who seeks the ruffed grouse for But the sportsman who seeks the ruffed grouse for the true sport of it has a more exalted pleasure than comes from shooting any other game bird. First of all, he must be skilful with the gun, and when he shoots, be he ever so skilful, he can only apply such skill as he can muster in a moment, the opportunities of ruffed grouse shooting being but mere fragments of the opportunities accorded to shooting in the open. When at length the bird is brought to bag it repre- When at length the bird is brought to bag it repre- sents a toiling through brush and bramble, wooded hill and dale, scrambling over ledges and floundering through swamps, all colored by constant expectancy, unavoidably lost opportunities, and seeking to circum- vent the birds by cunning woodcraft, supplemented by the powers of the dog—a degree of cunning, skill and persistent effort greater than that required in the shoot- ing of any other bird. RUFFED GROUSE SHOOTING. It is shooting pitched in the highest key, and that is why I think the shooter can justly feel a greater glow of pleasure when he makes a successful shot at ruffed grouse, and why he loves this sport above all others, since it tests to the utmost his skill, his wood- craft, his patience, his endurance and his dog; and of the dogs, if he owns a good one, he owns one of a thousand. SHOOTING THE WOODS GROUSE 383 Many sportsmen will not agree with Mr. Waters’ be- lief that a ruffed grouse in the open is as easily brought to bag as a prairie chicken. They even declare that the ruffed grouse flies three times as fast as his pinnated cousin, and is at least twice as difficult to hit. In his recent volume on “Life and Sport on the North Shore of the St. Lawrence River,’ Mr. Nap. A. Comeau says: “Ruffed grouse shooting in this section is not sport, “Ruffed grouse shooting in this section is not sport, and is not regarded as such by the residents, for the reason that neither the people nor the birds have been educated to it. I can count on less than the fingers of one hand all the men I know on this shore who will deliberately flush a grouse to shoot it on the wing. As for the birds themselves, unless they happen to be in an open spot, they will not fly any distance. In the woods, which are pretty dense here, when flushed they simply rise off the ground, perching in the nearest tree and stretching their necks to see you walking under them. If it happens that a covey is started they will frequently be all killed without any of the others around taking flight. Many a time when in the woods trap- ping we would not waste a shot on them, but simply go to work and cut down a small sapling, tie a noose or string at one end, slip it over their head and pull them off the branch. At other times, for amusement, we would go out with a bow and a blunt-headed arrow and whack them off the trees at twenty feet range, which is about the usual one that they are shot at here. “What a contrast to the educated ones! RUFFED GROUSE SHOOTING. Some years AMERICAN GAME BIRD SHOOTING 384 ago I received an invitation from C. Beatty, of Platts- burg, Lake Champlain, to go and have a few days of mixed shooting with him. It was late in September, but most of the leaves were still on the trees. The first day we had a grand duck shoot on Missisquoi Bay, and after that an outing for woodcock. The last day had been reserved for partridge and gray squirrels. We had breakfast at daylight and were off. We had not far to go to reach our ground—patches of hardwood trees with a good deal of underbrush. We soon heard some whirring off at our approach, but could not even get a glimpse of them. After a time I got a crossing shot at one, over fifty yards away, which I bagged, and that was the only bird we got. But we surely heard a dozen or more rising. I was simply astonished that such a bird could be so shy. When I came back here and told the natives about my experience, they thought I was pulling the long bow. I believe it will be many years before our birds get so highly educated.” DUSKY GROUSE SHOOTING To my mind, the most splendid of the many Ameri- can grouse is the dusky, or blue, grouse found in the Rocky Mountains, north to Alaska, and west, at vari- ous points, to the Pacific coast. To be sure, it is not as large as the sage grouse, yet it is a big bird, some- times weighing up to four pounds, and nearly two feet long. Its tender and delicate flesh is always good eating, and its habits of life, in underbrush and timber SHOOTING THE WOODS GROUSE 385 along the mountain side, make it seem much more a bird for sport than the larger sage grouse, which is found on the dry, hot, open prairie. In these days, when cities, towns, villages and farms In these days, when cities, towns, villages and farms are scattered all over the range of the dusky grouse, there must be a multitude of men who follow this bird with dog and gun, and shoot it much as people in the Eastern States and the Mississippi Valley shoot the ruffed grouse; yet, curiously enough, we hear very little of killing this bird in a sportsmanlike manner. Shooting the dusky grouse before the broods are Shooting the dusky grouse before the broods are full grown, and when they are more or less scattered out to feed, is but tame sport. The birds lie like stones, and fly straight and easily, dropping at .a touch of the shot. In September and October, when they are full grown, it is quite different, however. Then they are strong of wing and fairly well able to take care of themselves; all the birds are large, and while they still present a fairly easy mark, they fly with great swift- ness, and from the situations in which they are often found the shooting calls for readiness and care. In the old days, when no one in the West thought In the old days, when no one in the West thought of carrying a shotgun, it was often necessary to kill birds for food, and then the young broods of dusky grouse often gave one a little practice as they stalked ahead of one up the valley or stood on the branches of the trees of the mountain side. DUSKY GROUSE SHOOTING At a distance of twenty or thirty yards, provided one thoroughly knew his rifle, it was considered fair shooting to knock the heads off four out of five of these young birds. In- AMERICAN GAME BIRD SHOOTING 386 evitably there was a miss now and then, when a walk- ing bird would unexpectedly stop, or would move its head to one side just as the trigger was drawn; but as I say, we used to be able to cut the heads off four out of five. In the same way, when a brood of dusky grouse flew into a tree, and stood there unfrightened by the report of the rifle, a number of them could be secured, pains being taken always to shoot the lowest bird—in def- erence to an aged tradition—in order that others might not be alarmed by a fluttering body falling close to them. One of the best morning shootings that I ever had at dusky grouse was in northwestern Montana, on one of the high benches that overlook the St. Mary’s Lakes. It was a rounded knoll—an old lateral mo- raine—a mile or two long, once overgrown by aspens, which had been killed by fire and had now fallen and rotted. A new growth of aspens, just starting, reached only about up to the knee. Among these little aspens grew huckleberries, and the ground was more or less carpeted with the vines of the bearberry—the smoking weed called “larbe,” perhaps a corruption of the trap- per French word ’herbe. On these berries several broods .of grouse were feeding, and after camp had been made near the upper end of the knoll I took my shotgun and walked back over the ground where sev- eral birds had been started. It was not long before, with a thunderous roar, a It was not long before, with a thunderous roar, a full-grown bird rose but a few yards before me, and, ‘UIe}JUNOM 8q} UO asnoIg AYsNG = - , od sy ‘UIe}JUNOM 8q} UO asnoIg AYsNG SHOOTING THE WOODS GROUSE 387 scaling off on the fresh breeze that was blowing down the lakes, was thirty or forty yards off before the heel- plate touched my shoulder. Luckily, I held on the bird, and the center of the load hit it so that it fell quite dead. DUSKY GROUSE SHOOTING At the report two more rose near me, and this time I brought my gun to bear a little more quickly, and killed the second one. Two or three more rose at this report and flew down the lake, but they did not fly the whole length of the knoll, and I marked them down. It was evident that if this lasted I was going to have good shooting, and so it proved. I went on, more slowly and carefully, and tried to pick my shots. As I had no dog, only the birds immediately in my way got up, except that sometimes, when a shot was fired, several rose ahead or to the right and left, and most of these pitched off down the hill, and, scaling off on the wind, reached cover in the thick aspens of the lower ground, where I knew it would be useless to follow them. Before I had reached the lower end of the knoll I had more birds than I could carry, and I was not sorry to see my companion coming after me on horseback to relieve mc of the load. The shooting that he had heard had notified him of the sport that I was having, and he rightly concluded that I would need help. I was interested to notice, as he came toward me, that he put up a number of grouse, though his route was not far from the one I had followed. When we had tied our birds on his horse it was When we had tied our birds on his horse it was AMERICAN GAME BIRD SHOOTING 388 found that I had already ten, which I estimated to aver- age not far from three pounds apiece in weight. Just at the end of the knoll, and as we were about to turn back to go to camp, a grouse jumped up before me, at the foot of a clump of aspens, and dived into them, precisely as a ruffed grouse would pitch into a piece of underbrush. Just as I should have snapped at a ruffed grouse, so I snapped at this bird, and a moment later a loud splash in the water, and a muffled drum- ming, told that the shot had reached him just as he was about to cross the river. DUSKY GROUSE SHOOTING My companion went down, and riding out into the water, picked up the eleventh bird. A little later, on the way home, another grouse A little later, on the way home, another grouse sprang from some low aspens at some distance in front of me and pitched into a growth of pines, and this one I snapped at, again, but not with the success of the former shot, for the bird passed through the pines and flew a long way to a little island, where he seemed to go down. If I had had a dog and a good shooting pony I could undoubtedly have killed forty or fifty birds in this one place; but forty or fifty would have been in- excusable slaughter, since there were but two of us in the camp, and we could not have used anything like that number of birds. As it was, those that I got that day lasted us for quite a long time, and most delicious food they were. The white, juicy flesh, sweet, and well flavored from the diet of berries on which the bird had been fed, was most delicate. Properly cooked, no SHOOTING THE WOODS GROUSE 389 bird is worthy of higher commendation than the dusky grouse. This is especially true when the birds on the lower plateaus are feeding on the tiny red huckle- berry that grows in such profusion in the pine woods. While the females are down in the lower ground, attending to nest building, hatching, and the rearing of their young, the old males and the barren females resort to the higher land, often being found on the mountain sides far above timber line. From such places they are often startled by the goat or sheep hunter, and pitching down from these great heights take long flights, at last bringing up down among the timber, and flying so far that no one knows just exactly where they go. Nowhere, so far as my limited experience goes, 1s Nowhere, so far as my limited experience goes, 1s the dusky grouse pursued in so systematic and sports- manlike a manner as on Vancouver Island, near the beautiful city of Victoria. DUSKY GROUSE SHOOTING My shooting of them there dates back many years, and it may be that in recent years the sportsmen of other parts of the Pacific coast have taken to shooting this splendid bird over dogs, as in old times they did near Victoria. What good shooting there was at Victoria twenty- What good shooting there was at Victoria twenty- five years ago, and how varied the bags used to be! There were the pheasants rising like an explosion of fireworks, sometimes from under your very feet, and seeming—after you had ineffectively fired both bar- rels in the air in your fright—to wave at you in de- rision long brown tails that you almost felt you could grasp by reaching out the hand. There were blue AMERICAN GAME BIRD SHOOTING 390 grouse, big, and straight flying, getting up with a roar, and almost at once plunging into the dense thicket. There were ruffed grouse, simple birds, that you some- times saw walking about on the ground not far from your feet, but ready enough, after they had been pointed by the dog and kicked out from their hiding place, to practice all the arts that their cousins use three thousand miles away. Then, finally, there were the California quail, big flocks of them, more often heard running through the underbrush than seen, yet sometimes rising in thick flocks and darting off like little blue bullets through the timber. It was here that, in company with two or three Vic- toria sportsmen, I first saw dogs used on the blue grouse; not always with success, for two wild young puppies, blundering excitedly through the underbrush and the heavy green forest, flushed the birds, some of which took refuge in the branches of the tall cedars or Douglas firs, quite out of reach of the shotgun. There was one old white setter, however, which re- garded the younger dogs not at all, but trotted method- ically through the forest in businesslike fashion. To him and to his owner I attached myself, and during the day had the opportunity to see him point half a dozen birds in most workmanlike style. The grouse lay well, and did not run ahead of the dog, as an edu- cated ruffed grouse would have done. DUSKY GROUSE SHOOTING At the same time, when flushed, the birds displayed wonderful quickness in putting some object between themselves and the gun; though in this case, as there were two SHOOTING THE WOODS GROUSE 391 guns, the stratagem was not always successful. No great amount of wisdom was required to circumvent these birds. They had not been subjected to the con- stant pursuit suffered by the ever persecuted ruffed grouse of our eastern covers, and did not resort to his puzzling devices. They afforded great sport, but the shooting was very destructive to the birds. On the other hand, the thick cover which prevailed over much of the forest did not permit following up the birds, and if not secured on the first rise they were not seen again. No doubt at the present time the dusky grouse are frequently shot over dogs in the Rocky Mountains. Any dog which had been broken on ruffed grouse would be serviceable also in finding its larger relative. Yet as the dusky grouse has been, until recently, altogether without education in this respect, the extreme caution needed in a dog used for ruffed grouse shooting would scarcely be required for the dusky grouse. It would be interesting to know whether, in modern It would be interesting to know whether, in modern days, the ruffed grouse, or the dusky grouse of the north Pacific coast, had been sufficiently pursued to acquire a wisdom which men of the eastern part of the continent usually expect the ruffed grouse to possess. PTARMIGAN SHOOTING. Ptarmigan are never shot for sport, but only for food. Occasionally a few birds may be secured on the wing, but usually the ptarmigan is not found in situa- tions where the gunner is able to pursue it in a sports- manlike manner. Commonly he is traveling, is with- out a shotgun, and is thinking more of filling his pot for the night’s meal than of giving the bird a chance to escape. Practically the only exception to this rule is Newfoundland, where, under the name of partridge, the ptarmigan is commonly shot over dogs, and gives excellent sport. Mr. Comeau, in his “Life and Sport on the North Mr. Comeau, in his “Life and Sport on the North Shore,” speaks most interestingly of the migrations of the willow ptarmigan, and tells of the extraordinary numbers of them that are killed. He says: “During the last two migrations, taking the best “During the last two migrations, taking the best years, 1895 and 1904, I took some trouble to try and find out, approximately, how many birds were killed between certain points. During the first year men- tioned, between Mingan and Godbout, 175 miles of coast, 30,000 were killed; in the second (1904), 14,000; and I am sure that during 1885 nearly 60,000 must have been shot or snared. When a flight begins, every man, woman and boy able to handle a gun is out. To avoid accidents, which are very rare, indeed, 392 392 Willo Ptarm Wil Pta PTARMIGAN SHOOTING 393 each gun occupies a certain point or station, and shoots at all the birds that pass or alight in his vicinity. The ladies keep watch for those that may alight near the houses. The bags vary, of course, according to the skill of the shooter and his method of shooting. If he is there for business he will take all the pot shots. He can frequently get five or six in one shot. I have seen fourteen killed in a single shot. A few will only shoot on the wing, but there are many days when the wing shooter comes out ahead. The biggest bag I ever made (it was in 1885) shooting at flying birds was eighty-two brace in one morning. At Caribou Island, that winter, nets were tried, but they were not very successful, more being got by shooting. PTARMIGAN SHOOTING. Indians frequently snare them by setting their snares around willow clumps where the birds feed. It is a very sim- ple arrangement. A twig is stuck in the snow, a twine snare is tied to it, a very light support placed under it to hold it in position, and it is ready. In walking around the bird runs into it, then tries to rise on feel- ing the snare, only to tighten the noose. There is a little fluttering, and it is all over.” Such is the abundance of the Such is the abundance of the willow ptarmigan, and sometimes of the rock ptarmigan as well, though this last is not so numerous, and such is the way in which they are killed for food. Rarely it happens that Rarely it happens that the earnest ornithologist or the big-game hunter in search of a new sensation toils laboriously up to the home of the white-tailed ptarmi- gan, carrying his shotgun with him. When he reaches AMERICAN GAME BIRD SHOOTING 394 the haunt of these little birds he is very likely to be disappointed. Either, on the one hand, they trot along before him, refusing to rise on the wing, or else they are so shy that long before he is within gunshot they take wing, and fly off along the mountain side, only to repeat this operation when again approached. Like all the grouse, the white-tailed ptarmigan, when Like all the grouse, the white-tailed ptarmigan, when almost fully grown, are very gentle and unsuspicious, and may be approached within a few feet; but as the nights grow colder and the autumn storms begin, they grow more and more shy. Finally, in September or October, they are wild birds, often rising at good gun- shot distance, flying thirty or forty yards, and, alight- ing on the hillside or on some great rock, walk about with head and tail held high in air, in an attitude of great suspicion. If by chance they are closely ap- proached when on a steep hillside they often throw themselves into the air and scale downward with great swiftness, sometimes continuing their flight across a narrow valley, so that it is quite impossible to follow them. PTARMIGAN SHOOTING. In the days of their youth, before they have grown shy, many are killed in the mining districts of the high mountains with stones or clubs, by miners going to and from their work. In Newfoundland, the willow ptarmigan, and occa- In Newfoundland, the willow ptarmigan, and occa- sionally the Newfoundland form of the rock ptarmi- gan, used to afford superb shooting over dogs; but as with so many other game birds in many places, they have been so overshot there that it is now reported PTARMIGAN SHOOTING 395 that they are comparatively few in number and that good shooting can no longer be had. What it was in old times may be inferred from an article long ago contributed to Forest and Stream by M. Harvey, who, formerly wrote so much about Newfoundland, and, as one of the historians of the island, did much to make it better known to the world at large. The sub- stance of the article is as follows: The ptarmigan or partridge (“patridge” in our local vernacular) of Newfoundland is quite equal to the Scotch grouse, and, indeed, resembles it so closely that it is difficult to make out any specific difference be- tween the red grouse, gorcock or moorcock of Scotland and the ptarmigan of Terra Nova. They are a most delicious article of food, whether roasted, stewed; or in white soups. All visitors to our shores admit that the flavor of a plump partridge, well cooked, is unsur- passed in richness and delicacy. They are of respect- able proportions, a brace of them, in season, weighing from three pounds to three pounds and a half. When the shooting season opens, on the first of September, they are in prime condition after feeding on the wild berries, the partridge berry and cranberry being their favorite food. In certain localities they are very abun- dant, and to the sportsman there can be nothing finer than a day’s partridge shooting over our breezy bar- rens during the fine autumn weather. The air is then cool and exhilarating, and the skies bright. The weird and charming scenery is varied by countless lakes or AMERICAN GAME BIRD SHOOTING 396 ponds and by low, rounded hills, covered to the sum- mit with the tapering firs. PTARMIGAN SHOOTING. The lakelets are bright with the white and yellow water-lilies ; between the summits of the bold headlands along the coast glimpses are had of the restless Atlantic. All these natural beauties, combined with the excitement of the sport, furnish to the lover of nature a day of rapturous enjoyment. It is a thrilling moment to the sportsman when, gun It is a thrilling moment to the sportsman when, gun in hand and dog at foot, he finds himself among the partridge coverts. His faithful dog scents the game, and every nerve in his frame quivers as, step by step, he thoughtfully and cautiously advances toward the unseen covey; then, suddenly pausing, the right fore paw balanced lightly, and every limb and muscle tense and rigid, the beautiful animal is at once transformed into a marble statue. Presently a whirr is heard, and with a loud ca, ca, ca, a magnificent old cock rises on the wing. Crack goes the gun, and down tumbles the great bird, the scarlet tips over his eyes glistening like rubies, as with a thud that gladdens the sportsman’s heart he strikes the earth. Or perhaps a whole family —father, mother and children—trises at once, and the double barrels bang at them right and left, bringing down two or three brace. At times a late covey is started, the chickens of which are only two or three weeks old, just able to run smartly along the ground. It is a touching sight then to see the cock fearlessly exposing his life to save the lives of his offspring. He tumbles along the ground a few yards in advance of the dogs, rolling there in order PTARMIGAN SHOOTING 397 to decoy the sportsman from the brood, which the hen is anxiously calling into the thicket. No more touch- ing instance of paternal affection could be witnessed ; no more touching proof among the lower creation of self-sacrifice prompted by love. The poor feeble bird would almost attack dogs and men in his efforts to save his children. No true sportsman would harm a bird under such circumstances.. Only a brute would fire upon it. The dogs are called off, and father and mother ptarmigan are soon rejoicing over their res- cued family. PTARMIGAN SHOOTING. At times, the ptarmigan here are so tame that they can be killed with a stick, and at others so wild that they will not allow you to approach within gun- shot; and this is generally the case in winter, when the snow is hard and crusty, and the noise made in approaching them alarms them. They are shot at all times by our population in the distant districts, but a close time is now fixed by law, which will have a good effect when the law can be enforced. There is another species found in Newfoundland, There is another species found in Newfoundland, but it is comparatively rare—the rock ptarmigan, L. rupestris (Gmelin). It is a truly alpine species, and is seldom found below the line of stunted black spruce, except in the depths of winter, when they descend to the lowlands and feed on the buds of dwarf trees, some- times in company with the willow grouse. Our settlers call this the mountain partridge. SHOOTING THE PRAIRIE GROUSE The shooting of the prairie grouse differs widely from the same sport on the woodland species. In the one case the birds are. likely to dart into cover, dis- appearing almost at once, and snap shooting is the rule. In the other the bird rises in the open, seems to fly with deliberation, and the gunner has every opportu- nity to make all his calculations, take careful aim, and cut down the bird before he gets out of shot. Only in the case of the sharp-tailed grouse, after they have moved from the high uplands into brushy ravines or wooded hillsides, is there any similarity between the two styles of shooting. In September, after the sharp- tailed grouse are grown, they spend the middle of the day in wooded ravines or willowy bottoms, where cover is often thick, and a single gunner, who has no dog, and is trying to tramp the birds out of their places of concealment, may have to do more or less snap shoot- ing; but if he have a good dog that can be trusted to work through the bottom, the gunner can usually so place himself as to have clear and open shots. As the laws of most States now read, the prairie As the laws of most States now read, the prairie chickens—of whatever kind—are reasonably well pro- tected; but formerly, when shooting began, during the month of August, at which time the birds lie very close, it was not uncommon for two gunners to ex- 398 SHOOTING THE PRAIRIE GROUSE 399 terminate a brood, and in many places, a few weeks after the shooting season had begun, there were prac- tically no grouse left on the prairie. When, however, the shooting season begins in October, or even No- vember, as is now sometimes the case, the birds are shy, scarcely lie to a dog, and are strong of wing. To kill them in great numbers is not an easy matter under such conditions. PINNATED GROUSE SHOOTING To-day there are few places, except in Texas, where much pinnated grouse shooting may be had, but thirty or forty years ago things were very different. Then men would start out on foot, and after two or three hours of shooting would return to their homes bending under a heavy backload of birds. Even later than that, great shooting was to be had in Illinois, Iowa, Minnesota and Nebraska. One scarcely dare say how large the bags used to be, but they were unquestion- ably large. Men hunted from wagons, carrying relays of dogs, with abundant water to keep them fresh at all times. Driving from farm to farm, and from vil- lage to village, they covered an immense area of ter- ritory and killed a great number of birds. In fact, at one time, during the first days of amateur photography, it was common to see photographs of such wagons hung around with game, and with the well-armed gun- ners and their dogs standing near. Happily, the taste for such pictures is slowly changing, though the big- AMERICAN GAME BIRD SHOOTING 400 game hunter still delights to photograph his camp with half a dozen deer hanging up in it, or to have his own picture taken with his foot upon the body of the slain bear or moose. Twenty years ago, when there were still prairie chickens in Minnesota, and plenty of them, it was well for the shooter to be early abroad. Breakfast must be eaten, the dogs looked after, a drive of perhaps five or six miles made to reach the ground, and it was neces- sary to be called long before daylight to reach the shooting’ ground as soon as the birds began to move. It is hard to leave a comfortable bed in which it It is hard to leave a comfortable bed in which it seems only a few moments since one settled himself to sleep, but a strong heart and prompt action brings the gunner out on the floor, and a few minutes of activ- ity in putting on his clothes causes him to forget the comforts of bed. Then, how good breakfast tastes, and how eagerly he looks forward to the sport before him. The drive in the dark may be cold and uncom- fortable, but the growing light promises warmth, which exercise certainly will bring. PINNATED GROUSE SHOOTING In those days chicken shooting was lawful on the In those days chicken shooting was lawful on the 15th of August, and the nights, though often damp and chilly, were not cold. There was an opportunity when there bred in Minnesota many ducks which have long since been expelled by the farmer, to get an occa- sional shot at teal, shoveller, mallards, and other home- breeding birds; but, after all, the chickens were the important thing. When the ground was reached the horses were put in the stable, or perhaps were driven SHOOTING THE PRAIRIE GROUSE 401 through gate or bars into the extensive green stubble, where the dogs were turned loose and eagerly com- menced to range. On they go for some hundreds of yards, crossing each other at high speed, with heads high in air and merry tail action; but presently there is a pause, one of them checks himself, and makes game, quartering here and there with nose close to the ground, and then making a cast, circling to try to pick up the trail of the running birds. The other dog joins him, and with a little rivalry they press forward, first one and then the other in the lead, until at last, drawing more and more slowly, one stops, and the other backs him. The shooters hurry forward on foot, and presently are close behind the dogs, which are standing staunchly. One man pushes forward and walks up even with the leading dog, while his companion steps off a little to one side; and then, suddenly, just before the pointing dog, there is a tremendous hubbub, and three birds get up and hurry away on swiftly beating wings. Alas! they stayed too long. Two of them fall to the right and left of the man standing by the dogs, while his companion, a little to one side, accounts for the third. The rapidly succeeding shots start the rest of the bevy, which in a moment are on the wing, another falling to the last barrel of the man to one side. The dogs have sunk to their haunches, and are gazing eagerly over the stubble. In a moment of such excitement it is too much to expect them to drop flat. PINNATED GROUSE SHOOTING Presently the guns are recharged and the dogs called Presently the guns are recharged and the dogs called AMERICAN GAME BIRD SHOOTING 402 up and sent forward for the dead birds. They advance very gingerly, and, picking them up, bring them neatly to hand. One of the dogs brings his always by the wing or neck, while the other grasps his bird about the body, and seems to rejoice in the huge mouthful that he has; yet he never bites nor apparently even pinches the bird. Long before this both men had been watching the disappearing bevy, which flew far, and at last seemed to go down in a slough of high grass which inter- rupted the stubble. Here the young birds, lying like stones, were pointed by the dogs and walked up by the gunners, one after another, until almost the whole covey was down; and so, very likely, it went on all through the day, until the heat of the midday sun be- came too strong for walking or shooting or dogs, and a couple of hours’ rest was taken. Late in the afternoon this was repeated, and some- times a great bag was made; but, after all, while this was killing, and was exciting enough, it was not what in these days would be called sport. The young birds lie very close, they present a large mark, and are too easy to hit. The great destruction finally led to the almost total extermination of the pinnated grouse of the West in regions where it had always been most abundant, and to-day the sportsmen of that region are bearing the burden of the sins of their fathers. The following charming account of shooting prairie SHOOTING THE PRAIRIE GROUSE 403 chickens in Illinois, in the old days, is abbreviated from a sketch from the pen of Wilmot Townsend, whose charming contributions to Forest and Stream have been well known for so many years: The prairie has been likened to the ocean, and the The prairie has been likened to the ocean, and the simile is not altogether wide of the mark; and as one looks out over the boundless expanse of rolling grass land that spreads on every side until land and sky meet on the horizon line, it does not require a great effort of the imagination to see the resemblance. PINNATED GROUSE SHOOTING Though the waving grass gives life to the view, it lacks the mighty heave of the ocean; that ceaseless pulsing of the waters is wanting; the swelling waves of prairie land seem to have stiffened and died, deserted by the impulse which gave them birth. The mere turning of the sod reveals a rich dark The mere turning of the sod reveals a rich dark loam, that, without fertilizing, will yield immense crops of corn and produce a wonderful return in golden grain. In late autumn the chickens resort to the fields of standing corn to glean, and rich pickings they find. On a still, clear morning the scolding of the cocks On a still, clear morning the scolding of the cocks begins at sunrise, and the air resounds with their kek- kek! kek-kek! until long after the hoar frost has dis- appeared. Then you will see flock after flock leave the prairie and enter the corn, there to remain through- out the day, unless disturbed. A short hour before sunset they run out to the edge and spring into sturdy flight for the night bivouac on the open plain. At this season these camps of the prairie chicken often contain thousands of individuals, representing hundreds AMERICAN GAME BIRD SHOOTING 404. of coveys, that arrive from all quarters, coming some- times from a distance of miles to this favored spot. One may frequently see some belated party string across his path as he tramps along just after sunset. Silent as owls, they flit past on outspread wings, and vanish in the gloom, The shooting among the standing corn is, to me, the The shooting among the standing corn is, to me, the most exciting of any of the methods pursued in hunting this\ fine bird. \2) 20; It is early in the forenoon, and I will see if I can- It is early in the forenoon, and I will see if I can- not find some of the birds I saw enter that eighty-acre piece a while ago. We are presently in the thick of it, and save for the We are presently in the thick of it, and save for the rustling leaves on the tall stalks which rise above our heads it is very still. PINNATED GROUSE SHOOTING The chickens are running, as I see by the trailing of my dog, and it is necessary to step out briskly in order to keep him in view. There! He has struck a hot scent! Hear how his tail is beat- ing a tattoo on the stalks as he worms his way along, crouching close to the ground in his eagerness to locate the birds. At last, with head almost at right angles with his body, he stands rigid. There they go. A dozen chickens whizz up through There they go. A dozen chickens whizz up through the corn about us. I catch a glimpse of one just as he clears himself, and pull on him almost without sighting. Crash! thud! Down he comes, and quickly I have him in hand, a fine young bird, as I find by hold- ing him up so that his whole weight falls on the under mandible, which breaks under the strain. The differ- ence in plumage of these full-grown birds is very slight, Kansa ing in ken shooti irie Chic irie Pra Kan ing in ken shoo irie Chic irie Pra SHOOTING THE PRAIRIE GROUSE 405 and it is by this simple test that we may distinguish young from old. In an old bird the lower mandible always sustains the weight of the body without breaking. The old dog is pointing again. A single bird rises this time, and sails off uninjured, though I give him the contents of both barrels. How easy it is to miss them in this cover, in spite of their size. We move on. There has been a covey about here, as numerous There has been a covey about here, as numerous tracks tend to show. It will take careful work to put them up now, as they have doubtless taken the alarm ere this, and are keeping well ahead of us. There they go again, off to the left. If we flush another bunch in this cover they will probably get up all together, as is their habit after being so disturbed. Try to pick your birds, for though they rise with a thunderous roar of wings, and look very large, you will find, as in quail shooting, that pot shots can seldom be made. Here is the edge of the field. I think we will work Here is the edge of the field. PINNATED GROUSE SHOOTING I think we will work around to where we entered, and after trying that little patch between here and the house I will wait until to- ward sundown, when they come out, and perhaps I may get a little flight shooting. Chickens feed quietly among the corn, rarely be- Chickens feed quietly among the corn, rarely be- traying their presence, save to the keen nose of your dog. Once flushed, they lie very close for some time after alighting, and will not scatter until thoroughly satisfied that danger is past. This, however, is only in cover where they have been used to feeding undis- turbed. Later in the season, when constant gunning AMERICAN GAME BIRD SHOOTING 406 has made them wary, they will make for the open at first flight, and rarely return before next morning. Look here! See where they have been dusting in this dry loam, just like so many old hens. At four o’clock I walk out on the prairie to an old At four o’clock I walk out on the prairie to an old haystack, that seemed to be located near the center of their flight as they left the corn last evening, and lie down to await developments. High overhead a flock of wild geese is moving south; travelers, I guess, from the height at which they fly, and the silence and regularity of their action. Blackbirds in dense flocks are sweeping past, with incessant noise of harsh, rasp- ing cries. Close by, a miniature whirlwind is sucking up the stray heads of buffalo grass and careering with them a short distance, where it leaves them awaiting the next puff that shall waltz them off again. Keeping a sharp lookout in the direction of the corn- Keeping a sharp lookout in the direction of the corn- fields, I presently see a covey of chickens rise. They come down well to one side, far out of range, but af- fording me a fine opportunity to observe their manner of flight. Rising with a burst of strong wing strokes, they attain an elevation of from 20 to 30 yards, and scale off on extended pinions for long stretches; then with a renewal of wing-beats they acquire fresh impe- tus. In this manner they alternate, now scaling, now flying along, until lost to sight over the prairie. Here comes another covey. PINNATED GROUSE SHOOTING Now they are close Here comes another covey. Now they are close upon me. Bang! one—bang! again, as two cross each other in flight, and down they come. There are num- bers of chickens sailing by, but none venture in my SHOOTING THE PRAIRIE GROUSE 407 direction. It is nearly dark when I reach the house, as night shuts in very quickly. How cosy the firelight makes the room after the silence of the prairie. The luxury of field shooting is pointed out in the following aecount of shooting in the Indian Territory. It presents a striking contrast to the sorrows of the eastern gunner of old times, who spent his shooting days in wallowing through swamps, squeezing among alders and trying to break down cat-briars and grape- vines : Prairie chicken shooting is, par excellence, the sport of the lazy man. It is the easiest of all land shooting. lirst, because the field is always open, and if one is too lazy to walk he can shoot from a horse or wagon; secondly, because early in the season, before the birds are matured or have been too often disturbed, they lie in the tall grass as close and as long as one wishes; and thirdly, because they make a good big mark, flying true, and not too rapidly, and there is so much of them that one need have no fear of blowing them all to pieces, leaving nothing but feathers in the air. If they happen to get up too near for a shot you can measure your distance, knowing there is no bush or tree for them to dodge behind. Thus, in all respects, they make fine game for one not disposed to be in a hurry; and for these same reasons the gentle things are easy plunder for the unscrupulous market hunter. PINNATED GROUSE SHOOTING Later in the season, during the last of September, the birds are fully matured, have become stronger flyers, AMERICAN GAME BIRD SHOOTING 408 and have been made a little more shy from an occa- sional shot among them, even in this far-off locality, | and will not always allow a dog to approach so near them; and if a bird gets up twenty-five or thirty yards away one has to wink his eye pretty quick in order to stop him, for being strong, they will carry off quite a weight of shot unless winged, or hit in a vital part. We shot mostly on the Omaha Indian reservation, We shot mostly on the Omaha Indian reservation, which, here, is quite rolling, but excellent land, covered with a heavy growth of prairie grass; and there being no fences, we could drive at will in almost any direc- tion. The Indians had here a few fields of corn and other grains, but there were thousands of acres over which we could ride and shoot at pleasure. Our wagon was a comfortable two-seated spring vehicle, with a park top, which would carry four to six persons and our dogs, and we had a couple of ponies, somewhat larger than jack rabbits, for a team, which would walk or run all day, but manifested a most decided disin- clination to trot. We had two dogs with us, an Irish and an English setter, and our landlord had two Rus- sian setters which were at our service. Our mode of proceeding was about this: We would Our mode of proceeding was about this: We would get an early breakfast, load up our dogs, guns, ammu- nition, lunch, a big jug of water for ourselves and the dogs, and point the ponies for the Indian reserva- tion, when a ride of a little more than a mile would bring us to good.shooting grounds. We always drove to the leeward of the field over which we designed to shoot. Then we would get out, leave Miss S. to Our mode of proceeding was about this: We would get an early breakfast, load up our dogs, guns, ammu- nition, lunch, a big jug of water for ourselves and the dogs, and point the ponies for the Indian reserva- tion, when a ride of a little more than a mile would bring us to good.shooting grounds. PINNATED GROUSE SHOOTING We always drove to the leeward of the field over which we designed to shoot. Then we would get out, leave Miss S. to SHOOTING THE PRAIRIE GROUSE 409 manage the team, following slowly in our wake, and occasionally marking birds for us, which services she rendered in an admirable manner and with a new and delightful pleasure to herself. With the four dogs, the three of us keeping about two hundred yards apart and moving in line as nearly as practicable, each would generally find birds enough for his individual shoot- ing without disturbing the others or placing them in danger; and when one’s pockets became too heavy for comfort or convenience he would fall back to the wagon and deposit his load. Occasionally we met at the wagon, to water the dogs, eat lunch, smoke a cigar, look over our birds, and when we were thoroughly rested, started out for another tramp. Thus we would put in time until about 11 o’clock, when it was time to bundle ourselves and dogs into the wagon and drive back to the hotel for dinner, after which, about 3 o’clock, we would find ourselves again seated in the wagon and on our way for the evening shoot, which usually lasted far into the “twilight soft and gray.”’ To me there is a rare and indescribable delight in shooting on a still, quiet evening, watching the last rays of the setting sun, and the last faint glimmer of light as it quietly passes away under the gauzy curtain of night. We always found supper awaiting us on our arrival We always found supper awaiting us on our arrival home, when, after caring for the dogs and shedding our hunting traps, and taking a good bath, we gathered about the little round table, doing ample justice to broiled chicken and other good things. 410 AMERICAN GAME, BIRD SHOOTING Should you ask me how many birds we bagged I could not tell you. I kept tally till we got past 100, and then quit. We did not forget our friends, nor neglect ourselves, for we sent away a box each day, and kept a string hanging under the little porch of the hotel from which our table was supplied at each meal. PINNATED GROUSE SHOOTING Thus we passed the week, changing our route occa- sionally, always getting birds enough to make it en- joyable sport, never turning it into downright slaugh- ter, and leaving birds enough and to spare. And with it all we had a good time. At the approach of cold weather the birds gather At the approach of cold weather the birds gather together in large flocks and pack, as the. term is, re- maining in these packs all through the winter. At this time they are shy, will not lie for a dog, get up at a considerable distance and fly a long way. An interesting account of the birds at this time was An interesting account of the birds at this time was written by Latrobe in his “Rambler in America.” La- trobe accompanied Washington Irving and one or two other friends on an expedition to what was then the Far West—beyond the Missouri River, and like most Englishmen did not let pass the opportunity to get some shooting. In 1832 he wrote of his efforts to shoot some prairie fowl. This was in Indian summer, at a time when the squirrels were at work among the dead leaves beneath the hickory and pecan trees near the Osage Agency, and when countless bands of water fowls and flights of pigeons, which had been constantly observed ‘sneyyjso TH “gq Aq 3uyured WOlT ‘SUdHIIYD WO yseq siojuI0g iirc oncihoW HS iy ‘s TH “gq Aq 3uyured WOlT ‘S WO yseq siojuI0g SHOOTING THE PRAIRIE GROUSE AIT passing to the southward during a season of cold, had ceased to be a source of wonderment. He says: “The prairie fowl “The prairie fowl had now completely thrown aside their summer habits. Instead of keeping apart in dis- tinct families, scattered over a vast extent of country, like our grouse at an earlier season, they now appeared congregated in immense flocks in the immediate vi- cinity of the farms. I had plenty of opportunity of studying their habits, but to shoot a few brace, as they were extremely wild, required frequently hours of patient and wary exertion; whereas at an earlier season a sportsman, if aided by a dog, might bag any quantity from the pertinacity with which they will lie close until forced to fly. PINNATED GROUSE SHOOTING “Tt appeared that at this time of the year all the birds within an area of three or four miles square congre- gated together by consent at sundown on a given spot in the rank dried grass of the unburnt prairie to sleep. Many a time have I seen them coming at sun- set from every point of the compass with their re- markable level and even flight over the swells of the prairie toward the place of rendezvous, which a few days’ observation enabled me to determine upon within a quarter of a mile, and twice I was on the prairie early enough to hear and see them rise, and the sight was such as might make an English sportsman’s mouth water. The number must have amounted to many thousands, and the sound of their wings might be heard a very great distance. After rising for about half an hour they crowd the scattered trees on the edge AMERICAN GAME BIRD SHOOTING 412 of the prairie by hundreds at a time, after which they disperse. Their wariness at this time is extreme; and the slightest indication of the approach of man, even at a great distance, is noticed by the cock, who, perched on the topmost twig, elongates his neck and peeps first on one side and then on the other with the most pro- voking caution. How often have I been foiled, when, after the most cautious approach, either in serpent fashion like the Indian, dragging myself through the grass inch by inch, or in an upright position striving to counterfeit a tree stump, never stirring till the sen- tinel looked another way, and then, by imperceptible approaches and five more feet in five more minutes, would I approach the tree within range—the careful bird began to grow more and more doubtful and rest- less and finally set up that clear, tremulous crow which said: ‘There’s a rogue with a gun almost within shot,’ as plainly as though he had spoken English. The in- stant and complete dispersion of the whole covey to a great distance would be the immediate consequence.” SHARP-TAILED GROUSE SHOOTING Except for the seasonal movements already mention- ed, the sharp-tailed grouse is very local in habit. Half a dozen pairs of these grouse will build their nests and hatch their young along three or four miles of prairie stream, or the dry ravines which lead down to it. Mother and young will spend the summer on a range of prairie covering less than a square mile, and there, SHOOTING THE PRAIRIE GROUSE 413 in September or early October, one may find these broods of full-grown birds scattered over the hillsides at feeding time, and in the middle of the day gathered together in the shade of willows or box elders, or in a clump of rose bushes, from which, if disturbed, they rise with a startling roar and much cackling, to fly from seventy-five to two or three hundred yards, and drop in some convenient cover along the ravine or on a hillside. I have found them in southern Mon- tana under just these conditions, where, if one had had a good dog and a shooting pony, it would not have been difficult to exterminate the whole brood. It is soon after this, however, as the nights grow cold and frosty, that the birds get together in larger companies, several broods uniting, and in the early mornings take to the tops of the cottonwoods and wil- lows, where they keep that excellent lookout that Mr. Latrobe has so well described in another species. At that time they are shy and quite impossible of approach in these exposed situations. Though the sharp-tailed grouse rises from the Though the sharp-tailed grouse rises from the ground with noise enough to disconcert the gunner of slight experience, it is not a difficult bird to kill. The mark is large and the bird’s flight steady and straight. There is time enough to shoot carefully. Ata straight- away, low-flying bird one may shoot directly, but crossing shots demand that the bird should be led a little. After all, this is almost wholly a matter of experience, and as almost all the shots are in the open, one may fire deliberately, and can, usually, if he misses, AMERICAN GAME BIRD SHOOTING 414 explain to himself why he did so and at the next shot correct his error. SHARP-TAILED GROUSE SHOOTING Like all the prairie grouse with which I am ac- Like all the prairie grouse with which I am ac- quainted, the meat of the full-grown sharp-tailed grouse is dark, though the half-grown birds are white- meated. The flesh is tender and well flavored, as might be expected of birds that had passed the summer feed- ing on prairie insects and a variety of berries. In winter, when the character of the food largely changes, and willow and cottonwood buds constitute a large share of its dietary, its flesh is less toothsome, being much drier. The sharp-tailed grouse, owing to the character of country which it inhabits, brushy ravines, willowy bot- toms, and, at certain seasons of the year, hillsides over- grown with underbrush and some large trees, is much less easily killed than the pinnated grouse, and will probably remain with us much longer than that spe- cies. If it could be introduced to eastern covers, and protected until it had established itself, it would prove a grand bird for Atlantic coast gunners. So intro- duced, and so acclimated, it would, however, bear only the very lightest shooting, perhaps not more than three or four per gun in a season. More than that would soon result in their extermination, and the efforts put forth to introduce them would thus be wasted. In the early days of western field trials held on In the early days of western field trials held on prairie chickens in this country, the birds sought for were chiefly the pinnated grouse. These proved not sufficiently numerous, and were so readily killed that SHOOTING THE PRAIRIE GROUSE 415 they did not give the dogs an opportunity to show off the best that was in them. A little later, therefore, a change was made, and grounds were sought where the sharp-tailed grouse was abundant. This was in northwestern Minnesota and in Manitoba, and here, for a number of years, were held what used to be called the chicken trials. SHARP-TAILED GROUSE SHOOTING Before they have been much shot at the young sharp- Before they have been much shot at the young sharp- tailed grouse lie well, and offer easy shooting ; but when they attain their full growth, and the weather grows colder, they become much more shy, and while still spending most of their time on the upland prairies, seek refuge, when disturbed, in the ravines or the wil- lows of the stream bottoms. During the winter they get together in considerable During the winter they get together in considerable packs, and live largely among the underbrush of the river bottoms; yet often they may be seen sitting on the roofs of buildings or willow trees, or fences im- mediately about the ranch, and sometimes at this sea- son of the year, while roosting in such places, may be killed in large numbers with a rifle. When it will lie, the sharp-tail furnishes fine sport When it will lie, the sharp-tail furnishes fine sport with a dog, but often it runs for a considerable dis- tance, and so rapidly that it may rise far ahead of the dog. As soon as the weather has become cool a quick eye and arm and a strong lead to the gun are required to bring the bird down. In many respects the sharptail suggests the bobwhite In many respects the sharptail suggests the bobwhite quail, especially in the way in which it leaves the ground in flight and the speed with which it moves on AMERICAN GAME BIRD SHOOTING 416 the wing. In the late summer or early autumn it may be seen stalking about over the ground, walking quite differently from some of our other grouse, holding the head and tail erect and lifting its feet quite high, as if stepping over some obstacle before it. It is not easy to recognize in this seemingly absurd, affected bird the ball of feathers that a month later will rise from before your dog and speedily be out of range. In some parts of the country I have heard gunners In some parts of the country I have heard gunners speak disparagingly of the sharp-tailed grouse, per- haps for no better reason than that they fly more swiftly and rise with more tumult than the pinnated grouse; but I regard them as one of the finest of our game birds. SAGE GROUSE SHOOTING The sage grouse, which has its home on the high, dry plains, where grows the artemisia, harmonizes in color with the gray soil and gray stems of the sage brush to an extraordinary degree. When the sage grows high, and, as is often the case in river bottoms, is interrupted by frequent clumps of rye grass, one may even walk into a brood of the birds without see- ing them, and perhaps have them rise all about him; or, on a sudden, his eye may catch a dozen heads, on tall, straight necks, staring at him from every side. The sage hen, it must be confessed, does not convey to the observer the idea of a game bird. When tame it is so gentle and unsuspicious, and when shy so SHOOTING THE PRAIRIE GROUSE At. clumsy and lumbering in its efforts to escape, that one hardly gives it credit for game qualities. It is a splen- did bird, however, and its great size makes it a trophy of the shotgun eagerly to be sought after. The range of the sage grouse is commonly believed The range of the sage grouse is commonly believed to be coterminous with that of the plant whose name it bears. Though often seen in localities where no sage is actually visible, it never, we think, occurs far from the region where fhe plant is found. It is at home alike where the stunted shrubs of the wormwood grow only two or three inches high, or in valleys of the great central plateau where they are real trees, as high as one’s head. In cover such as this the traveler may often ride up on a great flock of the birds, which are usually so unsuspicious that they will stand for some time before taking alarm. When the birds do rise they get up heavily, with a When the birds do rise they get up heavily, with a lumbering flight; but after they have acquired momen- tum they go much faster than one would suppose, and it is easy to shoot behind crossing birds, whose great size often tends to make the shooter careless. SAGE GROUSE SHOOTING Inhabiting, as it does, a region of extreme aridity, and because the shooting season comes at the dryest period of the year, dogs would be at a great disad- vantage in the pursuit of this bird, and I personally have never seen them used in this shooting. The bird is large, it inhabits the open, and is readily seen at some little distance. Though often unsuspicious, yet as the season advances, or in places where they have been shot, or much disturbed, they will often rise at AMERICAN GAME BIRD SHOOTING 418 a distance of 125 or 150 yards, and then make such long flights that it is useless to think of following them. The adult sage grouse is tough and heavily feath- ered, and possesses great vitality. Unless properly hit it will carry away a heavy charge of shot. Young birds, on the other hand, drop to a mere touch. I have killed a great many on the wing with small charges of dust shot. Many years ago a correspondent of Forest and Stream declared that, when there is reasonable cover, sage grouse will often lie to a dog better than the pin- nated grouse. He adds what is now well known— that the peculiarities of its home do not give the dog a fair chance to work or to do himself or the game justice, since the artemisia grows only upon barren prairies some 4,000 to 10,000 feet above sea level, where the soil is hard and dry, with little vegetation save stunted shrubs, cactus and an occasional clump of wild grass, where rain rarely occurs and there is little moisture in the air or upon the ground. When thick, the tough sprawling crooked evergreen, or rather ever- gray, shrub is often difficult for man, horse or dog to get through. The sage cock is a good skulker and runner, and not easily flushed if it can hide. The same idea is suggested in Dr. Newberry’s ac- count of his first meetings with this great bird during one of the early explorations for a railroad route. It is in- Volume V lof Paciie R. R. Reports: “Coming into camp at evening, I had been attracted “Coming into camp at evening, I had been attracted SHOOTING THE PRAIRIE GROUSE 419 by a white chalk-like bluff, some two miles to the right of our trail, which I visited and examined. SAGE GROUSE SHOOTING Near it was a warm spring, which came out of the hillside, and, spreading over the prairie, kept a few acres green and fresh, strongly contrasting with the universal brown of the landscape. In this little oasis I found some, to me, new flowers, many reptiles, and a considerable number of sharp-tailed grouse, of which I killed several, the whole presenting attractions sufficiently strong—as we were to remain in camp one day—to take me over there next morning. I had filled my plant-case with flowers, had obtained frogs, and snakes, and chalky infusorial earth enough to load down the boy who accompanied me, and had enjoyed a fine morning’s sport, dropping as many grouse on the prairies as we could conveniently carry. Following up the little stream toward the spring on the hillside, a dry, treeless surface, with patches of sage brush (Artemisia tridentata), I was suddenly startled by a great flutter and rush, and a dark bird, that appeared to me as large as a turkey, rose from the ground near me, and uttering a hoarse hek fiek, flew off with an irregular but remarkably well-sustained course. “T was just then stooping to drink from the little “T was just then stooping to drink from the little stream, and quite unprepared for game of any kind, least of all for such a bird, evidently a grouse, but so big and black, so far exceeding all reasonable dimen- sions, that I did not think of shooting him, but stood with open eyes, and, doubtless, open mouth, eagerly watching his flight to mark him down. But stop he did not—so long as I could see him, now flapping, AMERICAN GAME BIRD SHOOTING 420 now sailing, he kept on his course till he disappeared behind a hill a mile away. “T was, of course, greatly chagrined by his escape, but knowing that, given one grouse, it is usually not difficult to find another, I commenced looking about for the mate of the one I had lost. My search was not a long one; almost immediately she rose from under a sage brush with a noise like a whirlwind, not to fly a mile before stopping to look around, as the cock had done, but, by a fortunate shot, falling helpless to the ground. SAGE GROUSE SHOOTING No deer- stalker ever felt more triumphant enthusiasm while standing over the prostrate body of a buck, or fisher- man when the silvery sides of a salmon sparkled in his landing-net, than I felt as I picked up this great, and to me, unknown bird. I afterward ranged the hillsides for hours, with more or less success, waging a war on these birds, which I found to be quite abundant, but very strong-winged, and difficult to kill. I repeatedly flushed them not ten yards from me, and, as they rose, poured my whole charge, right and left, into them, knocking out feathers, perhaps, but not killing the bird, which, in defiance of all my hopes and expectations, would carry off my shot to such a distance that I would not follow him, even did I know he would never rise again. Here, as elsewhere, I found these birds con- fined to the vicinity of the sage bushes, from under which they usually spring. “A few days later, on the shores of Wright and “A few days later, on the shores of Wright and Rhett lakes, we found them very abundant, and killed all we cared to. A very fine male which I killed there “A few days later, on the shores of Wright and Rhett lakes, we found them very abundant, and killed all we cared to. A very fine male which I killed there “A few days later, on the shores of Wright and Rhett lakes, we found them very abundant, and killed all we cared to. A very fine male which I killed there SHOOTING THE PRAIRIE GROUSE 421 was passed by nearly the whole party, within thirty feet, in open ground. I noticed him as soon, per- haps, as he saw us, and waited to watch his movements. As the train approached he sank down on the ground, depressing his head, and lying as motionless as a stick or root, which he greatly resembled. After the party had passed I moved toward him, when he depressed his head till it rested on the ground, and evidently made himself as small as possible. He did not move till I had approached within fifteen feet of him, when he arose, and I shot him. He was in fine plumage, and weighed over five pounds,” TURKEY SHOOTING. There are many methods of shooting the wild turkey, yet, after all, it is probable that it is more often killed by accident than by any one of the more approved methods. In other words, men traveling through the woods or fields stumble on the turkeys, flush them, and so get a shot. In the South, “calling” is the approved and most used means of killing these birds. The hunter con- ceals himself in a blind, imitates the call of the hen, and so draws to him the other members of the flock. This is practiced with especial success either in the autumn, when the young turkeys are still innocent, or in the spring, at gobbling time, when the gobblers are likely to respond readily to the supposed call of the female. In regions where turkeys are plentiful, it is common for hunters to go out in the afternoon, and, finding a brood, to flush and scatter it. If night is near, the turkeys are likely to try to get together before roost- ing time, and to begin calling almost at once. If, how- ever, it is too late in the day for them to do this, the birds roost in the trees, and in the morning are still more anxious to get together. The hunter, therefore, starts out long before daylight, repairs to the locality where he started the turkeys the night before, and con- cealing himself there, either by means of a blind con- 422 422 422 ye bk ee CES Sear eT TS Wild Turk in the Pines Wil Tur in the Pine TURKEY SHOOTING 423 structed the night before, or in some thicket, listens carefully for the first calling of the turkey, to which he responds. In this way he may succeed in drawing several turkeys toward him. The instrument used in callin The instrument used in calling turkeys is sometimes made of the bone of the bird’s wing, sometimes of a little box floored with a piece of slate, the cry being caused by the stroke of another piece of slate on the slate in the box. At all events, the noise must be a good imitation of the turkey’s cry, for the great bird is one of the most acute of fowls, and very little escapes either its eye or its ear. TURKEY SHOOTING. If the imitation of the cry is a poor one, answers will soon cease to come, and the birds will retreat. Some Some years ago a correspondent, Tripod, wrote from Mississippi a brief but vivid account of the kill- ing of a turkey by Miss Mary C. Breckenridge, whose experience with a shotgun was limited, but who, when the occasion arose, showed herself an apt pupil of her skilled instructor. She was new to the woods, and after traveling for some distance made up her mind that turkeys were a myth. Nevertheless, it was de- cided to call, and after a time, in the open woods, Miss Mary selected a good log for an ambuscade, while Tri- pod used the yelper, and at once received an answer from a nearby bird. The turkey kept approaching, ing, but before he had been seen a shot in the distance frightened him and he ceased answering. The turkey hunters sat quietly for a time, uttering now and then a little yelp, or cluck, when a twig snapped, and as AMERICAN GAME BIRD SHOOTING 424 Tripod carefully peeped over the log, there, not twenty- five paces from them, came a big gobbler, all fluffed up and strutting. He was coming nearer, so that prompt action was necessary. Tripod goes on: “ “Now, Miss Mary, don’t move a bit,’ I caution. “Her gun lay across the log, pointing in the direc- “ “Now, Miss Mary, don’t move a bit,’ I caution. “Her gun lay across the log, pointing in the direc- “Her gun lay across the log, pointing in the direc- tion from which the first answer had come, with its muzzle among some twigs, to free it from which the gun had to be withdrawn entirely from over the log and pushed forward again in a new place, as the tur- key was approaching from a different quarter, very much askew to the log’s position. This difficult move- ment in the face of the enemy was successfully accom- plished. “ “Now take the gun cautiously, aim carefully at his neck, low down, and shoot. Don’t hurry!’ “She did it just that way, and over rolled that big “She did it just that way, and over rolled that big bird. TURKEY SHOOTING. With great exultation we then bore down upon him, and saw him flutter, then quiver, and then lie still, On the lower part of his neck was much evi- dence of No. 5 shot. Miss Mary then looked much prouder than the turkey had looked a few moments before, and very justly so—and said turkeys were not a myth any more. I realized this myself as I bore this tangible specimen to where our horses had been left.” A successful turkey hunter, writing from Missouri A successful turkey hunter, writing from Missouri recently, expressed his views of turkey hunting, and what he says, which I give in substance, will apply to the pursuit of the bird over much of its present range: TURKEY SHOOTING 425 To be a successful turkey hunter one must know his country and conditions, for unless the ground be cov- ered with snow, and the pea crop has been a late one, the birds keep to the woods, where they are not easily seen in the tangle of hazel and blackberry. Plenty of turkeys means a good crop of mast, for Plenty of turkeys means a good crop of mast, for in autumn the chief food of the bird is the acorn, and the feeding grounds of the bird will be where the sup- ply of acorns is greatest. If the crop fails in the swamps, most of the birds move into the rough hill country, for when the mast fails in the lowlands the hills are likely to have a very good crop, and a failure in the hills means an abundant harvest of acorns in the swamp. To find where the birds feed, however, is not to get your game, or even to see it. Often the scattered birds may be easily called up, but on the other hand, some flocks of young birds refuse to respond to the call. To stalk the turkey requires more care and skill than to stalk the deer, and he who is successful in this description of hunting may congratulate himself. On the other hand, one may often blunder on turkeys without special effort, and may believe that he has mastered the art of turkey hunting; while the very next day he may not be able to get within long range of his game. TURKEY SHOOTING. Sometimes it is possible to walk close up to turkeys, so that one wonders if he has not fallen upon a flock of domestic birds. As turkeys commonly roost over a water-course, or As turkeys commonly roost over a water-course, or slough, as protection from danger, it is sometimes pos- AMERICAN GAME BIRD SHOOTING 426 sible, late in the evening or early in the morning, to pass under their roosts in a boat. If birds can be found in such a situation there is opportunity for good shoot- ing with a rifle. A very successful method of shooting them in Mis- souri is to ride through the timber on horseback, for the birds are not as shy of the approach of a rider as they are of a footman. On the other hand, turkeys alarmed are likely to make for swamps or down tim- ber, where a horse cannot go. If a flock of turkeys is scattered in the evening, at If a flock of turkeys is scattered in the evening, at sundown, or frightened from its roost at night, the hunter who can be at the place before sunrise is likely to be able to get a number of shots by calling up the birds. Shooting turkeys from the roost at night is a method that does not present many features of sport. Track- ing birds in the snow is good sport, and calls for work and endurance. It is a very certain method of get- ting turkeys, but is obviously to be practiced only where snow falls. An excellent description of the country in which turkeys are found in Arkansas, and of the opportuni- ties which used to come to the turkey hunters there, is found in an account written by J. E. London in the spring of 1900. He says: “While I was engaged in having some assessment “While I was engaged in having some assessment work done on some mining lands in Newton County, Ark., in December, 1899, I was informed by some boys TURKEY SHOOTING 427 who came to our camp that on the mountain north of us I could find a large drove of wild turkeys. “The mountains in that section are very rugged, and “The mountains in that section are very rugged, and heavily timbered, but the woods are rather open, the undergrowth being sparse and small. TURKEY SHOOTING. The timber is white, black and post oak, hickory, beech, walnut, cherry, and a half dozen other kinds, and such timber! Walnut six feet in diameter, and white oak the same. In fact, nowhere in the South does such a wealth of timber exist. “One bright afternoon, taking my shotgun, I climbed the steep mountain for about one mile, to where a beautiful spring bursts from the rocks. I stopped, and was enjoying the solitude of the gloomy, grand old forest, when I espied a yellowhammer on a small tree, and shot him. This disturbed two gray squirrels, that ran to the top of a large wild cherry tree, but two loads of No. 6 put them in my game bag. “I then started north along a path, but I had only “I then started north along a path, but I had only gone a short distance when I walked out of the path to look down into a grove of post oaks, when I beheld a sight that put my. heart to beating like a trip-hammer. Twenty-six as fine turkeys as I ever saw, not two hun- dred yards distant, were feeding as though nothing could harm them, and paying no attention to the noise I had made killing the bird and squirrels. Armed only with a shotgun and some No. 2 shot cartridges, I began the task of getting in reach of those turkeys. “Down on my knees and hands I went, and after “Down on my knees and hands I went, and after forty minutes of hard work I was within seventy-five AMERICAN GAME BIRD SHOOTING 428 yards of them. Selecting two big gobblers that were close together, and in line, I gave them both barrels, and maybe there wasn’t a racket on that hillside. One of them began to flop and whirl around like a chicken with its head cut off, and the other flew about two hun- dred yards at right angles with the remainder of the drove, that had gone over a high bluff into a ravine about one-quarter of a mile from where they rose. He came to the ground, staggered a few steps, rose, and when he had gone perhaps one hundred yards he let go and came tumbling down dead. TURKEY SHOOTING. I ran to the first one, and hanging him up in a small tree, followed the other, and did the same with him. “Just then I heard some dogs on the opposite side “Just then I heard some dogs on the opposite side of the mountain begin to bark, and a boy encouraging them. I knew this would turn the other turkeys back, so I hurried across a small draw and around to the north of where they came to the ground, and climbed on some large rocks, where I had a good view of the mountain side. In a minute or two I saw two hens coming for me on a dead run, and waited till they were within forty yards. I gave one of them the left-hand barrel, and as the other rose I knocked her a clean somersault. “T now had four as fine turkeys as I ever saw, and “T now had four as fine turkeys as I ever saw, and not caring to hunt any more that day, I hung the two hens over my back and carried them to the gobblers. In sections where turkeys still exist, and where snow falls in winter, a favorite method of hunting these TURKEY SHOOTING 429 birds is to take a track and walk the bird down. Usu- ally, for several hours the turkey keeps well ahead of the hunter, the man steadily following the track and obliterating the turkey’s trail as he goes. After a time the turkey is likely to grow tired, and at length to hide, when it can be flushed and shot on the wing. Sometimes, to be sure, if the ground was unfavorable for hiding, the turkey might fly, but usually it did not go far, and its track could be taken again. A short but well told story of tracking turkeys in the snow was printed in Forest and Stream, October 26, 1907, from a correspondent signing himself “Brinkstine.” It took place in Missouri: “We walked two or three hours, and not a sign of “We walked two or three hours, and not a sign of aturkey. Another hour, and yet no sign. We rounded a deserted field with no success, further than seeing plenty of rabbits and a few squirrels, but we did not want to set the hills quaking by shooting such game. “We were tired. TURKEY SHOOTING. Walking in a foot of snow four “We were tired. Walking in a foot of snow four or five hours steadily, and as silently as possible, 1s not conducive to a hilarious feeling, as one grows cold and hungry. We built a fire, ate our lunch, rested and warmed ourselves. We were almost discouraged, but started on with some hope of success. We walked two or three hundred yards, and found a turkey track, and a huge one. We had gone a quarter of a mile when the tracks of three other turkeys joined the one we were tracking. One turkey would not satisfy us both, so we were glad to know that there were four turkeys in the woods. Just as we were ready to start AMERICAN GAME BIRD SHOOTING 430 again we were chagrined to see George coming down the hill, trailing the three that had joined ours; but as age deserved consideration, we let him go in advance. age deserved consideration, we let him go in advance. “Thus we came to a bottom covered with a growth of hazel brush, grape-vines and weeds, an ideal hiding place for game. A cautious approach, and George looked over a brush pile and saw the turkeys sitting seventy-five or eighty yards away. It was impossible to approach closer, and George saluted them with both barrels, but got nothing. We marked the direction they took, and followed, George alone, and Riley and I after a huge one I had spotted. We struck a lively canter and ran quite a distance, as we knew he would run a long way before he tired and tried to hide. His tracks indicated that he was getting tired, so we slowed up and proceeded cautiously. Every fallen tree-top was closely scanned. The tracks led down hill toward a monster tree blown down during the summer, and which still had its leaves on, forming a splendid hiding place. We both recognized this immediately, and started to go one on each side of the tree-top. I stooped to go under the body, which lay three or four feet off the ground, when out burst the turkey. I ‘dropped to get a view of him, and fired one shot as he pitched down hill. TURKEY SHOOTING. We ran across the ravine, and at the top of the next bluff found tracks where a turkey had alighted and departed in hot haste. A run of half a mile down the backbone of the hill, and we found the turkey had flown. “Sadly we retraced our steps, while I caressed a “Sadly we retraced our steps, while I caressed a “Sadly we retraced our steps, while I caressed a TURKEY SHOOTING 431 weeping eye, through which a hazel twig had dragged, and we mused on what might have been. “ What on earth are you fellows doing, anyway ” “ What on earth are you fellows doing, anyway ” “We looked up, and there stood Uncle Will, giving “We looked up, and there stood Uncle Will, giving us a rather tantalizing smile, we thought, as we re- membered his caution as we left the wagon. We were not particularly glad to have him see us just then, as we came back panting and hatless. The best we could do was to tell him the straight of it, and as he was an experienced hunter maybe he could help us out. At the risk of being guyed, we even hinted that the turkey had limped as it ran away. “ “Are you sure you hit it?’ he asked. “ “Are you sure you hit it?’ he asked. “ “Think I did. I know that I tried aw “ “Think I did. I know that I tried awful hard.’ “ “Well, step here and see what this is, and tell m “ “Well, step here and see what this is, and tell me how it got there,’ pointing to a big gobbler lying in a hole which it had made in the snow when it fell. How quickly I grabbed it up, admired its beauty and guessed at its weight. “ ‘Flere,’ said Riley, ‘you’ve got your turkey. Mine “ ‘Flere,’ said Riley, ‘you’ve got your turkey. Mine has just gone over the hill, and is on the other bluff. Come along, and we will get him, too.’ “We separated, seventy-five or one hundred yards “We separated, seventy-five or one hundred yards apart, in order to cover more ground. We followed the tracks ten minutes, when we saw the turkey rise in open ground one hundred and fifty yards away. TURKEY SHOOTING. We marked the direction and hurried on, found the tracks, and followed briskly along the top of a broad, flat ridge. We had taken only a few steps through the tall grass when the turkey rose, twenty-five yards away, AMERICAN GAME BIRD SHOOTING 432 but directly between me and the sun. Quicker than thought my gun went up, a flame of fire leaped out, and was answered by the thud of the turkey as it fell. “*“Confound you!’ said Riley good-naturedly, walking over and picking up the turkey, a young hen. He took the gobbler and made me take the hen, weighing only about one-half of his load, and we set out for the wagon, four or five miles away. ‘I am glad you got them, if I didn’t get any,’ said he. ‘You got them both, and that is just as good.’ ““No, it isn’t,’ I answered. ‘You claim that you killed one, and [ll stand by you.’ “No, you won’t. You killed them both, and you’ve “No, you won’t. You killed them both, and you’ve got to have the credit of it.’ And he was as good as his word.” The story of an oddly secured Thanksgiving turkey, killed in 1893, in the Alleghany Mountains, was inter- estingly told in Forest and Stream, some years ago, by Mr. Edw. Banks, now of Wilmington, Del. A party of three, of whom Mr. Banks was one, had been shoot- ing ruffed grouse near Bellwood, Pa., but without any great success. There was a little snow on the ground and many tracks of turkeys in the snow. Presently the party went to a cold spring and sat down to eat lunch. While doing so one of them remarked that he wished he could get a Thanksgiving turkey. Mr. Banks says: “Lunch was about over when we heard a gun go off, with a regular old black-powder dr-ro-o-oom. It was not more than three or four hundred yards away from TURKEY SHOOTING 433 us, judging by the sound. Somebody said turkeys. The rest of us agreed. By common consent we jumped up and went to a pair of bars where an old woods road came out into the orchard, and stood looking in the direction of the sound. TURKEY SHOOTING. “All at once out into the open, over the little valley, “All at once out into the open, over the little valley, away up above the trees, sailed a turkey. At first it looked as if it were coming toward us, but almost in- stantly we saw that it had turned down the valley and was flying directly away from us, along the hillside. Our eyes followed it as it kept on its way for about a quarter of a mile, when suddenly up went its head and down it came, ‘deader’n a mackerel.’ “ “He got it, after all,’ said some one. ‘He did,’ said Bill. “Did you ever see anything like it?’ said Jake. “Then we returned to our lunch, and talked over the wonderful thing we had seen. “ “He got it, after all,’ said some one. ‘He did,’ said Bill. “Did you ever see anything like it?’ said Jake. “Then we returned to our lunch, and talked over the “Then we returned to our lunch, and talked over the wonderful thing we had seen. “Bill was quiet for a while. Presently he broke the silence with, ‘Say, do you suppose that fellow who shot at that turkey saw it fall? Do you think he had any idea that he hit it?? That was quite a new idea to us, and we caught at it at once. It would be easy to follow the road in the woods and find out whether the man with the gun had turned off to the right or left anywhere. His tracks in the snow would tell us. We had marked where the turkey fell, right alongside of a monster pine which had been left when its breth- ren were taken by the lumbermen. Some three hundred yards along the road we saw where a man had come down into the road, and saw, also, that his tracks, as ’ “Bill was quiet for a while. Presently he broke the silence with, ‘Say, do you suppose that fellow who shot at that turkey saw it fall? Do you think he had any idea that he hit it?? That was quite a new idea to us, and we caught at it at once. It would be easy to follow the road in the woods and find out whether the man with the gun had turned off to the right or left anywhere. TURKEY SHOOTING. His tracks in the snow would tell us. We had marked where the turkey fell, right alongside of a monster pine which had been left when its breth- ren were taken by the lumbermen. Some three hundred yards along the road we saw where a man had come down into the road, and saw, also, that his tracks, as ’ AMERICAN GAME BIRD SHOOTING 434 well as those of his dog, led along the road in the direction the turkey had flown. “Following the tracks, we finally came to the big pine, which was down hill, on the right of the road. The turkey had fallen to the right of the pine, and so was further from the road than the pine was. Jake and I turned off into the woods, which was open enough for us to have a good view of the ground. Bill went down the road a bit, to make sure that the man with the dog had not turned off further along, then he, too, turned to the right into the brush, and began to investigate. “To Bill belongs the honor of finding that bird, a “To Bill belongs the honor of finding that bird, a gobbler, too, wattled, bearded and bronzed as only an old gobbler can be. He was a beauty, with not a feather damaged and not a drop of blood on the snow. ... Was it our turkey, or did it belong to the man who had shot at it? By this time he was a mile or more on his way, and that turkey certainly did look good. “At all events, we took the bird back with us, and if I remember rightly, it weighed just a fraction under twenty-two pounds at Bellwood. There was not a fresh shot mark on the body when I removed the skin for the taxidermist, who found, however, when he was taking the head from the neck, that a single small pel- let, hitting the bird in the head, had cut a large vein in the throat, and the bird had bled internally.” In Kansas, the Indian Territory and Texas, where the turkeys necessarily roost in the trees growing alone the river bottoms, the practice of shooting them on TURKEY SHOOTING 435 the roost was long a common one. TURKEY SHOOTING. If a roost was found, the hunters sometimes approached it during the afternoon, and concealing themselves, awaited the re- turn of the turkeys, and perhaps the rising of the moon, so that the great birds might be seen against the sky. At all times, and especially in sections where turkeys had not been much hunted, it was easy to ride one’s horse beneath a roosting-tree, and there dismounting, to fire into the birds above. Stories are told of hunt- ing parties sent out from the forts in Texas to get fresh meat for the men, who, after a few days, returned with a wagon loaded with turkeys. On the far-stretching a On the far-stretching and often level plains of Texas turkeys were frequently coursed with greyhounds. A troop of riders, with a number of dogs, rode in a long line over the prairie, beating up an extent of country half a mile or more wide. Minally, from some little hollow or clump of bushes a brood of turkeys was started, which took wing and flew, fast and far, over the prairie. The dogs, accustomed to the sport, fol- lowed some particular bird, and all the horsemen, plying whip and spur, followed the dogs at top speed. The turkey’s flight was swifter than the speed of the dogs, and after it had gone half a mile or more it slanted slowly down to the ground and then raced away with great swiftness; but the eager dogs soon overtook it, and forced it to wing again. This time it once more gained distance on the dogs, but now its flight was much shorter than before, and once more it came to earth. The third time, tired by its previous AMERICAN GAME BIRD SHOOTING 430 flights, it rose with difficulty, and the dogs were almost under it, looking up, and now and then one of them springing high in the air in an endeavor to seize it, which at last one of them did. A graphic account of this sport, written by the late A graphic account of this sport, written by the late Elliot Roosevelt, is given by Theodore Roosevelt in his very charming book, “Hunting Trips of a Ranch- man.” In old times, when turkeys were plentiful, and espe- cially in the South, they were trapped and slaughtered in considerable numbers. TURKEY SHOOTING. The commonest form of trap was one formed of rails piled up, crossing at the corners so as to make an ordinary rail pen. From this a deep trench was dug out, slanting up to the level of the ground. This trench was deep enough and wide enough for a turkey easily to pass through. Within the trap a little brush was thrown over the trench, close to the rails. Then through the trench, and running out some distance into the woods, was laid a trail of corn. Turkeys wandering through the woods found the corn, began to pick it up, and fol- lowed it to the trench, and down the trench into the pen, where more corn was scattered. In this way half a dozen turkeys, or even a whole brood, might enter the pen. When they had eaten the corn, and wished to go away, they walked around the walls of the pen looking for a place to get out, walking or jumping across the trench, but never seeming to think of going out the way they had come in. In this manner large numbers of turkeys used to be caught. 437 TURKEY SHOOTING Audubon says that sometimes, after people had be- come surfeited with the flesh of turkeys, they failed to visit the traps which they had baited for some time, and so the poor birds were allowed to starve to death. Another means of slaughtering turkeys, of which we read, is by laying a trail of corn in some place where turkeys congregate; then, hiding near at hand, the gunner prepares to shoot at their heads and necks when all are busily engaged in picking up the corn. In April, 1900, E. W. Nelson, the distinguished field naturalist of the Biological Survey, described a new sub-species of wild turkey from Arizona, under the name Meleagris gallopavo merriann. This bird is found in the mountains of Arizona, western New Mex- ico, and south as far as the Mexican border, extending north into southwestern Colorado. Little has been written about this bird, which pre- Little has been written about this bird, which pre- sumably does not differ in habits from its relatives. Because it is so little known, we are glad to quote an account written by E. A. Goldman, who secured for the Biological Survey the specimens from which Mr. Nelson described the subspecies. TURKEY SHOOTING. He has been Mr. Nelson’s companion on many He has been Mr. Nelson’s companion on many expeditions, and in the Auk, in 1902, described a trip he made into the Mogollon Mountains of northern Ari- zona to secure a series of wild turkeys. He started in January, 1900, and was very successful. The weather was cold, and promised snow, which raised the collector’s spirits, since, of course, with snow on the ground turkeys would be more easily tracked and AMERICAN GAME BIRD SHOOTING 438 found than if the ground was bare. They camped upon a ridge separating Clear Creek Canyon and some of the upper branches of Canyon Diablo, and from here we will let Mr. Goldman tell his own story: “The first turkey tracks were seen in spots of soft “The first turkey tracks were seen in spots of soft soil among the pinons, soon after noon, and others we crossed at intervals along the road. Late in the afternoon, when near the upper edge of the pinon belt, we entered what was evidently the feeding ground of a large flock, for many tracks were seen crossing the wagon road in several places. This encouraged us to camp near here, and when we came to a trail lead- ing down into Clear Creek Canyon, and indicating ac- cessible water, we did so. After a few hasty prepa- rations for the night the camp man was sent to the bottom of the canyon, over a mile away, to water the horses and fill casks for camp use, while the hunter and I started out in opposite directions to look for turkeys. I walked slowly and as quietly as possible through fairly open pine and pifion woods, following a half-circular course in order to avoid going too far from camp so late in the day. Many tracks were seen, but none of them had been made that day. At the end of an hour or so I came to the head of a small side canyon, about a mile from camp. The sun had just set, and all nature seemed to have gone to sleep. Not a sound broke the silence, except the slight, unavoidable rustling of my own footsteps among the dry leaves. TURKEY SHOOTING. I stopped a moment to listen, and had about decided to cut across to camp, when suddenly, from down the TURKEY SHOOTING 439 canyon, came faint but unmistakable turkey notes, which started me on a run in that direction. A few minutes later I cautiously approached the place, where probably over a hundred and fifty turkeys, all females and young of the year, were noisily trying to settle themselves for the night. They occupied the tops of tall pines for about two hundred yards along one of the steep walls of the canyon. In many of the trees there were only two or three turkeys, but some of the larger ones, and especially those with many dead branches, contained from five to ten birds. Many of them sat as closely together as possible, and constantly craned their necks about, squawking, crowding each other, and struggling for places. They flew frequently from tree to tree, and sometimes a bird alighting clum- sily on a crowded branch would knock off one or two others, which would fly noisily to other places. At first the disorder seemed to be general, and most of the birds were crowding, or being crowded, and were uttering loud cries of quit, quit, quit, with many modu- lations, depending apparently upon the degree of ex- citement. They rapidly became quieter, however, until by the time it was dark they were settled for the night. When all was still I rose from the cover where I had been concealed, and carefully noting the locality, left the birds undisturbed, and picked my way across sev- eral small canyons into camp. The hunter came in soon afterward, and a comparison of notes showed that we had located the same roost, he having come up he canyon, while I went down, and each had decided AMERICAN GAME BIRD SHOOTING 440 to watch until dark, and not to begin to fire without the other. About nine o’clock we returned to the place. The first few shots, fired rapidly, created a great com- motion, and the air seemed to be filled with turkeys, flying heavily off in all directions; but there were no outcries, and in a few minutes all was quiet and no more birds could be found. TURKEY SHOOTING. I was satisfied, however, that we had secured as many as necessary for speci- mens, though we did not know the exact number, for some of them went thumping down to the bottom of the canyon and others fell at some distance. “At daylight next morning we were again on the “At daylight next morning we were again on the ground, and found the flock broken up into small par- ties, which soon left their roosts and went off in vari- ous directions. On leaving the trees the turkeys usu- ally flew two or three hundred yards and then ran rapidly until out of sight. During the following days it became evident that all the tracks seen for several miles about our camp had been made by this flock. No more fresh ones were found in the vicinity, show- ing that the frightened birds had left the locality at once. “Our lucky night hunt having given us a fine series of females and young of the year, we then devoted our attention entirely to the old gobblers. We hunted steadily day after day, covering the country for miles in all directions, without seeing any of the old fellows; but they were in the country, and it could only be a question of time until we found them. From the tracks of the different flocks it appeared that the old males 441 TURKEY SHOOTING were living apart from the females and young. Fe- males and young were seen several times, but were not molested. Evidently these birds wandered far and wide, for tracks a day or two old were often found along some ridge, and no fresh ones were seen in the vicinity for days. It did not appear that the birds returned regularly to the same roosting place. Several old roosts were found, usually among tall pines, near the head or along the walls of some side canyon, which were evidently occupied occasionally. None appeared to have been used very long, and at least two had only been occupied once. The birds spent the day wander- ing over the broad and gently sloping ridges between the canyons, and as evening approached worked to- ward one of the canyons and roosted wherever night overtook them. TURKEY SHOOTING. “Finally, on the ninth day, soon after noon, I came “Finally, on the ninth day, soon after noon, I came upon some big gobbler tracks, which were evidently only two or three hours old, and decided at once to follow them. There were about fifteen of the old fel- lows, and in crossing patches of soft soil they left a broad trail, which became very indistinct, or disap- peared altogether, on rocky ground. I had gone only a short distance when my hunter, whom I supposed far away, came up. He had found my moccasin tracks following the turkey trail, and quickly overtook me. I was very glad to see him, for it was difficult work, and even with our combined skill at trailing we made slow progress. Sometimes we had no trouble for sev- eral hundred yards, then suddenly we came to places AMERICAN GAME BIRD SHOOTING 442 where the birds had paused to feed, and found they had wandered about in all directions, spreading among the leaves. At such places the trail became so in- volved that it was difficult to find the direction taken when the birds left. When we came to hard or stony places a few misplaced leaves or an overturned stone or stick were the only things to guide us. Where the trail became dim one of us usually went a trifle to the right and the other to the left, so that unless the flock changed its course abruptly, one or the other was pretty sure to find some signs every few yards. Occasionally we lost the trail altogether and had to go ahead and to ‘cut other tracks’ in softer ground. For a while the course followed was very crooked, and several times it even doubled back and crossed itself, but late in the afternoon it became evident that the flock was working toward a branch of Clear Creek Canyon. Shortly be- fore sunset the trail became so fresh that we kept a sharper lookout ahead, expecting to sight the flock at any moment. It was still proceeding in a leisurely manner, as was plainly shown*by the number of places where birds had paused to scratch out deep pits in search of food. At sunset we were quite close to the canyon, and I began to fear they would be able to roost before we could overtake them. TURKEY SHOOTING. With the idea that I could hear for a considerable distance the heavy wing strokes they would make in rising to the roost, I decided to go ahead and listen, leaving my companion to follow the trail as best he could. I had only ad- vanced about two hundred yards, to some higher TURKEY SHOOTING 443 ground, when I suddenly saw the flock, only about forty yards to my left. The birds had not seen me, and were walking quietly along in single file, following a course directly parallel to the one I had taken. They presented a fine sight, and I was strongly tempted to shoot, but on second thought decided to follow them until they roosted. Moving quietly out of sight in a small arroyo, I ran back a short distance and gave a low whistle, when my companion soon overtook me. Together we followed the birds, using great care not to show ourselves. Food had ceased to interest them, and they were evidently looking for a place to roost. They continued in single file, pausing occasionally to look warily about, until they reached some high ground overlooking a small canyon, along the slopes of which stood several tall dead pines. The leader, a fine old fellow, of unusual size, stopped, and the rest of the flock came trailing up, and gathered in a group facing the canyon. Several low, tremulous single notes, quir-r-r-rt, quir-r-1-rt, quir-r-r-rt, were uttered, and suddenly they took wing together and flew almost hori- zontally out to the branches of the dead trees. After leaving the ground no sound was heard except the heavy flapping of wings. A few moved into the tops of live trees a short distance farther down, but most of them remained in the dead ones. Their behavior was in marked contrast to that of the females and young. There was no crowding and no confusion, and in a remarkably short time they were settled for the night and all was quiet. From where I was lying AMERICAN GAME BIRD SHOOTING AAA their bodies appeared in the gathering darkness like enormous black fruits outlined sharply against the glowing western sky. “When it became thoroughly dark we cautiously ap- “When it became thoroughly dark we cautiously ap- proached the trees and took a stand almost under one containing several birds. TURKEY SHOOTING. They were perched two or three feet apart, so that only one could be shot at a time. “As we knew the old fellows were very wary, we held our guns in readiness as soon as we came within range, and prepared to shoot at the first sign of alarm. I sent my companion to the next tree, and told him to give a low whistle when he was ready. I held my 10-bore gun leveled at one of the birds, and it began to feel very heavy before the signal was given. When it finally came I fired both barrels in quick succession, and was much gratified an instant later to hear a crash- ing noise among the branches as two fine old gobblers came tumbling down, landing before me with a loud thump. Instantly heavy wing strokes could be heard in all directions as the frightened birds left their perches. My companion had also made successful right and left shots into his tree. After some search we each located and killed another turkey, after which no more could be found. We then gathered the big birds together, swung them well out of reach of prowl- ing coyotes or mountain lions, and started for camp. When about a quarter of a mile from the roost I saw by the dim moonlight a turkey form among the branches of a big pine. A chance shot was TURKEY SHOOTING 445 made at it, and I was more than half surprised when another big turkey came crashing down. ‘To reach camp we had several deep, dark canyons to cross, with- out trails, and floundered about, finding the usual ob- stacles, which are unconsciously avoided in daytime, but are always encountered at night. We were in a cheerful frame of mind, however, and above noticing small bruises and other mishaps. “The next forenoon we spent taking pack horses to the roost by a circuitous route and bringing our game to camp. In the afternoon we started on our return to the railroad, and camped in the pifions. The snow- storm we had hoped for until it could no longer serve us came during the night, and the morning found everything white and cold outside our tent. We made a hurried breakfast, and after a long drive reached Winslow in the afternoon. TURKEY SHOOTING. “The result of the trip was a series of thirteen speci- mens, including adults of both sexes and the young of the year. When specimens reached Washington Mr. Nelson found they represented an undescribed sub- species, which he named Meleagris gallopavo merriami. Merriam’s turkey ranges in summer over the higher Merriam’s turkey ranges in summer over the higher slopes of the Mogollon Mountains. In winter, and especially when the snow lies over the summits, the birds move down to the pifion belt, where food is abun- dant. I found them feeding largely on the nuts of the pifion (Pinus edulis). According to some of the old hunters, they also eat the berries of the cedar (Junt- perus utahensis), but none were found in the stomachs AMERICAN GAME BIRD SHOOTING 446 examined, although the turkeys, just before being killed, had been wandering through the upper edge of the cedars, where the ripe berries were exceedingly abundant.” AIDS TO SHOOTING Many excellent books have been written on the art of shooting, and yet it will be obvious to any one who takes the trouble to think a little, that no one can learn to shoot merely by reading books about it. The act of shooting is a complex operation, calling for close attention and the coordination of a variety of func- tions. Comparatively few people become really skilful, because few practice shooting enough to bring about this co-ordination. Most of us remain beginners all our lives. Many parts of the body are called into action in discharging a shotgun: hand, arm, frame, eye and brain must all act together to make the operation per- fect, or nearly so. If any one of these factors fails the act is incomplete. The way to learn to efficiently perform a complex operation is to repeat it many times. In other words, we learn how to do things by doing them over and over again. A player of instrumental music must prac- tice many hours a day before he can play swiftly and correctly. A person learning how to use a delicate tool must work with it a long time before he can accomplish satisfactory results. The professional shooters—men hired by the great arms or ammunition companies to go about to all the shooting tournaments and use the products manufactured by these companies—devote 447 447 447 AMERICAN GAME BIRD SHOOTING 448 their lives to shooting, and are as skilful as any men can be; yet, in fact, it is the rarest possible exception when these men make perfect scores. I suppose that there is no shooting practice so good I suppose that there is no shooting practice so good as to take the gun, say twice a day, for five minutes in the morning and five minutes in the evening, and throw it up to the shoulder, aiming at different objects near at hand, and trying to catch the sight as quickly as possible; in other words, to try to throw the gun up as nearly as possible in the line of sight. If this is done often enough it will prove the greatest aid in shooting, and will tend to make shooting more rapid and more accurate. Upland shooting may be divided into two branches, Upland shooting may be divided into two branches, open shooting and snap shooting. AIDS TO SHOOTING In the first you see your mark clearly, and aim at it; in snap shooting you use only the “eye of faith,” shooting at a shadow, a sound, or a moving leaf, and trust to Providence that your gun is so aimed that the bird which you know is there shall fly into the circle of the shot. Obviously, open shooting grades into snap shooting, and no hard and fast line can be drawn between the two. Some men, who are especially talented, practice snap shoot- ing at all times, in the open as well as in cover. In open shooting, as, for example, where the prairie In open shooting, as, for example, where the prairie grouse are found, or quail are scattered in the open fields, there is almost always plenty of time to take de- liberate aim and kill your bird before it is out of reach. In the brush, however, where snap shooting must be practiced, because the game is hardly seen, and its AIDS TO SHOOTING AAQ whereabouts, distance or direction must often be guessed at, there is no time at all. The writers on shooting of many years ago laid undue stress on snap shooting, and their books seem to urge the sportsman just entering his novitiate to get his gun off first and learn how to aim at his game last. This, of course, makes a quick shot, but in these days, unless one shoots constantly at the traps, the opportunities for practicing shooting are so very few that a man might live beyond the allotted three score years and ten without ever shooting at birds enough to learn how to hit anything. I believe the way to learn the fundamentals of shoot- I believe the way to learn the fundamentals of shoot- ing is to practice with an empty gun until you have learned to throw the gun up in the line of sight; in other words, make your body handle your gun mechan- ically, so that when you put it to your shoulder it will point in the direction in which you are looking. If you can do that you will have advanced far on the road toward making yourself a good shot. In open shooting there is usually abundant time to In open shooting there is usually abundant time to take aim. AIDS TO SHOOTING If the bird gets up anywhere near you, you have time enough to fire both barrels at him and then to stand and watch him for a second or two before he is out of range. Now, if you had given one-quarter of a second more to the effort to secure a good aim on each of these shots you probably would have killed the bird with the first or second barrel, instead of en- during the mortification of a miss. The operations of throwing the gun to the shoulder and pulling the trig- ger take very little time, and although the bird appears AMERICAN GAME BIRD SHOOTING 450 to be going very fast, and seems likely to get out of shot before you can pull the trigger, this apparent swiftness of flight is due partly to the nervousness of the shooter. Where the shooting is open there is al- ways, or almost always, abundant time to point your gun just where you want it. Most of the shots that are missed are due to overeagerness, which means undue haste. The shooter may feel perfectly certain that he will miss his bird unless the gun is pointed very nearly straight at it. The shot spreads over a circle of three or four feet in diameter, and sometimes, even if the gun is not pointed straight at the bird, the edge of the charge of shot will hit it and perhaps knock it down; but this is a chance not worth considering—the bird should be hit with the center of the charge. It was, perhaps, Frank Forrester who spoke of “deliberate promptitude,’ and this deliberate promptitude is one of the secrets of being a successful and a good shot. Aiter all, this merely means coolness and training, the keeping of one’s wits about him all the time, and never being disturbed or flustered by any event of the field. Many of the best and most experienced shots never reach the point where they can hear birds get up all about them without quickened heart-beats and jumping nerves, yet many of them never show by their shooting that they are discomposed by the noise. AIDS TO SHOOTING It has been my experience, on a number of occasions, It has been my experience, on a number of occasions, to go out with people who had done little or no field shooting, and I have found that a word of caution, the mere warning, “Take your time,” when a bird rose, AIDS TO SHOOTING 451 or was expected to rise, helped many a man to retain his self-possession and to kill birds that otherwise he would have missed, firing the gun when they were so close to him that if the charge had hit them they would have been torn to pieces. In ways such as this the man of experience may greatly help the novice with- out in the least hurting his feelings or in any way offending him. In his adm In his admirable little book, entitled “Hitting vs. Missing,” Mr. Hammond recommends practice by the novice at stones skipped over the smooth surface of a body of water. The suggestion is an excellent one, for the striking of the shot on the water enables the shooter to see precisely where he is holding with rela- tion to the mark at which he is shooting. This, of course, is what each gunner wishes to know when he shoots at any object. Why did he miss? Did the charge go above, below, or to one side of the mark? The shooter who could tell where each charge of shot went, and had intelligence enough to apply this knowl- edge to each subsequent shot, would speedily attain a high degree of skill. It is generally It is generally known that a person properly placed can see a charge of shot flying through the air toward a target. The charge of shot, as it flies through the air, is said to look like a long shadow composed of many lengthwise strips. It is a common practice for a trap shooter, when he finds that he is frequently miss- ing his targets, to ask some friend to stand near him and watch where the shot is going with relation to the AMERICAN GAME BIRD SHOOTING 452 target, whether under or over, before or behind. Given this information, the shooter corrects his aim accord- ingly. AIDS TO SHOOTING The many books that have been written on shooting The many books that have been written on shooting give hints and suggestions which are of great value to the young gunner, explaining what he should do, and, more important than all, cautioning him against the mistakes he is likely to make. This is helpful, but, after all, nothing can take the place of practice. Each beginner must necessarily go through the school of preliminary practice, finally coming to clay targets, thrown from the trap, which afford the best practice of all. He may feel sure that the more practice he has the better he will learn to shoot, but I believe that the best practice of all, if it be faithfully continued, is that taken with an empty gun, for five minutes at a time, two or three times a day, throwing up the gun on different objects and pulling the trigger the instant the sight is caught; or throwing up the gun and then swiftly swinging it on to some mark and pulling the trigger just as this mark is reached. A point that must be remembered, and that has much to do with success in shooting, or the lack of it, is the disparity in strength and skill which exists in the two arms of all men. Most men are right-handed, and are constantly performing various tasks with the right hand and arm, which thus becomes far more efficient and much stronger than the left. A few people are ambidextrous, and can use either hand indifferently. A few men are left-handed, and can shoot from the shoul- AIDS TO SHOOTING 453 der, drive a nail, or write a letter with the left hand, but cannot use the right hand efficiently. Now, with the average right-handed man, shooting from the right shoulder, the gun is supported, and, more important still, is guided by the left hand; yet, as the man is unac- customed to performing tasks with that hand, he has far less control over its movements than he has over the right hand. If he swings his gun to try to point it at a certain object, he is likely to swing it too far, or not far enough, or above or below the object. AIDS TO SHOOTING It becomes important, then, for the man who is train- It becomes important, then, for the man who is train- ing himself to shoot well to endeavor to strengthen and to train the left arm so that he may have it con- stantly more and more under control, and thus may make it do more exactly the things which he wishes. Such strengthening and training may be begun by the use of dumbbells or the swinging of Indian clubs, which will give strength to the muscles, and then by the performance of simple tasks with the left hand, which will teach it to respond better to its owner’s will. Professional shooters, who are among the very best shots in the country, find it necessary to the perform- ance of their work that they should keep themselves in the very best physical condition. Many of them practice constantly at gymnasium work, including dumbbells and Indian clubs, are careful about their eating and drinking, and are, in fact, trained athletes. In order to do the work for which they are paid they must be in good physical condition, and if they are AMERICAN GAME BIRD SHOOTING 454 unable to continue to do this work their services are no longer required by the people who hire them. In old times the best shots that we used to know were the market shooters. Many of them were men who worked at some trade when they were not shoot- ing for a living, their muscles were hard, their health good, and they were in the pink of physical condi- tion. When they took up their guns in summer or autumn they shot all the time, and their constant prac- tice, together with their strength and hardiness, made them most destructive of the game they followed. In the same way to-day, we find that the best shots along the south Atlantic coast are the gunners who shoot ducks for the market. Farming during the spring and summer, fishing in late summer and early autumn, and gunning through the autumn and winter, they are always in splendid condition, and always in good practice. Very different is the case of the city or office man, who perhaps spends fifty weeks of the year at his desk, walking each day only a few blocks. AIDS TO SHOOTING When his vaca- tion comes he sets out on his shooting trip, and is sur- prised and disappointed to find that he can neither tramp nor shoot. Why should he be able to do either ? It is not by doing a thing half a dozen times a year that one acquires skill in it. The man who is to under- take tasks requiring skill, muscular effort, long wind, or endurance of any kind, must prepare himself for the task which lies before him. As I wrote some years ago, in “American Duck As I wrote some years ago, in “American Duck AIDS TO SHOOTING A55 Shooting,” “Skill in shooting is not born in any one. Just like reading and writing, it must be learned; and, like reading and writing, the more practice one has the more easily and better it is done. Many a pro- fessional gunner who is a wonderful shot would find it labor of the hardest kind to sit down and write a four-page letter; and many a business or professional man, who goes gunning perhaps once in two or three years, finds that killing the fowl that give him shots is something that he cannot accomplish. Many men have noticed that sometimes at the end of a season they can shoot very well; and then, if for two or three years they do not go shooting, they find that they can- not hit anything, and have to begin at the beginning and learn it all over again. They have, perhaps, for- gotten how to hold on their birds, and besides, their muscles, through disuse, refuse at first to act with the brain as they formerly did. This reflex action, so called, can only be regained by practice.” From the books on shooting much may be learned From the books on shooting much may be learned about how to hold on straightaway birds, incomers, quartering birds, and cross shots. It is well to remem- ber that at twenty-five or thirty yards the charge of shot from a cylinder-bore gun covers a circle two or three feet in diameter, and that on quartering birds, and those flying across one at such a distance, a cer- tain allowance may be made for the great area of the charge. AIDS TO SHOOTING In other words, if the shooter fires one foot ahead of a crossing bird it will be quite certain to fly into the load. If the shooter fires a little behind the AMERICAN GAME BIRD SHOOTING 456 bird, very likely it will be missed altogether, or only the edge of the load will prick it. Birds fly fast, but shot flies much faster, and while almost every one is likely to shoot behind wild ducks, there is very much less dan- ger of doing so on cross shots at upland birds. In the brush shooting which prevails over many parts In the brush shooting which prevails over many parts of this country the birds usually get up very near te the gunner, and the shooting is quick and close. A cylinder-bore gun and a charge of small shot will give better results for this shooting than a choke-bore gun and large shot. I usually begin the season by using one ounce of No. 10 shot for woodcock, partridges and quail, but as the weather grows colder and the leaves fall, the birds are likely to be wilder and to be a little more heavily feathered, and for the very last of the season an ounce of No. 8 shot may be used for quails and partridges alike. In prairie shooting, toward the end of the season, when the chickens have become wild and get up at long range, heavier shot and a choke-bore gun may profitably be employed. Sage grouse, being very large and—the old ones—very tough birds, may be shot at with No. 6 shot all through the season. In all shooting there is really nothing more impor- In all shooting there is really nothing more impor- tant than that the gunner should know just what his arm can do. If he thoroughly understands this, if he will practice as recommended, and if he has his nerves and impulses under good control, he can very soon make of himself a more than average shot. CLOTHING The hunter’s comfort and endurance are largely de- pendent on being properly equipped in the matter of footwear and clothing. The average novice is more than likely to be ill furnished in these essentials, even though he be expensively furnished. For instance, clothing which is too tightly fitted, or too heavy in weight, adds unnecessarily to the strain of walking or riding, thereby inducing premature fatigue, and mean- while cramping the activity and lessening the enjoy- ment. The clothing should always be loose enough to admit of the freest movements of the limbs, the matter of style being a secondary consideration from a common-sense viewpoint. Proper footwear is also of prime importance, for while the walker may proceed passably well with a hampering fit as to clothing, he is at a constant and painful disadvantage if he wears ill-fitting shoes. The latter are sure to strain, chafe or blister the feet, so more or less crippling the wearer and marring his en- joyment. When the feet are sore the walker, in the attempt to save his feet, is thrown out of his usual stride, and this lack of balance throws strain on weaker muscles, bringing on a general soreness of the body, with the result that what should be a recreative pleas- ire becomes a painful labor. 458 AMERICAN GAME BIRD SHOOTING The hunting field is no place for the breaking in of new footwear. ‘The latter should be purchased months ahead of its actual use afield, and should be worn often enough to be thoroughly broken in, so that it may be worn with comfort in actual hunting. The footwear should be as light in material as is The footwear should be as light in material as is consistent with practical use. If the going is easy and dry, the hunter may advantageously use some of his old cast-off shoes, assuming, of course, that they have some wear left in them. About two dozen hobnails, driven in proper distribution in the soles and heels, add greatly to their efficiency in walking, and protect them from the grinding and cutting effects of rocks in rough going. It should be observed that the hob- nails are an indispensable adjunct to any hunting foot- wear. CLOTHING In dry weather the soles of the boots or shoes take on an exceedingly slippery polish, so much so that walking on dry leaves or grass in shoes without hob- nails presents the same difficulities as walking on ice. The specially manufactured hunting boots and shoes The specially manufactured hunting boots and shoes are made of various kinds of excellent leather, heavy and light, and in styles to suit the fancy of the pur- chaser. The best makes have tough, pliable uppers, strongly reinforced on the sides, double-soled from toe to heel, hobnailed; soft and elastic as a whole, yet so closely constructed, withal, as to be nominally water- proof, comfortable and durable. The boots, as a rule, are simply the shoes with a leg added, and they weigh more accordingly. After all, lightness of weight in foot gear is a most CLOTHING 459 important consideration, and many sportsmen of great experience wear only low shoes with canvas leggings of the cavalry type. Some men wear tennis shoes with leggings, and rejoice greatly in the ease with which they can get about, walking down their more heavily shod companions. Of course, tennis shoes wear out, but on the other hand they are much less expensive than heavy boots or shoes. I have seen them used in big game hunting in the Rocky Mountains. The footwear should be selected with a considera- The footwear should be selected with a considera- tion of the surface features of the ground on which it will be used. On comparatively smooth ground a light shoe can be worn with advantage, but on ground where rocks and ledges are numerous a heavier shoe is neces- sary to withstand the increased rough usage. The only advantage of the boot is that the leg serves The only advantage of the boot is that the leg serves as a legging; but it has the disadvantage, which the regular legging has not, that in warm weather, owing to the tightness, and absence of ventilation, it causes overheating and much discomfort. It also hampers the free movement of the ankle. The combination of shoe and legging is much lighter The combination of shoe and legging is much lighter than the boot, is better ventilated, and does not hamper the leg action in the least. CLOTHING The moccasin-shoe is an excellent article of foot- The moccasin-shoe is an excellent article of foot- wear, particularly in such hunting as enjoins the most silent activity. It is a kind of compromise between the moccasin and the shoe, being the former with a light but tough sole added thereto. The material, how- AMERICAN GAME BIRD SHOOTING 460 ever, as to uppers, is of the lighter and tougher leath- ers, such as calfskin, etc. These, in the main, are the essentials of hunting foot- wear, though presented in many varied styles and of varied materials. A city dweller, accustomed to the use of light foot- wear day after day, seriously handicaps himself if he wears unnecessarily heavy footwear afield, particu- larly if he has reached an age which can no longer be recognized as young. An excellent leather-top rubber shoe serves admir- An excellent leather-top rubber shoe serves admir- ably for still hunting, for hunting in wet or winter, and are strictly waterproof, but they draw the skin on per- spiring feet to an injurious extent, and therefore, for certain persons, are not a proper article of footwear. As to clothing, there is even a greater variety as to style and material from which the hunter may select. Of the materials offered, corduroy, moleskin and can- vas have held conspicuous place in the garments used in upland shooting, though severahother kinds of mate- rial also have strong claims to excellence. However, in upland shooting, any material having However, in upland shooting, any material having a woolen admixture is quickly picked to pieces by brush, briars or heavy grass, and does not possess the peculiar advantages of the aforementioned cloths, which are of cotton material, hard in finish, and of great durability. Khaki and Duxbak are very superior cloths for hunt- Khaki and Duxbak are very superior cloths for hunt- ing garments. Advantages of canvas are that it dries 461 CLOTHING quickly, keeps out heat and cold, will not hold burs, and is cheap and durable. made of various kinds of skins are excellent Jackets made of various kinds of skins are excellent protection in cold weather. They are light, pliable, impervious to wind, have a smart appearance, though, as a rule, the upland shooter rarely needs overclothing for the sake of warmth. His exercise keeps him warm. CLOTHING Still, at the end of the day, if the hunter rides home, he needs protection from the chill of evening, and should have an overcoat in reserve for his comfort. One weight of outer clothing will serve for all seasons, hot or cold. all requirements as to weather The shooter can meet all requirements as to weather by selecting his underwear, light or heavy, according to the temperature. If the shooter desires to provide against stormy If the shooter desires to provide against stormy weather, he can find a large variety of storm coats, light and heavy in weight, made of rubber, oilskin and Duxbak, the latter being a canvas partially waterproof. If the shooter desires a raincoat for use on horseback, he should be careful to so specify, as one not divided in the back to a proper height so that it may spread over the rider’s legs, will not be a protection from rain. GUNS AND LOADS. The selection of a gun is a matter which requires careful consideration. Weight, bore, and shooting qualities are not to be determined arbitrarily. As a rule, the novice desires the closest shooting As a rule, the novice desires the closest shooting gun obtainable, regardless of whether it is appropriate to the shooting he expects to have, or whether his skill is equal to the exactions of such gun, be the shoot- ing what it may. Even superior marksmen are prone to use guns of too close pattern and loads of unneces- sary power. If the shooter cares to own but one gun, and uses it for all-round shooting, a compromise as to weights, loads, ete., is desirable, though for cover and open shooting special guns are quite the best, a modified cylinder for the one and a closer choke for the other. Whatever the bore, the gun should be as light as is consistent with safety. An extra pound or two of gun counts heavily in added weariness toward the end of a day’s shooting, and at all times in the way of slower manipulation. Quickness of execution is one of the prime essentials in the practical use of the gun in wing shooting. In the closing years of the past century the popu- larity of the 10-bore rapidly waned, till at last it be- came obsolete as a gun for upland shooting. In weight 462 GUNS AND LOADS 463 it ranged from 8 to 12 pounds, and, as a rule, was chambered for heavy loads; that is, from 4 to 5 drams, sometimes more, of black powder and 1% ounces of shot. The advantages of the 10-bore were in its capability to handle heavy loads, and it is an excellent gun for duck shooting and for prairie chicken shooting late in the season. A light 10-bore, shooting light loads, is opposed to the prin- cipal reason—that is, the capacity for shooting heavy loads—which justifies its use. It was demonstrated, after a number of years, that the ro-bore had very little advantage in ballistic properties over the 12- bore, but had the very serious disadvantage of extra weight and more expensive ammunition. At present At present, bores smaller than 12, as 16, 20 and even 28, have their warm advocates, who present many cogent reasons for their faith. GUNS AND LOADS. However, for all round shooting purposes, the 12-bore is still the popular gun. In weight it runs from 6 to 8 pounds, or more, with barrels of 26, 28, 30, 32 and 34 inches, 30 inches being the favorite length. It is chambered for from a 2% to a 34-inch shell, the commonest measurements, how- ever, being 25% and 2°/, inches. The lightweight gun uses The lightweight gun uses a lighter load, there being always a proper relation of load to weight of gun. If the gun is excessively light, then its advantages in the matter of handling a heavier load are lost, and it is better to get a gun of smaller bore. The load for the 12-bore ranges from 2 to 4 drams of bulk pow- der, according to the weight of the gun and the AMERICAN GAME BIRD SHOOTING 464 kind of game bird on which it is employed. Four drams, however, is an excessive load, used chiefly by pigeon and duck shooters, and by very few of them. Besides the punishing recoil, the excessive load strains a gun seriously and shortens its life. The most desirable all-round load for a 12-bore is 3 The most desirable all-round load for a 12-bore is 3 drams of powder and 1% ounces of No. 7% chilled shot. This is a very killing load, and is in general use by target shooters at the traps. However, for special shooting, different sizes of shot are essential. Some shooters use 3% or 3% drams of powder, but the recoil is much greater, relatively, than that of 3 drams, and punishes the average shooter. The recoil may be kept within reasonable limits by reducing the weight of shot as the load of powder is increased; thus, 3% drams with 1 ounce of shot gives a high velocity, and is very effective in duck shooting, at the same time being free from excessive recoil. The only advantages of bores smaller than the 12 are their lightness, and the consequent greater ease in carrying them and greater quickness in aiming, because of their greater lightness. The ammunition for them is somewhat cheaper, but not enough so to be a mat- ter for serious consideration. GUNS AND LOADS. While the smaller bores will deliver the load of shot with as much velocity as the larger bores, it should be noted that the killing circle is less, and that, therefore, a higher degree of skill in pointing is a necessary correlative. If the bore is such as to make the killing circle equal, then the pat- tern is thin. Thus there is reason, while giving con- GUNS AND LOADS 465 sideration to the powers of the gun, to consider there- with the capability of the shooter. Some shooters have a false pride concerning the Some shooters have a false pride concerning the ownership of a cylinder bore, which is often assumed to indicate inferior shooting qualities in the gun or lack of skill in the shooter; but it is far more sensible for the shooter to use a gun in accord with his shooting capabilities, and the conditions as to cover shooting, etc., and thereby achieve reasonable success in the field, than it is to make a ridiculous failure with a gun which shoots too close. A full-choke gun puts about 70 per cent. of the shot in a 30-inch circle at 40 yards, a half-choke about 60 per cent., a quarter-choke about 50 per cent., an im- proved cylinder 45 per cent., a cylinder—so called; it commonly has a trifle of choke in it—35 per cent. It is a safe assumption that by far the greater num- It is a safe assumption that by far the greater num- ber of birds, in upland shooting, fall within 25 yards of the shooter; therefore, the pattern made at 40 yards is not the criterion by which to judge of the gun’s powers at 25 yards. In cover shoot- ing, a full-choke gun mutilates many birds to a degree which renders them worthless. Some shooters have a 16-bore set of barrels fitted Some shooters have a 16-bore set of barrels fitted to a 12-gauge frame, the barrels being correspondingly heavy, the greater weight necessarily being about the chambers; thus this greater weight negatives all the advantages of the 16-bore in the attempt to shoot heavy loads. GUNS AND LOADS. Again, the 20-bore, mounted on a 16-gauge frame, 466 AMERICAN GAME BIRD SHOOTING is another freakish perversion of the smaller bore, though the fads and fancies of shooters are of much trade value to the gun makers and repairers, The smaller bores can rarely be used to advantage The smaller bores can rarely be used to advantage with less than a quarter choke, it being obvious that three-quarters of an ounce of shot should be held closer together than an ounce and a quarter to insure a proper distribution in the pattern. The 28-bore is a wonderfully effective gun for up- land game shooting, and its lightness makes for quick execution; but the killing circle is relatively small, and a higher degree of skill is a requisite in its success- ful use. The 16-bore, 26, 28 or 30-inch barrels, may weigh from 5 to 634 pounds, and the 20-bore may have about the same range in weights, with 614 pounds, per- haps, as the maximum. Two sets of barrels, one set of modified cylinder bore, the other a close choke, add immensely to the efficiency of the gun. There is no doubt but that in the matter of de- structiveness the repeating shotgun, “pump” and auto- matic far surpass the double gun. Fortunately, at the rise of a bevy, in upland shooting, the average sportsman using such gun is not sufficiently expert to fire more than two shots with any degree of cer- tainty. Yet, having extra loads at his command, he can take advantage of any slothful or timid birds which rise after the main part of the bevy has flown, hence is hardly ever caught unprepared. An expert with the repeating shotgun is terribly An expert with the repeating shotgun is terribly GUNS AND LOADS 467 destructive when in action afield. He can fire with wonderful quickness and precision, and may destroy a small covey at the first rise. Except for that feature it has no advantages over the double and single-bar- reled guns in such sections as have a legal limitation to the number of birds any one shooter may kill in one day. However great the destructiveness of any gun, it may not be properly employed to exceed the statu- tory limit. GUNS AND LOADS. It is urged as a plea against the repeater that, its capacity to kill being so great, there is always a temptation to kill more than the legal limit; but the same argument can be urged against any other kind of gun in the hands of a skilful but lawless shooter. Men have broken the law many times while using guns other than repeaters, therefore there is nothing inher- ent in the kind of gun which makes for good citizen- ship. That is a point of ethics which refers to the mari. Factory-loaded ammunition has been so admirably improved that it is practically perfect. The average shooter, in the attempt to kill, is prone to attribute fail- ure to faulty ammunition or to a gun which does not shoot straight or does not fit. If the shooter could calmly persuade himself to look upon his failures with the same unprejudiced opinion common among his com- panions in respect to himself, he could safely place the blame for all failures without any risk of creating an argument. In any event, when afield, it is not an un- wise policy to keep all explanations of one’s failures to one’s self. Nine men out of ten do not care to listen AMERICAN GAME BIRD SHOOTING 468 to hard-luck stories, which oftenest have their origin in incompetency; and the other man, who might be sympathetic, right or wrong, is more than likely to have troubles enough of his own. There are shooters who have a belief that they can There are shooters who have a belief that they can produce better cartridges loaded by hand than those made by machinery. Such belief is fallacious. Given like materials, the machine-loaded cartridge is likely to be the better of the two. To a certainty it will have more uniform loading, as the pressures on the wad- ding will be automatically alike. This uniformity is beyond the compass of the hand. The kind and quality of wadding, however, is an important factor in the ef- fectiveness of any load, hence a well wadded hand load may surpass a poorly wadded machine load; but this is a matter of material and not of methods. GUNS AND LOADS. For the 16 and 20 gauges the loads of shot should For the 16 and 20 gauges the loads of shot should be comparatively light, from three-quarters of an ounce to one ounce, with from two to two and a half drams of bulk powder, or a corresponding quantity of dense powder. There should be no attempt to arbitrarily make the smaller gauges equal to the 12 in weight of ammunition. As already suggested, the advan- tage of the smaller bores is in their lightness, and therefore in their fitness for upland shooting, which does not exact such hard killing qualities in a gun as does wild fowl shooting. The gun should be selected with a view to its fit- The gun should be selected with a view to its fit- ness for the special shooting of the owner, his physical strength and skilful capabilities, which, summed up, GUNS AND LOADS 469 are quite apart from the matter of reducing all the factors to an arbitrary standard. Any gun that is overloaded, be it light or heavy, will kick unpleasantly, and sometimes harmfully, as shown by a bruised shoul- der, a contused cheek, and perhaps a cut trigger finger. As a consequence of a punishing recoil, the shooter As a consequence of a punishing recoil, the shooter is quite likely to flinch or fudge, which, in turn, with continued punishment, is quite likely to become habit- ual, thereby greatly impairing success. The lighter the gun, the more violent the recoil from overloading. If the shooter is physically so hardy that he can If the shooter is physically so hardy that he can withstand the punishment consequent on excessive re- coil, he nevertheless is quite likely to lose control of the kicking gun in delivering the second shot. No argu- ment is required to show that a man in the act of firing both barrels, one quickly after the other, can main- tain a perfect poise and do quick, accurate execution with a gun which does not kick, and that he cannot do so equally well with a gun which kicks so hard that it throws him off his poise. There is no arbitrary rule. Each case must be considered by itself. As a rule, a large man will withstand the recoil better than a small man. GUNS AND LOADS. Again, some men have the ability to take most of the recoil on the hands and arms, which ab- sorb the shock as would a spring, thereby relieving the body from the hard shock which is communicated when the gun is held firmly against the shoulder. Apart from all this, the average gun, when overloaded, does not equal its performance with its proper load. DOGS. Setters and pointers are the breeds of hunting dogs which are chiefly used for work afield by American sportsmen. These breeds display especial usefulness in the pursuit of the various kinds of game birds, such as quail, grouse, snipe and woodcock. Sport of this kind is known as upland shooting, in contradistinction to wild fowl shooting, a sport of marsh and water. Spaniels also have appreciative recognition, in a rela- Spaniels also have appreciative recognition, in a rela- tively limited way, by certain sportsmen whose domi- cile happens to be situated in a locality favorable to spaniels’ use. But even at that, this breed holds such an insignificant place in relation to the gun, in America, that it may be considered as a negligible factor. In this country the areas in which the spaniel can In this country the areas in which the spaniel can be worked to advantage are so few and unimportant, as compared with the vast areas favoring the work of the setter and pointer, that it is obvious that these natu- ral conditions dominate and determine the sportsman’s choice to an important degree. As few sections favor the spaniel’s use, few sportsmen own him. His physi- cal powers, compared with those of the pointer and setter, are exceedingly limited, and he is thus at a dis- advantage in the comparison with them. Nevertheless, in certain areas containing much cover and irregular ground, in which the view is constantly obstructed, the 470 470 470 Setter standi Ruffed Grous Sette stan Ruff Grou DOGS 471 work of the spaniel fits in admirably, assuming that game is present in reasonable abundance. The spaniel’s working metho The spaniel’s working methods, natural and educa- tional, are quite distinct from those of the pointer and setter. The two latter hunt mute, and, having found the birds, indicate the find by pointing them, the flush being left to the shooter’s choice of time and manner. The spaniel trails the birds to their place of conceal- ment, and, the opportunity offering, presses them to a flush without any attempt at pointing, if he acts properly; hence the term “springer.” Some spaniels whi Some spaniels whimper, babble, or give tongue mer- rily while trailing; others work mute. Giving tongue at all is an objectionable trait, as in upland shooting man and dog should work as silently as possible. DOGS. This is especially important when the birds are wild and seek safety in flight at the slightest alarm. Thus the need of silence in upland shooting would seem to be quite obvious. Howe However, this is contrary to some of the traditions and usages governing the use of the spaniel afield. Stonehenge (J. H. Walsh), in his work “Stonehenge on the Dog” (1873), in respect to this matter speaks as follows: “A good and somewhat musical tongue was, by the old school of sportsmen, considered a desid- eratum, in order not only to give notice that the dog is on game, but also the particular kind which he is ‘questing,’ and which many good spaniels enable their masters to distinguish by a variation in their notes. Formerly this was thought so important that if a AMERICAN GAME BIRD SHOOTING 472 spaniel happened to be mute he was hunted with a bell around his neck, as is sometimes done with the setter when used in covert. In the present day a very fash- ionable breed (the Clumber) is invariably mute; but as these dogs are chiefly used in aid of the battue, there is not the same necessity for them to give notice of their approach as in the case of spaniels used either in wild pheasant shooting or for cocks, hares or rab- bits. It will, therefore, appear that for every kind of covert shooting but the battue we require a strong, use- ful spaniel, capable of bearing exposure to the weather, and neither too large for the runs nor too small to bear work. Added to these qualities we want an ex- quisite nose, and a musical but not noisy tongue, which is all the more valuable if it will distinguish by its note the various kinds of game.” While giving tongue might charm the ear of the While giving tongue might charm the ear of the English sportsman, and indicate the kind of birds ahead of the spaniel, in this country, with its rough, irreg- ular coverts, much of which are in the condition that nature’s hand shaped them, a noisy dog is a detriment to the sport. As to the advantages of flushing, there are many qualifications. Considered in its proper relation as a spaniel trait, the spaniel must be trained to work well within gunshot of the sportsman. DOGS. He also must con- duct his efforts at all times with a reasonable degree of steadiness, to the end that the gun shall constantly have favorable opportunity. This intelligent co- DOGS 473 ordination of effort between dog and gun is absolutely essential to success. When on a trail, the spaniel should time his speed to that of the shooter. When pressing in close on the birds he should refrain from flushing till the shooter is properly placed and ready for the shot. If he is specially intelligent, accomplished and helpful, he may get the birds between himself and the shooter before flushing them, so that they may be driven toward the shooter, thus enhancing the chances for a successful shot. It is self-evident that a lawless spaniel, ranging out of gunshot, and flushing the birds regardless of the shooter’s interests, is in no sense an aid to the gun. Such misbehavior, on the contrary, insures that even if the game be plentiful the opportunities of the shooter are constantly ruined, and he then is much worse off than if he had no dog at all. As the spaniel is not required nor desired to point, As the spaniel is not required nor desired to point, his training is correspondingly simpler than that of the setter and pointer. When trained, he beats out a very limited area of ground about the shooter, keeping at all times well within gunshot, flushing the birds with- out any preliminary point as an entirely proper act. In thick cover, or in ground so rough that the dog is much hidden from view, the advantages of this manner of work are manifest. In the case of the pointer and setter, which fre- In the case of the pointer and setter, which fre- quently find birds far out of gunshot, the advantages AMERICAN GAME BIRD SHOOTING 474: of pointing, and of stanchness to the point till the gun arrives in position, are also self-evident. By feathering, and a general change of manner and By feathering, and a general change of manner and intensity of action, the spaniel indicates his recogni- tion of the foot scent or the whereabouts of the birds. By the observation of similar mannerisms, displayed when he catches the body scent, the shooter discerns when the spaniel is close to the birds or is about to spring on them. DOGS. Thus, while his preparation for effi- cient field work requires but a moderate degree of training, as compared with the more complex educa- tion required for the setter and pointer, it is impera- tive that his education, so far as it goes, shall be complete. It is obvious that in the interest of good sport he must conform to the gun’s requirements. He should promptly obey the commands, “Heel,” *Go on,” ‘Drop, ‘Fetch,’ “Come uns? + Seekidead. or “Find,” and “Steady,” the latter command being of special importance, since it comes into frequent use in many relations, as when the spaniel is roading too fast, or is intent on a premature flush, or when he is disposed to become riotous and generally disobedient. A proper observance of this order makes in many ways for the better success of the gun and the pleasure of the shooter; therefore, the importance of establishing thorough obedience to it. As to methods of finding the birds, the spaniel, as a rule, follows the foot scent, differing in this respect from the best working pointers and setters, which carry a high nose to catch and follow the body scent direct to DOGS 475 the birds. However, as the spaniel works in a narrow area, while setters and pointers work over considerable territory, each method, in its proper relation, has its special fitness and value. In the matter of speed the spaniel is at opposites In the matter of speed the spaniel is at opposites with his pointing brothers. Huis range, necessarily so limited in scope, requires but a moderate degree of speed to meet the requirements of the gun. Even with moderate effort he has ample time to search out every nook and corner within gunshot of the shooter, as the latter meanders along. With a team of spaniels —a team may be any reasonable number which the fancy or needs of the shooter requires—nothing is overlooked by them. If up to the proper requirements, the spaniel works merrily, diligently and intelligently. He delights in the quest. A team of well broken spaniels, working in a section adapted to their peculiar methods and limi- tations, affords excellent sport. Unfortunately, in the United States such sections are few and far between. DOGS. In England, before the battue and driving had In England, before the battue and driving had largely ousted the pointing dog and spaniel, the latter, as a worker afield, held an important place in upland shooting and in the esteem of sportsmen. There were many good reasons for this choice—good reasons as they pertain to English shooting, but otherwise in re- spect to shooting in this country. On the English game preserves the dog trainers, the gamekeepers and the owners know every cover, every range and every runway. They know where the birds most fre- AMERICAN GAME BIRD SHOOTING 476 quent, and, therefore, know how to work out every inch of ground to the best advantage. For cover shooting the spaniel is a superior assist- ant. His shorter leg and smaller size enable him to pass readily through or under cover which would offer a real obstacle to setter and pointer, even if it did not deter them from attempting it. As he works within gunshot he meets an essential of close cover shoot- ing. Flushing the birds under such conditions is a distinct advantage. Thus, then, the springer may be considered as a better worker than the pointing dog for rough and cover shooting exclusively. It is obvious that a setter or pointer, when standing stanchly in dense cover, out of sight of the shooter, is, as an assistant, inferior to the spaniel, which flushes the birds at once. To overcome this disadvantage, some shooters tie a small bell to the collar of the pointer or setter, and when its tinkle ceases, the dog being hidden from view, the inference is that he then is pointing. To overcome the disadvantage of pointing stanchly To overcome the disadvantage of pointing stanchly in dense cover, some trainers school their setters or pointers to flush to order, but such a practice tends to disorganize the dog’s training as a pointing dog, as he is likely to become unsteady and unreliable in this most valuable particular. As showing the great popularity of the spaniel in As showing the great popularity of the spaniel in England, the following is taken from “British Dogs,” written by the late Mr. DOGS. Hugh Dalziel, and published about twenty years ago: “The spaniel is not only DOGS 477 the oldest breed we have that has been kept to the hunting of fur and feather, as a help to hawking, net- ting and the gun, but he is still the most generally, useful of our game dogs, as he is the most universal favorite. In field or covert, no dog works so close as a well bred and well broken spaniel; neither fur nor feather can escape him; no hedgerow is too thick, no brake too dense for him to penetrate and force out to view of the sportsman the reluctant game; he is a most active, ardent and merry worker. His ‘wan- ton tail,’ ever in motion while he quests, increases in rapidity of action with that tremulous whimper that tells so truly that he is near his game, and says to his master, in terms that never deceive, ‘Be ready—it is inenesa Referring to the ancient history of the spaniel, the same author remarks: ‘‘Spaniels were, in olden times, also known by the name of the game they were kept to, as ‘a dog for the partridge,’ ‘a dog for the duck,’ ‘a dog for the pheasant,’ as in our own day we still have the cocker, or dog for the woodcock; but at what date the term ‘springer,’ or ‘springing spaniel,’ was introduced, I do not know, but presume it must have been when the qualities of the setter, or ‘setting span- iel,’ became fully developed and permanently fixed by breeding setters from known setting spaniels only, and keeping the breed of questing spaniels distinct; the term ‘springer’ was probably given to them on account of their natural disposition to rush in and flush or spring the game.” 478 AMERICAN GAME BIRD SHOOTING In view of his special aptitude for work in cover and rough country, the spaniel should fit in usefully in many sections of Canada, New England, Pennsyl- vania, etc., for woodcock and ruffed grouse shooting. Yet, even in those sections, the stretches of open game country are so numerous, though contiguous to cover, that much of the best shooting ground is beyond the capabilities of the spaniel. Thus the advantages and disadvantages average up in such a way that in the compromise as to methods the setter and pointer have the preference. DOGS. In open country, where range is an essential to suc- In open country, where range is an essential to suc- cess, the spaniel has no standing as a worker. As he ranges close, from compulsion, and flushes his birds when he finds them, it is quite obvious that in a prairie or open country he could not meet the requirements. In bench show competition, and as a house dog, the In bench show competition, and as a house dog, the spaniel shines as a favorite. Some years ago his ad- mirers made a vigorous attempt to organize spaniel field trials in the United States, but the effort resulted in failure. However, in England, spaniel trials have been held with more or less success. The foregoing remarks, in the main, apply to field The foregoing remarks, in the main, apply to field and cocker spaniels, but there are several other breeds used for field work, chief of which are the Clumber spaniel, a dog of heavy physique, slow and mute in his work, and easily broken to work to the gun. The Sussex spaniel is a dog of hardy constitution, a good all-round worker, much prized at one time in England for his superior usefulness afield. The English springer DOGS 479 is said to be identical with the old Norfolk spaniel, a compactly built dog, standing much higher on his legs than do some of the other spaniel breeds, and quite disposed to give tongue on occasion. There is also the Welsh springer, of comparatively recent classifica- tion, and there are several others as yet unnamed. Of setters, three breeds—English, Irish and Gordon Of setters, three breeds—English, Irish and Gordon —are recognized in America. In England, some authorities affect to recognize the In England, some authorities affect to recognize the Russian setter, though everything purporting to be de- scriptive of that breed is meager, and seems to rest largely on hearsay. DOGS. Edward Laverack, in his work, “The Setter,’ describes them as white, lemon and white, liver and white, and black and white in color, yet naively remarks: “I have never seen but one pure specimen, which was in the possession of the late Lord Grantley, at Rannoch Barracks, head of Loch Rannoch, Perthshire.” He also stated that “these dogs are but little known in this country.” As a matter of hearsay, he relates that the head keeper to Her Majesty, many years ago, had a beautiful breed of Russian setters, and that ‘“‘they were good, but most determined, wilful and cbstinate dogs, requiring an immense deal of break- ing, and only kept in order and subjection by a large quantity of work and whip; not particularly amiable in temper, but very high-couraged and handsome; an enormous quantity of long, silky white hair, and a lit- tle weak lemon color about the head, ears and body; and their eyes completely concealed by hair.” AMERICAN GAME BIRD SHOOTING 480 In “Stonehenge on the Dog” special attention is devoted to this setter. By way of illustration, a pic- ture of ‘a Russian setter slightly crossed with English blood” is presented. In many points it resembles the griffon. Stonehenge remarks: “They are now very scarce in this country, of pure blood, and even the cross with the English setter is seldom seen.” Many years ago Mr. R. L. Purcell Llewellin, who was sceptical concerning the existence of this breed, offered a prize for any Russian setter shown at the Birmingham dog show. None was shown in response to the induce- ment. The breed, if it ever existed as a breed, was thought to be extinct many years ago. More likely it was merely a cross-bred strain. The probabilities are that it never was known in Russia. The product of a cross between the setter and pointer is called a dropper. It is considered a mongrel cross, and therefore is not recognized as a breed. However, this cross has produced useful working dogs, though even at that far inferior to the best straight-bred point- ers and setters in respect to high-class performance. In certain quarters, which are without any recognized authority in matters pertaining to breeding, there is a belief that the dropper averages well and pleasingly with the setter and pointer as a worker. DOGS. Well in- formed, practical sportsmen know that while there may be a passably good dropper now and then, their average as workers afield is not above the mediocre. There is an abundance of data concerning field work and field-trial competition which establishes the fact DOGS 481 that the dogs of pure blood are far superior. In the matter of beauty the dropper has no standing. In any event, it is admitted that after the first cross In any event, it is admitted that after the first cross the dropper deteriorates physically and as a worker. In any generation he does not breed true to any fixed type. Perhaps he may have all the externals of the setter or of the pointer, or he may show modified char- acteristics of both. For instance, a pointer coat may be accompanied by a rudimentary flag on the tail, or the latter may have a bushiness foreign to true pointer type. This cross is never attempted or approved by careful breeders, and is repugnant to all true sportsmen. As a rule, the dropper is the result of ignorance in breeding matters, or of cheap pot-hunting proclivi- ties, or he is a makeshift of indigence. The origin of the setter is unknown. As a matter of conjecture, it is held by certain writers that his origin is in a spaniel ancestry. There is much of plausibility in the arguments advanced to sustain the assumption. In many respects, some breeds of spaniels and setters bear a striking physical resemblance. They possess many instincts in common, though differing in some important particulars as to methods of pursuit. Some spaniels at times display rudimentary attempts to point, while on the other hand there are many set- ters which display a natural inclination to flush birds without pointing them at all, or with but a mere pre- tense of pointing them. Nevertheless, the evidence ad- duced in support of a spaniel origin is too fragmentary and too speculative to furnish actual proof; and, more- AMERICAN GAME BIRD SHOOTING 482 over, in the ancient writings there are conflicting opin- ions on this point. DOGS. In “The Dogs of the British Islands” (edition of In “The Dogs of the British Islands” (edition of 1867) Stonehenge, from such evidence as was available, reached a vague conclusion, as follows: “As some dif- ference of opinion appears to exist with regard to Setters, we have determined thoroughly to satisfy our- selves as to their origin and best form, and we have called the best authorities to our assistance.” His ipse dixit is that the Romans, after the invasion, “‘in- troduced the Land Spaniel, if not the Water Dog also, into this country” (England). Hecontinues: ‘When used either with hawks or for the net (especially in the latter case), a far heavier dog answered the pur- pose than what we call a ‘High-ranging Setter.’ The net enveloped a whole covey in its meshes, and few manors would allow of many coveys being taken in a day; while the disentangling the birds and securing them allowed time for a heavy dog to rest and regain his wind.” This is a labored, far-fetched attempt to account for the origin of the setter. All experienced trainers know that any kind of dog that will hunt can be trained to drop to shot, wing, order or signal, and teaching a dog to drop to scent or sight of the birds is not a difficult educational feat to encompass. But Stonehenge states that a far heavier and less enduring dog than the modern setter was used for the net, thus proving—so far as he proves anything at all—unlike- ness, instead of likeness. Stonehenge candidly confessed that he personally did Stonehenge candidly confessed that he personally did DOGS 483 not know the origin of the setter, in which respect he differed to an important degree from some other writ- ers, who make greater pretensions without possessing any greater knowledge. He continues: “But in after years, as we shall see, dogs were required to point,” in proof of which he quotes in part as follows, from the writings of Richard Surflet, who wrote in 1600: “There is another sort of Land Spannyels which are called Setters, and they differ nothing from the former (the springer) but in instruction and obedience, for these must neither hunt, range, nor retain, more or less, than as the master appointeth, taking the whole limit of whatsoever they do from the eie or hand of their instructor. DOGS. ‘They must never quest at any time, what occasion soever may happen, but as being dogs with- out voices, so they must hunt close and mute. And when they come upon the haunt of that they hunt, they shall sodainely stop and fall down upon their bellies, and so leisurely creep by degrees to the game till they come within two or three yards thereof, or so neare that they cannot press nearer without danger of re- trieving. Then shall your setter stick, and by no per- suasion go further till yourself come in and use your pleasure. Now the dogs which are to be made for this pleasure should be the most principall, best, and lustiest Spannyels you can get, both of good scent and good courage, yet young, and as little as may be made acquainted with hunting.’ The late Rawdon B. Lee, a much better authority than Stonehenge, writing about the origin of the setter, states: ‘However, other AMERICAN GAME BIRD SHOOTING 484 writers, and perhaps more reliable ones, including Delabere Blane (1840), say that Robert, Duke of Northumberland, as early as 1555, is said to have trained a setter to the net; and that other authorities of antecedent dates notice the sitter, or setter, as a dog used for sporting purposes.” Stonehenge, referring to the foregoing, states: Stonehenge, referring to the foregoing, states: “Soon afterward the Setter was produced, either by selection, or by crossing the Talbot Hound with the Spaniel.”” The inconsistency of Stonehenge—that is, asserting in one relation that the setter is of spaniel origin, and in another relation that its origin may be in a cross of hound and spaniel—is apparent. Lee quotes quite extensively from Dr. Johannes Lee quotes quite extensively from Dr. Johannes Caius, a Doctor of Physic of the University of Cam- bridge, who wrote about the year 1570, the following being directly to the point: ‘The dog called the Set- ter, in Latin Judex.—Another sort of dog there be serviceable for fowling, making no noise with foot or tongue whilst they follow the game. These attend diligently upon their masters, and frame their condi- tions to such becks, motions and gestures as it shall please him to exhibit and make, either going forward, drawing backward, inclining right hand or yielding to the left. In making mention of fowl, my meaning here is of partridge and quail. DOGS. When he hath found the bird he keepeth sure and fast silence, and stayeth his steps and will proceed no further, and with close, covert, watching eye, layeth his belly to the ground and so creepeth forward like a worm. When he approach- 485 DOGS eth near to the place where the bird is, he lays down and with a mark of his paws betrayeth the place of the bird’s last abode, whereby it is supposed that this kind of dog is called Index-setter, being, indeed, a name both consonant and agreeable with his quality.” Numerous other ancient writers refer to the set- Numerous other ancient writers refer to the set- ting dog as a land spaniel, but it appears that the term spaniel was generic in its application, being applied indiscriminately to several distinct breeds, thereby hopelessly beclouding the subject. The term, as used at the present day, frequently may have a distinctly different meaning from that of the same term as used centuries ago, and confusion and inaccuracy are sure to arise in any discussion which does not take cog- nizance of that fact. On this point, the following, taken from “The Gentleman’s Recreation” (1697), is specially pertinent: ‘The dog which you select for setting must have a perfect and good scent, and be naturally addicted to the hunting of feathers; and the dog may be either Land Spaniel, Water Spaniel, or mongrel of them both; either the Shallow-flewed Hound, Tumbrel, Lurcher, or small bastard mastiff.” To the ‘Field Book,” a sportsman’s encyclopedia, To the ‘Field Book,” a sportsman’s encyclopedia, published in London in 1833, is ascribed the following definition: “The English setter (Canis index, Variety B) is a breed between the Spanish pointer and the large water spaniel, and was famous for his steadiness and exquisite sense of smelling. The hair on his body was much more curled than the present breed, which has been lengthened by the additional cross of the springer. 486 AMERICAN GAME BIRD SHOOTING He was also much more steady than the improved va- riety, but then he has not the same speed to recom- mend him. . . . DOGS. He has an elegant figure, and is of a very pleasing diversity of color; added to this, his skin is covered with a coat of beautifully curled hair, very rilious on the lower margin of the tail, being alto- gether an extremely handsome dog, and quite unri- valed by any of the canine species.” These views, in a way, are a summation of the evi- These views, in a way, are a summation of the evi- dence bearing on the setter’s origin. For him whose fancy or prejudgments favor a spaniel origin there is much evidence to humor them. In like manner, there is evidence to console him who prefers a spaniel and pointer origin. For him who can impartially consider the pertinent facts, there also is ample evidence that we do not know the origin of the setter. It is lost in the mists of the ages. What are considered as finalities, at best are merely shrewd speculations concerning it. While recognizing that the act of pointing, in a more or less rudimentary form, is exhibited by span- iels and several other breeds of dogs, and, indeed, by cur dogs, under certain circumstances, it is never like the intensity and efficiency of that displayed by setters and pointers. Asa matter of speculation, it is much more plausible that the spaniel was crossed on the old Spanish pointer, which was introduced into England centuries ago, and that the cross-bred product might have been the setter. Here was the pointing instinct ready to hand to breed into the spaniel, an exceedingly simple matter as compared to the far-fetched theory d to hand. ir b ing Deliver d to hand. ir b ing Deliver DOGS DOGS 487 that it was implanted by education. This is sustained by the admitted fact that while the setter has infinitely more pointing instinct than has the spaniel, he in turn has much less than has the pointer. It may be ob- jected that the setter does not show any physical re- semblance to the pointer. As a matter of fact, he shows a great deal, the chief distinction being in the coat. <A cross of setter and pointer—the dropper— may appear to be pure setter or pointer. DOGS. By selection, the physical attributes of the pointer could soon be bred out, while the instinct to point would be added to the many others which the two breeds, spaniel and pointer, possessed in common. As before remarked, the As before remarked, the act of pointing is not the exclusive attribute of pointers and setters. The point seems to be a necessary factor in the successful pursuit of birds, and is many times developed by the dog from observation, from a realization of its helpfulness in effecting a capture. That a dog will change his meth- ods of pursuit to meet its conditions is well shown by the differences of method employed in following the trail of a rabbit and that of birds. In the one case he gives tongue vociferously, in the other he is cautious and silent. While the point no doubt has its origin in an instinctive impulse, as, indeed, the impulse to hunt is instinctive, a high degree of intelligence in its practical application is an essential of its useful- ness to man. The modern setter The modern setter, as owned by the majority of sportsmen in the United States, is the Llewellin AMERICAN GAME BIRD SHOOTING 488 setter, a term which denotes merely a certain line of breeding, of mixed English and Gordon setter blood. This family of setters originated in the ‘60s of the last century, and was a product of Barclay Field’s Duke and Thomas Statter’s Rhcoebe, or either of them or their progeny crossed on any Laverack setter. The Duke-Rhcebes had a large percentage of Gordon set- ter blood, and so were cross-bred. However, the cross produced some grand working dogs and field-trial win- ners. So great was the success of the Duke-Rhcebes and Duke-Rhcebe-Laveracks in field work and field-trial competition, that the superiority of this family was generally conceded. It was known for a time as “The Field Trial Setter,” and was so known when the first importations to this country took place, in 1874. Al- though there are many good native setters, so called, in this country, the Llewellin is far away the most popular with sportsmen. After Mr. Llewellin took up the field-trial setter, and bred it for a short time, it was called the Llewellin setter. DOGS. The Laverack setter was a strain of dogs bred by The Laverack setter was a strain of dogs bred by the late Edward Laverack, who averred that they were descended exclusively from Ponto and Old Moll, pur- chased by him of Rev. A. Harrison in 1825, and sup- posed to have been bred pure during thirty-five years prior to that time. However, the Laverack pedigrees have been subjected to much hostile criticism. There is much good evidence which shows that Laverack in- troduced outside crosses into his kennel, and the pedi- gree table, published in his work, “The Setter,” con- 489 DOGS tains internal evidence tending to show that it is spu- rious; that is to say, there are only four and five gen- erations given to cover a period of about forty years, showing that much of the breeding was done when the dogs and bitches were old, and ordinarily, in many instances, past the years of reproduction. Neverthe- less, the Laverack setter, when at his best, was beauti- ful in physique and a grand worker. The pure Laverack setter, in the years near the end The pure Laverack setter, in the years near the end of Laverack’s life, became very delicate and difficult to rear. The mortality was extremely high. At the time of Mr. Laverack’s death—he died in 1877—he had nearly lost the breed. There probably is not a pure Laverack setter in existence to-day, but its blood predominates in the Llewellin setter. The colors of the English setter are orange and The colors of the English setter are orange and white, liver and white, black and white with tan mark- ings, roan, lemon and white, black and white ticked with large black splashes, known as blue Belton—a des- ignation first employed by Laverack, naming it after a village in Northumberland County, England—and solid colors, liver, black, white, orange, lemon, though, as solid colors are difficult to distinguish when the dog is working afield, parti-colors are more desirable. Dogs should weigh from forty-five to sixty-five pounds, and bitches a few pounds less. A dog, when too light in weight, finds great difficulty in heavy going, whether mud or dense vegetation. The origin of the Irish setter is unknown. He un- doubtedly originated very far back in the past. While The origin of the Irish setter is unknown. He un- doubtedly originated very far back in the past. DOGS. While AMERICAN GAME BIRD SHOOTING 490 the Irish setter color, to conform to the standard, should be a deep red, or, as the Irish Setter Club of England has it, “a rich golden chestnut,” in ancient times the color was red and white, and the red then was not so deep and rich in color. The insistence on color is a bench show qualification. However, a trifle of white on toes, forehead, chest or face is not a dis- qualification. In English field trials and field work the Irish setter has succeeded far better than he has in the same line of effort in this country, though this applies to his status of some years ago. The Irish set- ter is but little used by American sportsmen, and in field-trial competition is rarely seen. The latter-day Irish setter seems to be lacking in The latter-day Irish setter seems to be lacking in speed, range, endurance and general capabilities, as compared with his more popular congeners, the pointer and English setter. Physically, like the English set- ter, which, in the main, he resembles closely, he is the embodiment of grace and beauty. Many things point to the conclusion that the black Many things point to the conclusion that the black and tan, or Gordon setter, is of comparatively recent origin. Ancient writers are silent concerning him, which would indicate that he was unknown to them. No one knows where the Duke of Gordon obtained his dogs. It is supposed that this breed is a composite of setter, collie and bloodhound, the latter showing strongly in the generous dewlap, the coarser skeleton, the larger, heavier head, the haw, and the tendency, when at work, to use on the trail the methods of a hound rather than those of the setter. The collie cross DOGS 4QI is shown by the tail and its carriage, and by the ten- dency to head off birds in manner similar to that employed in herding sheep. In color the Gordon set- ter is supposed to be deep black and tan, though there is good proof that the dogs at Gordon Castle were black and tan, black, tan and white, and liver and white. DOGS. Laverack, in “The Setter,’ wrote of them as follows: “As far as my observation goes, they lack the endurance of the other breeds that I have named; they are coarser and heavier made, and have not the light and agile action of the blacks, the black and whites, the black-grays, or blue and lemon and white Beltons I have tested. . . . Black-tans, as a rule, have sour, coarse heads; shoulders loaded, heavy, and too upright; are heavy and thick-limbed; large feet, often too straight and tilty in the hind quarters; tail thick and ropy. Many of the black-tans have obstinate, stub- born tempers, and are not particularly easy to break.”’ The modern pointer is supposed to be a descendant The modern pointer is supposed to be a descendant of the old Spanish pointer which was introduced into England in the early part of the eighteenth century. The Spanish pointer was a coarse, slow dog, sadly in- efficient in the matter of speed and range, to improve which a foxhound cross was introduced. The much racier modern type is said to be the result. In the late ’70s and early ’80s of the last century many pointers— dogs and bitches—of excellent blood, and fame as workers, were imported from England; but, strange to relate, only an exceedingly small percentage of their offspring proved to be noteworthy as workers afield, AMERICAN GAME BIRD SHOOTING 492 and quite as small a percentage of these imported dogs and bitches showed the excellent field form with which they were credited abroad. Many of them were first class in bench show competition, but as a whole they were lamentably inferior as workers. However, dur- ing the past two decades there has been wonderful im- provement in this breed, quite a number of pointers showing ability equal to that of the best setters in field work and field-trial competition. The comparative qualities of setter and pointer have been long debated without any final conclusions being reached. It is held that the pointer, having much the shorter coat as a rule, suffers much less from excessive heat than does his longer-haired brother; but that dic- tum has so many exceptions that it is far from being accepted. DOGS. The shorter coat of the pointer, in compari- son with that of the setter, counts for very little; for the setter, when properly conditioned by actual field work, loses nearly all his feather, and his coat, also, is much thinned and shortened from contact with the cover. Unless fully conditioned and acclimated, both suffer much distress in hot weather, and then, if set at work, soon become exhausted. It is claimed for the pointer that he can do without water better than the setter. I myself have shot over both breeds exten- sively when the weather was hot, and where water was scarce, and I never observed this difference. As a rule, both breeds require an abundance of water in hot weather. Occasionally, one—either pointer or set- ter, as the case may be—will show phenomenal en- DOGS 493 durance without the usual supply of water, but this is the rare exception, and furnishes no data on which to formulate any rule applicable to either breed. It is claimed as a point of superiority for the setter, It is claimed as a point of superiority for the setter, that his longer, protecting coat enables him to take punishing cover much better than the pointer can. The claim is fallacious. Many setters are quite intent on saving their precious skins when working where there is brush or bramble; on the other hand, many pointers will face punishing cover without flinching. This phase of work is dependent on the dog’s individual courage and his love of hunting, naturally possessed, much more than upon the physical attribute of coat. Some dogs can go through cover without much suffer- ing, while others are punished severely. Some dogs are continually suffering injuries under any condi- tions. It all resolves itself into whether the dog tries to save his skin, or is so stupid and awkward that he cannot do so. As to the general traits of the two breeds, the pointer As to the general traits of the two breeds, the pointer is much the greater glutton on opportunity, is much more likely to go stale, and so remain indefinitely, from overwork, and to lose his dash and industry in middle life. As a breed, the pointer does not display the fire, endurance and sharp quickness in his bird work that the setter does, and so is greatly handicapped in field- trial competition. DOGS. However, he takes to pointing much earlier in life, and more naturally, than the setter, and therefore is much easier to train, and retains his train- ing much better. AMERICAN GAME BIRD SHOOTING 494 Nevertheless, the pointing instinct, if exercised be- yond useful limitations, may be a serious impairment of a dog’s usefulness, as, while it fundamentally is an instinct, its usefulness to man depends entirely on its intelligent application in meeting special requirements. The pointing instinct of the pointer and setter, com- instinct, its usefulness to man depends entirely on its intelligent application in meeting special requirements. The pointing instinct of the pointer and setter, com- bined with their propensity to hunt birds diligently, establishes their chief claim to excellence in upland shooting. We do not know whence the pointing in- stinct was derived. Even if we concede that it was a natural evolution from education to dropping for the convenience of the net, this in no wise accounts for its origin in the old Spanish pointer. It is more reasonable to assume that the instinct to point is pres- ent in all breeds of dogs which will hunt birds. Of course, it differs much in degree of intensity and man- ner of application in breeds other than pointers and setters, but it is there, nevertheless. Cur dogs which have been used for all round pot-hunting purposes, acquiring thereby a knowledge of the best methods in the pursuit of birds, oftentimes learn to trail the latter and make use of the point when the birds are located. As a matter of course, they are far inferior to setters and pointers in every respect, in upland shooting, but that is aside from the matter under consideration. When roading birds the cur dog observes the stealthy, mute methods of the setter and pointer, although when in pursuit of fur he may give tongue frantically, as may also the setter and pointer. This seems to indicate that it is an instinct common to all dogs, and exhibited DOGS 495 with more or less intensity under certain conditions, as already mentioned. The exigencies of the pursuit of different animals The exigencies of the pursuit of different animals determines in the main the choice of methods. DOGS. It is quite obvious that a noisy, impetuous method, which might be successful in the pursuit of an animal that trusts for escape to its wits and legs, would wholly fail if employed in the pursuit of an animal which can fly swiftly away. If we assume that the point is an in- telligent act, useful to the dog in securing a food sup- ply, there then is no more difficulty in accounting for it than there is in accounting for any other phenomena of canine life. Foxes and wolves draw on their prey and make the point, which is merely the pause to accurately locate the exact position of the prey, pre- paratory to the final spring to capture. The feathering and bristling of the hair on the back are not exclusive traits of bird dogs. All dogs, on the eve of a bloody attack, exhibit it. Every one at all familiar with the habits of dogs has seen even cur dogs, strangely met, draw toward each other with hair bristling, stopping betimes in the attitude of a point, all concluding with a final rush which may end in battle or friendship, accordingly as the principals are fighters or bluffers. Cats, in stalking their prey, exhibit many of the char- acteristics of drawing and pointing. Whoever has seen a setter or pointer make a mighty spring after pausing a motnent—that is, pointing—in an effort to capture birds, has noted the amazing swiftness of the act, and that to make such a supreme effort the dog AMERICAN GAME BIRD SHOOTING 496 must set himself at the start, so that all his muscles would co-ordinate to accomplish it. This pause, so useful in the dog’s natural efforts to secure a food supply, serves a double purpose in such attempt: it enables him to locate the birds accurately before mak- ing the final spring to capture, and it enables him to spring powerfully and accurately. Sometimes, withal, the dog makes a false estimate, and springs wide of the mark, thus totally failing in his attempt. The point, however, does not always indicate the The point, however, does not always indicate the presence of game. Under such conditions the act is called a false point, and it may be a rarity in the field work of a dog, or it may be of frequent occurrence, or may happen irregularly. DOGS. A dog which habitually false points is next to worthless for field work. The fault is displayed by some dogs under unfavorable weather conditions, which affect the scent. The ma- jority of dogs will false point when fagged out or when stale from overwork, day after day. There is no cure for habitual false pointing. It probably is the result of a faulty brain or nose, or both combined, or, it may be, of overcaution. The standard of field work is precisely alike for The standard of field work is precisely alike for pointers and setters. In their methods of work, natu- ral or acquired, there are no particular differences which can properly be classed as peculiar exclusively to either breed. Yet each pointer or setter, in per- forming identical tasks, may have his own individual methods and mannerisms, no two dogs working or planning alike, just as no two men think or act pre- DOGS 497 cisely alike. Thus, while the main essentials of field work, such as ranging, judgment, pointing, road- ing, etc., are constants, there may be an infinity of variable detail in accomplishing like results. cisely alike. Thus, while the main essentials of field work, such as ranging, judgment, pointing, road- ing, etc., are constants, there may be an infinity of variable detail in accomplishing like results. For the better comprehension of the terminology of For the better comprehension of the terminology of upland shooting, some explanations will be of value. The terms pointing, standing, and setting, denote The terms pointing, standing, and setting, denote the pause, generally very tense and spirited, which the pointer or setter makes when either has located birds definitely, or which, when trailing them, he makes in an attempt to locate their direction or whereabouts. The dog, when pointing, may be quite close to the birds or a long distance from them, variable circum- stances entering into the consideration of this mat- ter, such as whether the wind brings the scent directly from the birds to the dog’s nose; whether the dog’s nose is keen enough to locate the birds without press- ing in close to them; whether at the time the birds are in thick or thin cover, or no cover at all; or whether it is at a season when they are either reluctant to take wing, or will flush instantly at the slightest alarm. DOGS. All these different circumstances the wise, capable, ex- perienced field dog recognizes and observes. The functional powers of the dogs’ noses, in respect The functional powers of the dogs’ noses, in respect to pointing, vary greatly in the matter of efficiency. There is nothing remarkable in this if we consider that there is much variability in sense organs—as in the eyesight of man. Supplementary to a dog’s efficiency in locating and pointing birds are his intelligence, as shown by the AMERICAN GAME BIRD SHOOTING 498 judgment he displays in conducting his efforts and his honesty of purpose and cheerful effort in working to the gun; these qualities, in sporting parlance, being known by the comprehensive term “bird sense.” But a dog which has a knowledge of approved meth- But a dog which has a knowledge of approved meth- ods in work to the gun, and skill in combining them successfully according to ever varying circumstances, combined with endurance, cheerfulness and loyalty, is a rarity indeed. He is the genius of his kind. Never- theless, however excellent a dog may be naturally, un- less he has an affection, or at least a tolerance, for the shooter, he will not perform at his best, even if he will perform at all. So much for the trained dog. Left to his own un- hampered will, the natural proclivity of the untrained dog is to impetuously follow his own impulses when in pursuit. At first he trusts to his speed alone. After a certain amount of experience he begins to use his intelligence. When hunting for himself he displays great enthusiasm and industry. This natural method, called self-hunting, is not in accord with the best inter- ests of the gun. The true theory of training is to preserve the dog’s best natural methods, enforcing only such restraint as the needs of the shooter require. No trainer can force a dog to hunt if the latter is disinclined to do so. The dog’s natural inclination, after locating the The dog’s natural inclination, after locating the birds, is to spring in to capture, but this being against the interests of the shooter, the dog is trained to check his impulse to spring while still encouraged to main- DOGS 499 tain the point. This is not always an easy task to accomplish. DOGS. While the act of pointing is commonly considered as a marvelous exhibition of the dog’s scent- ing powers, it really is not any more so than is any other organic function. Drawing and roading are terms used to denote the Drawing and roading are terms used to denote the manner in which a dog approaches the birds by means of their scent. Drawing has reference to the approach by the body scent. The dog, when drawing, carries his nose well up above the ground, and, if he performs in the best manner, goes to the birds with quickness and precision. The dog which follows the birds by roading may The dog which follows the birds by roading may do so with reasonable quickness and accuracy, or with such awkwardness, slowness and inefficiency that he is next to worthless. In respect to locating the birds, all that the trainer In respect to locating the birds, all that the trainer can do to assist is to afford the dog proper opportuni- ties for practice. Each dog selects his own methods, and arbitrary methods cannot be forced on him. There is a vast difference in the functional powers There is a vast difference in the functional powers of dogs’ noses and intelligence, therefore all dogs can- not work up to the same standard. Drawing is far away the best method of locating Drawing is far away the best method of locating birds, although in certain conditions of weather, when the scent lies low, the dog which carries a high nose on the trail encounters a difficult problem. But, un- der favorable conditions, when he strikes a trail he may follow it at top speed and locate the birds with marvelous quickness and sharpness. Such a performer AMERICAN GAME BIRD SHOOTING 500 would have the birds located before the dog which picks his way painfully and slowly along by the foot scent had well begun. The advantage of drawing as compared to roading is thus apparent, and it is of spe- cial advantage in field trial competition. Some dogs have the ability to draw or road, adapting Some dogs have the ability to draw or road, adapting their efforts to the scenting conditions, and acquitting themselves creditably. However, every dog which car- ries a high nose in the attempt to locate is not neces- sarily a good performer. DOGS. ‘There are wide-ranging, high-nosed dogs which are poor finders. The only test by which a dog’s merit can be properly determined is his actual work afield. Ranging is a term which denotes the casts taken Ranging is a term which denotes the casts taken by the dog, to and fro, while beating out the ground in the search for birds. It may be done in many quite different but successful ways, or it may be done in a way which at once demonstrates that the dog has no hunting intelligence whatever so far as the needs of the gun are concerned. Inability, due to stupidity, cannot be overcome by training. Neither a foolish man nor a foolish dog should be expected to surpass his natural limitations. A good nose is an aid of inestimable value in sup- A good nose is an aid of inestimable value in sup- plementing industry and good judgment. It com- mands a great area, thus enabling its possessor to search out much more territory with fewer casts than is possible to his fellow with the poorer nose. The good ranger maintains constant observation The good ranger maintains constant observation of the wind. He skirts promising stretches of cover DOGS 501 on the leeward side, so that, if there are therein any birds in hiding, he will catch their scent. He recog- nizes the likely places for birds, and, planning with them in mind, he conducts his range after a well-laid scheme, thereby economizing time and effort. On the other hand, the dog possessing a poor nose is quite likely to range here and there, hither and thither, hit or miss, as it may happen, working the same ground over repeatedly while leaving other places, perhaps the most likely ones for game, unnoticed. A fast dog, if he uses his nose properly, is the best, A fast dog, if he uses his nose properly, is the best, provided he can maintain his pace uniformly for a con- siderable time. It is obvious that such a dog can cover a certain area in a certain time much more thoroughly than a slower worker can. Many dogs will start at high speed, slowing down gradually thereafter, till they practically quit. A lot of stamina and pluck are required to maintain a fast pace while hunting during several hours in succession. DOGS. A moderate degree of speed, uniformly sustained, is much to be preferred over high speed of uncertain duration. Some slow dogs, of surpassing cunning in their work, are excellent bird finders. Though a dog may be slow he nevertheless may take wide casts, and if his nose and judgment are good, combined with honest work to the gun, he has the essentials of a hunt- ing dog. As to methods of hunting out any game area, the dog, if given the opportunity, learns them best when left to his own devices, without interference from AMERICAN GAME BIRD SHOOTING 502 his trainer. The latter can hamper the dog’s progress by interference, but he can do very little to assist. With age and experience, all intelligent dogs improve in their hunting methods. Many of the best finding dogs are those which have Many of the best finding dogs are those which have indulged in self-hunting, a term which denotes that the dog hunts independently, as when he sneaks off alone, or in the company of other self-hunting dogs, or as he pleases when with his trainer. Under such conditions he hunts till wearied or till satisfied, which may be in a few hours or a few days, according to the endurance, longing, and the food supply. Commonly, after a vagrant self-hunting trip, the dog returns mere skin and bone, the exposure, pro- longed exertion and short rations producing their natu- ral results. The vagrant, under such circumstances, regains his flesh as quickly as he lost it, if properly fed. Vagrant excursions should be strictly suppressed. Vagrant excursions should be strictly suppressed. They afford unlimited opportunity for the formation of bad habits, such as harrying sheep, killing fowls, robbing nests, etc. The self-hunting proclivity mani- fests itself sometimes when working to the gun. The self-hunters will apparently work under control for a longer or shorter time, gradually ignoring the shooter, till at last they go where and when they piease. This insubordinate stage lasts according to the whim of the dog. It may be for a brief while, or it may last a day or two. Meanwhile the dog is learning a great deal in respect to ways and means of finding birds, though of no service whatever to the gun while so absent. DOGS. ‘sneuysO ‘H ‘A Aq Burjured & urto1g ‘ddississiq@ ur 3u00ys ‘s ‘H ‘A Aq Burjured & urto1g ‘s ‘H ‘A Aq Burjured & urto1g ‘ddi ur 3u00ys ‘s ‘H ‘A Aq Burjured & urto1g ‘ddi ur 3u00ys ‘H ‘A Aq Burjured & urto1g ‘ ur 3u00ys DOGS 503 In ranging, the dog should adjust his efforts to fit the circumstances of cover and open, casting closer in a broken cover country than in an open country, and he should come into view of the shooter at reasonable intervals to note the proper course as a base line to guide him in keeping within proper bounds. When hunting in cover exclusively, as in hunting ruffed grouse and woodcock, his proper range should not much exceed that of the spaniel; that is, gunshot. Back, back s Back, back stand, or back set, is when one dog stands stanchly at sight of a pointing dog, the attitude of the backing dog being much like that which he assumes on a point, though in many instances it is more or less relaxed. In any case, as the dog advances in years, he backs with less intensity. If the pointing dog has pointed If the pointing dog has pointed false a few times, the backing dog, observing that the act is deceptive, loses confidence in his fellow worker, disregards his points, and goes on about his own affairs, or breaks in to see what it is all about. Dogs quickly comprehend Dogs quickly comprehend the significance of each other’s movements. Some deferential dogs will back when they observe their bracemate drawing on game, being satisfied to take a subordinate position at every opportunity. Other dogs, on the contrary, may cast in ahead at top speed when they see a dog roading or drawing, being ambitious to secure the point. Some will back well for a moment, then move forward a few steps at a time, playing craftily to steal the point. Some dogs, again, will back or point honestly when AMERICAN GAME BIRD SHOOTING 504 under the eye of the shooter; out of sight, they are lawless. Again, some dogs which point well refuse to back at all. So far as work to the gun is concerned, the purpose of the back, apart from its spectacular features, is to guard against interference with the pointing dog. DOGS. If a dog refuses to back he can be taught to drop instantly to oral command or to signal or to his bracemate’s point. This detracts from the spectacular beauties of shooting, but a good dog should not be condemned because of failure in this respect. Quartering denotes a system of beating out the Quartering denotes a system of beating out the ground in substantially parallel casts at right angles to the course of the shooter. This method has never been esteemed in this country, although it was viewed with favor in some sections of England. It fitted in usefully in English shooting, as on the English pre- serves the birds, in many fields, were quite as likely to be in one place as another; therefore, it was an essential that every bit of ground be searched out thor- oughly. In this country, there being much unpromising area in ratio to likely area, the really serviceable dog uses his brains in hunting only in the likely places. These conditions are ever changing. The local con- ditions must always be the guide of action; no arbitrary rule is possible. Before attempting to shoot game over a dog’s points with a serious purpose to secure enjoyable sport, it is better to give him a thorough schooling in the dif- ferent commands, and their significance, and therewith DOGS 505 sufficient discipline in obeying them, so that, when afield, there will not be too many conflicting ideas in his head. Added to all this, the dog should have suff- cient experience in working on birds and working to the gun, so that there will be sport instead of chaos, Beyond a perfunctory feeding of his dog, the aver- Beyond a perfunctory feeding of his dog, the aver- age dog owner, in times past, gave little thought to the animal’s general well-being. If given an abundance of food, without any consideration as to whether it met the dog’s bodily needs, he was considered to have received all the attention necessary. In recent years, however, there has been a better understanding of the need of promoting the comfort and general welfare of the dog, and to this understanding bench shows and field trials have contributed incalculable missionary work. Much valuable literature has become available on all subjects of canine interest, and so a better gen- eral knowledge of the dog’s requirements. DOGS. There are various kinds of excellent proprietary dog There are various kinds of excellent proprietary dog foods on the market, so cheap as to price, though ex- cellent as to quality, that they are within the reach of the most slender purse. Table scraps, assuming that they are other than potato skins and bare bones, make a good dog food. Milk, sweet, sour, or buttermilk, may be given to the dog in abundance. The latter is a specially good summer drink for the dog, as it also is for man. Contrary to a popular belief, milk does not generate worms. If an owner does not care to purchase proprietary foods, and the table scraps are insufficient for the needs AMERICAN GAME BIRD SHOOTING 506 of his kennel, scraps of mutton, beef, or goat meat, stewed with cabbage, onions, beets, turnips, and other vegetables—observing that all are strictly sound and good—make an excellent food during the idle months. When a dog is used for field work the food should be almost wholly meat. Sheep’s heads and tripe make an excellent food, preferably in a stew. The former should be skinned, and boiled till the bones can be readily removed from the flesh. Corn meal, though quite commonly used, is one of the poorest of dog foods, much of it being undigested, as can readily be known by an examination of the dog’s droppings. For working dogs, one feed after the day’s work is ended is quite sufficient. They then should have all that they will eat. If fed in the morning, digestion is suspended while the dog is ranging about, and he is carrying with him the equivalent of so much dead weight, with no nutritive qualities for the time being. If fed on a meat diet when working, the dog is al- If fed on a meat diet when working, the dog is al- most exempt from bowel troubles, while if fed much corn meal such troubles are frequent. For the latter there is no better or prompter remedy than lean beef, cut into small pieces, and heated through in a hot oven, thus being only blood rare. If a dog’s stomach is loaded when he is at work his powers of scent are seriously impaired. It matters not whether he is fed on meat or vegetables. DOGS. Nature has wisely provided that while digestion is in progress the dog’s sense of smell is dulled. Even man, whose sense of smell is incomparably inferior to that of the dog, DOGS 507 can smell a dinner a long way off when he is hungry; whereas, after dinner, the same gratifying fragrance is unrecognized. Man’s own requirements are no data on which to Man’s own requirements are no data on which to judge of the food needs of the dog. The latter, being carnivorous, requires food far less frequently than man, while the latter, in turn, feeds less frequently than the ox, which is wholly herbivorous. Next to a good food supply, the dog needs a com- Next to a good food supply, the dog needs a com- fortable, well-ventilated, sanitary place in which to sleep. Any man who cannot afford such a place, and neglects his dog’s comfort in this respect, cannot af- ford to own a dog, consistently with humane require- ments. With this, the dog should have plenty of exer- cise. It is best that he should have entire freedom, but as this is not always possible, particularly within city limits, regular times each day should be allotted for that purpose. PART III THE SHOOTING OF THE FUTURE ‘aspoH “WY °=C Aq poydeis0,0yg “plo S¥99M 991G} ‘yoryo eIGMqog ‘aspoH “WY °=C Aq poydeis0,0yg “pl S¥99M 991G} ‘yoryo eIGMqog ‘aspoH “WY °=C Aq poydeis0,0yg “pl S¥99M 991G} ‘yoryo eIGMqog A LOOK BACKWARD When the white men came to America they found wild birds and animals very abundant. The repro- ductive energy of indigenous species more than made up for their destruction by natural enemies. Human beings were not numerous in proportion to the area of the land, and took only what they needed. The balance of nature was preserved. It was rudely disturbed by the arrival of civilized It was rudely disturbed by the arrival of civilized man with his firearms, though for a time he made little impression on the life of the great continent to which he had come. Birds and animals continued abundant, though close to the settlements they were soon driven away or destroyed. Thus Josselyn, writing in 1671. tells, as already said, how thirty years before turkeys were very abundant at Black Point, now Scarborough, Maine, where many broods of young might be seen in a morning, but that the English and the Indians have destroyed them, “‘so that ‘tis very rare to meet with a wild Turkie in the Woods.” This was only about fifty years after the landing of the Pilgrim Fathers. Gee Gee Gee 512 AMERICAN GAME BIRD SHOOTING In the early days of the settlements game was killed for food, and not for sport. The arm carrying the single ball comes first into a new country, and the shot- gun follows at some distance of time. The musket or rifle is a necessity; the shotgun a luxury. The rifle protects life and property, supplies food for the family, destroys the wild beasts that would prey upon the settler’s stock. The shotgun is used in hours of leisure and recreation. Food captured by its aid is a delicacy. It is the implement of sport. In the settlement of America this has been everywhere the case. The hardy pioneer disdained to kill his game with The hardy pioneer disdained to kill his game with more than a single ball, which it was his pride to plant wherever he chose, and in his expert hands the old crooked-stock pea-rifle was his dependence for support, as it was the terror of the savage tribes into whose ter- ritory he pushed his fearless way. A LOOK BACKWARD Many, many years later, when the land had been cleared and waving fields of corn and wheat had taken the place of the wild grasses that once grew rank and thick along the valleys and over the prairie, when the large game had almost entirely disappeared, the children or the grandchildren of the rifleman began to use the shotgun. Geese and ducks, the noisy grouse, the brown quail, the whistling woodcock and the twisting snipe became the objects of pursuit to those whose fathers had killed the elk, the moose and the buffalo. The fathers hunted for meat. With them it was a fight for life. Each ball and each charge of powder was to be accounted for and must do its work. The sons and grandsons inherited the hunt- A LOOK BACKWARD 513 ing spirit from their sires, but no pressing need drove them to the field. They shot—for pleasure—wild fowl and game birds when these were to be had, but if not these, too often they destroyed in their ignorance tiny songbirds which were their best friends. Many a man has seen with his own eyes the expul- Many a man has seen with his own eyes the expul- sion of the rifle by the shotgun from more than one section of the land. Little more than a generation ago the shotgun was scarcely known in central Nebraska. Then it was necessary on occasion to fight off Sioux or Cheyennes or Arapahoes, while the buffalo, elk, deer and antelope were needed for food. The years have rolled by; the riffle has disappeared. Either it went westward to the mountains with its owner, or, rusty and dirty, it stands to-day neglected in a corner of the garret. All the young men and boys have shot- guns, and in the season the geese that visit the river bottoms, the ducks of the sloughs, the prairie chickens on the upland and the quail in the tow-heads are con- stantly pursued. So, in the settling up of a country the weapon adapts itself to the game to be killed. As long as large mam- mals are abundant the rifle holds its own, but when these are gone it must give place to the weapon of shorter range. A LOOK BACKWARD It was not until toward the middle of the nineteenth It was not until toward the middle of the nineteenth century that the birds of the United States began to be shot for sport. Before this the markets offered induce- ments to the thoughtless or the greedy to destroy birds with great recklessness. Game birds were taken in nets, AMERICAN GAME BIRD SHOOTING 514 snares and traps in vast numbers, and as early as 1855 and ’56 a writer said: ‘The havoc and slaughter, not only by the mercenary and cruel, but by the thoughtless and improvident, have consequently been very great, both of birds and animals, thousands of buffaloes, deer and hares, hundreds of wolves, panthers and bears, as well as myriads of partridges and hecatombs of grouse, have been slain and trapped. The markets of all our principal cities have been most bountifully supplied with every description of game—in fact, filled to repletion with partridges and grouse; not, however, as many sup- pose, with birds alone coming from the far, far West, but by incredible numbers of half-starved victims snared in our own and the neighboring States or mercilessly shot down, whole coveys at a ‘blow,’ as they sit huddled together on the treacherous snow, stupefied and be- numbed with the chilling blasts of the north.” Some of the sportsmen of that day were Englishmen, Some of the sportsmen of that day were Englishmen, and of these Wm. Henry Herbert—Frank Forester— was one of the most famous. Herbert wrote in charm- ing style, and professed high ideals of sport. His writ- ings were read by all who were fond of shooting and fishing, and exercised a good influence. Birds at that time were so extremely abundant—from the point of view of to-day—that no one realized the importance of moderation in killing, and the bags made were very large. Herbert inculcated good views of sport, and his teachings were far in advance of the practice of his day. Nevertheless, it was then a point of pride, as indeed it still was at a very recent day, to do a little better—that A LOOK BACKWARD 515 is, to kill a few more birds—than your neighbor or your rival. A LOOK BACKWARD Although all the larger game birds had been exter- minated in the Eastern and Middle States by the time of the Civil War, it was not until the advent of the breech-loader in America, about 1868 or ’69, that bird shooting began to be excessive in nearby localities. Yet, by the middle of the following decade, complaints began to be heard of the scarcity of game. People who a few years before had had no difficulty in killing thirty birds in a day, grumbled when they could get but fifteen, or perhaps ten, and found vacant covers long known to be depended on for a certain number of birds. These were the first warnings of the scarcity of game birds, which, since that time, we all have known. In those days, too, the gunners interested themselves much in the question as to which was the most useful arm, the breech-loader or the muzzle-loader. Those who had adopted the new weapon vaunted its conven- ience and general efficiency, while the more conservative declared that the breech-loader did not throw shot with the force of the muzzle-loader, and declared that they would never change their weapons. It was about this time that the first periodicals de- voted exclusively to shooting and fishing were estab- lished. These were, first, the American Sportsman, which lived but two or three years, and then Forest and Stream, which subsequently absorbed the successor of the American Sportsman, the Rod and Gun. These journals were immediately successful, because of the AMERICAN GAME BIRD SHOOTING 516 interest felt in their subjects by the sportsmen and field naturalists of that day. Sportsmen had much to say on various topics, and naturalists wished to discuss many matters which could hardly be brought up in pub- lications devoted strictly to science. Many sportsmen were careful and accurate observers, possessed of ex- perience and education, and writing delightfully. Later a large crop of periodicals devoted to these sports sprang up, most of which, before very long, perished for lack of support, their places being taken by others. These earliest periodicals, being directed by men of foresight and experience, urged upon their readers the importance of preserving the natural resources of the country. They preached game protection and forest protection, and while converting not a few of their readers, at first made little or no impression on the general public. A LOOK BACKWARD Such educational work goes slowly at first, especially when the teaching tends to limit the recreation and pleasures of the public by urging them to practice self-control. It was thus many years before any definite results could be seen from these teachings, and in the mean- time the works of destruction went on. Perhaps the most radical and most important step ever taken in game protection in this country was in the year 1894, when Forest and Stream announced its platform plank, “Forbid the Sale of Game.” Meantime a constant, though slow, improvement was taking place in the game laws, and a few sportsmen, by earnest work with ‘trend ueadoing ‘t ueadoing A LOOK BACKWARD ig the legislatures, backed up the work which Forest and Stream had set on foot. Meantime, sportsmen at large discussed with much earnestness the question of what should be done to re- stock the game covers. The first efforts took the direc- tion of importing bobwhites from the South and turn- ing them loose in covers that had been shot out. This was done, but the birds were soon killed off, for no one seemed to think of stopping shooting. Then came the idea of importing foreign birds, and about 1878 or *79 some one suggested the importation to America of the Old World quail (Coturnix), a migratory bird which breeds in Central and Northern Europe, goes south in autumn, crosses the Mediterranean and spends the winter months in Africa. These birds were easily obtained and were inexpensive. Considerable numbers of them were imported. They appeared to take readily to their new home and many of them bred. Nevertheless, after a time they disappeared without any apparent cause. It was reported that flocks of them had flown out to sea and been drowned, and it is quite possible that, migrating to the shores of the Gulf of Mexico, they attempted to cross that body of water and perished. At all events it is now many years since one of these birds has been authentically reported as taken in America. Attempts were made to acclimatize the English Attempts were made to acclimatize the English pheasant in the United States a hundred years ago, but the birds turned loose in New Jersey all disappeared. Another attempt was made early in the nineteenth cen- AMERICAN GAME BIRD SHOOTING 518 tury, near Belleville, N. A LOOK BACKWARD J., but again the birds disap- peared. It is said that between 1820 and 1830 pheas- ants were turned out near Baltimore, Md., but by 1830 the last of the birds seems to have vanished. In 1880, Hon. O. N. Denny, then U. S. Consul at In 1880, Hon. O. N. Denny, then U. S. Consul at Tien-Tsin, China, shipped some ringnecked pheasants to Oregon. Most of these died, but the following year, according to the report of the Fish and Game Protector of Oregon for 1895-96, another shipment was made which did better, and these were set free on the ranch of Mr. John Denny, in the Willamette Valley, in Lynn County, Oregon, where they did well and increased rapidly. In the late winter or early spring, 1885, an important importation came to Portland. These were again from Consul Denny, and were sent to the people of Oregon in care of a sportsmen’s association of Port- land. They included several species. Efforts were made to induce the legislature to enact a law protecting them and to make a small appropriation for their care until they could become established. The legislature laughed at these requests and treated them with so much scorn as to create quite a little sympathy for the sportsmen’s association, and incidentally for the pheas- ants. The owner of Protection Island, in Puget Sound, offered to give the birds a home and protect them if desired, and they were turned out there. In 1882 two hundred pairs of English pheasants were In 1880, Hon. O. N. Denny, then U. S. Consul at Tien-Tsin, China, shipped some ringnecked pheasants to Oregon. Most of these died, but the following year, according to the report of the Fish and Game Protector of Oregon for 1895-96, another shipment was made which did better, and these were set free on the ranch of Mr. John Denny, in the Willamette Valley, in Lynn County, Oregon, where they did well and increased rapidly. In the late winter or early spring, 1885, an important importation came to Portland. These were again from Consul Denny, and were sent to the people of Oregon in care of a sportsmen’s association of Port- land. They included several species. Efforts were made to induce the legislature to enact a law protecting them and to make a small appropriation for their care until they could become established. A LOOK BACKWARD The legislature laughed at these requests and treated them with so much scorn as to create quite a little sympathy for the sportsmen’s association, and incidentally for the pheas- ants. The owner of Protection Island, in Puget Sound, offered to give the birds a home and protect them if desired, and they were turned out there. In 1882 two hundred pairs of English pheasants were In 1882 two hundred pairs of English pheasants were brought to New York from England to stock Pierre Lorillard’s game preserve in Monmouth County, N. J. They did well there, and with others imported later by _ ‘yueseayd-asnorg Aysng pruqAy ‘y Aysng pruqAy A LOOK BACKWARD 519 Pierre Lorillard and Rutherford Stuyvesant and turned out at various points in New Jersey and along the border between that State and New York, stocked in a limited way a considerable territory west of the Hudson River. In the year 1881, nineteen Chinese pheasants were imported to Victoria. Vancouver Island, and set free there, while a law was enacted giving them absolute protection for five years. They increased astonish- ingly, so that when the closed time was ended there was excellent shooting in the neighborhood of Victoria, and it was estimated that during the first season not less than 3,000 pheasants were killed. During the winter the birds could be seen, sometimes along the roadside and often in the cultivated fields, and were evidently very abundant. The introduction of the pheasants in Oregon and The introduction of the pheasants in Oregon and California was generally welcomed, and public feeling protected the birds. It appeared that the climate was well suited to them. They increased rapidly, and now for many years have been abundant on the coast, but chiefly in the region of great precipitation. They have apparently not spread greatly to the south, into the dry region of California, although in that State there are believed to be some persons breeding pheasants in semi- confinement in a small way. The birds are popular on the coast. Some fruit-rais- The birds are popular on the coast. Some fruit-rais- ers, to be sure, declare that they injure the crops, but on the whole they are regarded as a welcome addition AMERICAN GAME BIRD SHOOTING 520 to the fauna of the region. In a few instances they have interbred with the dusky grouse. A LOOK BACKWARD During the last thirty years a number of different During the last thirty years a number of different exotic species have been suggested as useful birds for American shooters. Pheasants have done fairly well in certain eastern localities besides those mentioned. Major W. A. Wadsworth introduced them into the Genesee Valley, and Dr. W. Seward Webb has a large preserve in Vermont, where birds are hand-reared and turned out to be shot in battues once or twice a year. The Blooming Grove Park Association in Pennsylvania has successfully raised pheasants, and each year the members are permitted to kill a certain number, while many other parks and associations, to say nothing of some State farms. and a multitude of commercial people, have been engaged in the work of rearing these handsome birds. It has been demonstrated that the pheasant will endure the rigorous climate of the north, at least as far as central or northern New York. The pheasants are not without their opponents. It has been alleged that they destroy or drive off our native game birds; that they injure certain crops; and that while they themselves are immune to certain dis- eases, they yet may communicate these diseases to our native birds. The latest candidate for public favor on the roll of foreign game birds is the so-called Hungarian par- tridge—the gray partridge of Europe. It is a bird nearly as large as the ruffed grouse, and is hardy and offers good shooting. It is only within three or four ‘aspiijizg uvadoing ‘a uvadoing A LOOK BACKWARD 521 years that it has been imported in considerable numbers, the earliest importations having taken place in 1905 or ’06. It is reported to have done well, and by many is believed to be the coming game bird. Besides those named, few Besides those named, few foreign game birds have been introduced, and those in small numbers only. Mr. W. W. Thomas many years ago was instrumental in importing and liberating in Maine some capercailzie and black game. Black game was also imported and turned loose on the Island of Newfoundland. So far as known, nothing was ever heard of the birds. Much later, some capercailzie were imported and turned out in Algonquin Park, in Canada, whence for several years reports have come of young broods seen. A LOOK BACKWARD Some European grouse, including, we believe, ryper, black game and capercailzie, were turned out on Grand Island in Lake Superior, but these have all disappeared. Most of the efforts at r Most of the efforts at restocking our covers with exotic birds have been carried on in an unintelligent and aimless way. The belief seems to prevail that because our native species have disappeared—while game birds in Great Britain are still so abundant that good shooting can generally be had—therefore these foreign species are more prolific and better fitted to survive than our native birds. People who reason in this way forget the widely different conditions prevailing in the two coun- tries—the great size of the land holdings in Great Britain compared with those in this country, the fact that trespass laws are there strictly enforced, and the further fact that game is there regarded as the personal AMERICAN GAME BIRD SHOOTING 522 property of the owner of the land; while in the United States up to within a few years the game has been considered the property of whomsoever might re- duce it to possession. On one side of the water are large estates to which the public is not admitted, while game birds are arti- ficially reared and carefully protected to prevent de- struction by natural enemies, as well as by poachers. The result of this is that at the end of a season a crop of game is harvested and sold. In the United States are a series of small land holdings, over which until within a very short time, everyone, man or boy, citizen or alien, was at liberty to roam freely and to destroy at will; the game is left to reproduce itself, is exposed to attacks by its natural enemies as well as by all human beings who may wish to take it, and is only protected by laws which exist on the statute books, but are inefficiently enforced. Here each gunner wishes to shoot, and in practice may shoot, from daylight to dark, seven days in the week. Add to this that a large portion of the public here is accustomed to the use of firearms, and that we have the best guns and ammunition in the world, and it is not difficult to see why game is very scarce in most sections of North America. A LOOK BACKWARD In the thickly settled districts of the Eastern and Middle States matters have gone so far that it is diffi- cult to suggest a means by which a stock of birds for field shooting may again be obtained, except the one means, which gunners will adopt last of all, the practice of self-control in their shooting. One result of this A LOOK BACKWARD 523 state of things is that a considerable proportion of the men who formerly followed the dog afield, and a very large proportion of their descendants, have taken to shooting targets at the trap, for the reason that within the reach of most of us there are no birds to be had. I have never advocated the introduction into this I have never advocated the introduction into this country of foreign species of game, believing that our native species answer our requirements better than any foreign birds, and believing also that there is great danger in introducing exotic species into any country. I have believed that by the establishment of game refuges in various sections of the country the native game might be preserved and increased to such an ex- tent that it would overflow into adjacent territory, and that thus in each game refuge we should have a perma- nent source of supply which would at least prevent the extinction of species for any locality. I feel about the introduction of exotic species much as does Mr. Brews- ter, who, in his ‘Memoir on the Birds of the Cam- bridge Region,” says, speaking of the pheasant: “From the standpoint of the naturalist, the introduc- “From the standpoint of the naturalist, the introduc- tion of most exotic forms of animal life must be a matter of regret rather than of satisfaction, and these pheasants, despite their undeniable beauty of form and coloring, and reported value as game, seem deplorably out of place ina New England landscape. A LOOK BACKWARD Even if they did not crowd out our quail or ruffed grouse—as it has been feared they may—or devastate our cultivated crops—as they are already accused of doing—it would have been much wiser to expend the time and money AMERICAN GAME BIRD SHOOTING 524 which have been devoted to their naturalization in fos- tering and increasing our stock of native game birds.” The turning loose of foreign birds to take care of themselves in a climate to which they are unaccustomed and among conditions more or less different from those in which their ancestors have lived, is not likely soon to make much difference in our shooting. A few hundred birds turned loose in a township or a county would have to increase enormously before they would be suf- ficiently numerous to make the shooting good. Every- one killed would reduce the breeding stock, the process of reproduction would be slow and the final results, even if favorable, might not be important for a generation. Besides this, there are serious possible dangers in Besides this, there are serious possible dangers in the turning loose of foreign birds. There are some reasons for thinking that these foreign birds carry with them the germs of certain diseases to which they them- selves are immune, but which may be communicated to our native birds with fatal results. It is believed by some investigators that the domestic fowl carries with it the germs of a disease which is fatal to the turkey, and to our quail and grouse, although the young of the hen do not suffer from it. Many examples might be cited of the danger of intro- ducing into a new land an animal, harmless in its own home, but which when transported to a country where conditions are especially favorable to its existence and it finds few or no enemies, has increased to such an ex- tent as to become a nuisance, if not a public menace. The cases of the rabbit in Australia, of the English spar- A LOOK BACKWARD 525 row in North America, and of the mongoose in some of the West Indian Islands, suggest themselves at once. ly, within the past two or three years, the ex- Happily, within the past two or three years, the ex- periment of hand-rearing some of our native game birds has apparently advanced well along the road to success. A LOOK BACKWARD If it cannot be said that any of the public establishments for rearing such birds have been successful in any great degree, it is yet true that they hatch and partly rear many birds. Almost always it has happened that before the birds reached maturity, certainly before they had reached the breeding age, death in some form or other has overtaken them. It was left to a private individual, a professor in a New England university, to take up this matter of rearing native birds as a hobby and to succeed in it beyond the expectation of anyone, possibly beyond his own hopes. Certain it is that Prof. C. F. Hodge, of Clark University, at Worcester, Mass., has succeeded in rearing from the egg a considerable num- ber of ruffed grouse and quail, which birds are no more timid and fearful of the members of their owner’s family than they are of their fellow birds. Not only are they tame in this way, but they manifest no fear what- ever of strangers. Within their enclosures they carry on the operations of their daily life with the same un- concern that they would manifest if they were hidden in the depths of one of their native swamps, and this whether human beings are in the vicinity or not. Broods of quail which Professor Hodge has reared and turned out to shift for themselves will come at his whistle, flying from all directions, expecting to be fed. 526 AMERICAN GAME BIRD SHOOTING Gradually legislators are awakening to the increas- ing scarcity of bird life about us, and to a compre- hension of the invaluable services performed by the birds for agriculture. It has taken the public a long time to begin to comprehend what these services mean, and it is yet, standing only at the threshold of this knowledge. But having made this small beginning, our faith may be strong that this interest will increase, and that before long the birds will be generally recog- nized as a natural resource of this country which should be conserved. In a number of the States and provinces the law now provides that there shall be no spring shoot- ing; that non-game birds shall not be killed nor their nests and eggs disturbed, and that the shooting seasons shall be short—not more than two, or at most three months. A LOOK BACKWARD In some States the laws provide that only a limited number of birds may be taken during one day or during one season. This provision should obtain everywhere, as well as the now very generally accepted view that game birds shall not be sold. In many States we have excellent laws, which, however, lack some- thing of satisfactory enforcement. TRANSPLANTING AMERICAN GAME BIRDS. It was between the years 1885 and 1895 that the greatest interest was felt in trying to restock the covers of the Middle States with bobwhites imported from the South. They were turned out in large numbers, but, as I have said, did not increase the numbers of local birds because it did not occur to sportsmen to stop shooting. A few men paid for the birds in any locality, and the general public turned out and killed them all, very often without so much as saying “thank you” to the men who had paid the bills. But bobwhite was not the only native bird which sportsmen tried to introduce on the Atlantic Coast. Efforts have been made at various times to introduce the pinnated grouse of the Mississippi Valley to the old home of the heath hen. This might easily have been done, and could be done to-day, provided only the birds were introduced into game refuges where abso- lute protection would be assured them. The birds for- merly submitted readily to confinement, bore railway journeys well and speedily adapted themselves to new conditions. While most of these attempts have been forgotten. While most of these attempts have been forgotten. or remain only as tradition, we have fortunately a record of one of them. This account is from the pen of Mr. N. H. Bishop, well known a generation ago as 527 527 527 AMERICAN GAME BIRD SHOOTING 528 a traveler and canoeist, and was printed in Forest and Stream in December, 1874. It tells of the introduction of the pinnated grouse and two species of Pacific Coast quail in eastern Maryland. The essential parts of his letter are as follows: “About five years since a resident of Philadelphia sent to! Dr.) Py J) Purnell, near Berl) Worcester County, Maryland, a few pairs of prairie chickens and a covey of both the valley and mountain partridge, or quail. Dr. Purnell has an estate of fifteen hundred acres lying along the banks of Newport Creek, which stream flows into Sinepuxent Bay, on the eastern shore of Maryland. Since the war, this estate has been worked for the Doctor by his tenants. Much of it is woodland and salt meadows. The partridges were for some time kept confined in the house and then were set at liberty. TRANSPLANTING AMERICAN GAME BIRDS. They soon disappeared, except one pair, which returned daily to the kitchen door to be fed. For some cause the pair went to a neighbor’s house, on the same estate, and for some weeks were fed from the kitchen door. This pair of birds nested in the garden near the house, and raised a brood of young birds. The covey left their old quarters, and were heard from but once after their departure. A person reported that he saw the covey of ‘California quails on the other side of the creek.’ This was two years since. It is now sup- posed that these partridges have been shot by gunners or have died from natural causes. “The prairie chickens adapted themselves to their “The prairie chickens adapted themselves to their new home with but little trouble to the proprietor of the TRANSPLANTING AMERICAN GAME BIRDS 529 estate. Their nests filled with eggs were found along the fences of the fields near the meadows. The birds became tame, visiting the cattle yards, and feeding near the buildings of the farm. They multiplied rapidly. A law was passed by the Maryland legislature protecting them from gunners. The birds seemed to like the large salt meadows of the estate, and exhibited but little fear of strangers. Unfortunately for the birds, a number of terrapin hunters from New Jersey ascended the bay and river in their small vessels. Seeing these tame birds on the meadows, the Jerseymen commenced a war of extermination upon them, which soon resulted in the destruction of almost the entire lot. A work- man on Dr. Purnell’s estate informed me that he had seen eighteen prairie chickens in the cornfield, near the house, in November of the present year. It was the only covey left by the Jersey terrapin hunters, who came up from Chincoteague Inlet. The same gentle- man who sent these fine birds to Dr. Purnell is about to send down from New Jersey the ruffed grouse, called in that State and Pennsylvania, the pheasant. There are no ruffed grouse on the Peninsula.” In Forest and Stream of Nov. 25, 1880, Mr. C. S. In Forest and Stream of Nov. 25, 1880, Mr. C. S. Wescott reported that a half-grown prairie chicken had been brought to Mr. TRANSPLANTING AMERICAN GAME BIRDS. John Krider, of Philadelphia, for mounting, the bird having been sent from lower Dela- ware or Maryland with a lot of quail to a Philadelphia game dealer. The bird was not preserved. Mr. Wes- cott believed—and no doubt he was right about it—that this was a descendant of the birds liberated in Mary- AMERICAN GAME BIRD SHOOTING 530 land about 1869 by Dr. Purnell. If this is true, it would seem that these birds had survived and bred for a period of ten years, indicating that the conditions were then very favorable in that locality. About the year 1870 a number of birds are said to About the year 1870 a number of birds are said to have been imported from the West and set free on Long Island, where they did well, and reared broods, but finally disappeared. Within two or three years after that the late Shepard F. Knapp, of New York, long a member of the Southside Club and an enthusi- astic sportsman, told me of having seen on Long Island two prairie chickens that had recently been killed some- where near Islip. 1) 0. In the year 1874 a number of sportsmen strongly urged the restocking of certain barren areas in New Jersey with pinnated grouse, which had been extermi- nated there not very long before. Their idea was to import the birds from Illinois, where they were then numerous. That same year there was talk and correspondence in That same year there was talk and correspondence in England and in the United States as to the desirability of importing the pinnated grouse to England and at- tempting to establish them on the Scotch moors. The price put upon the birds seems to have prevented any action in this direction. Dealers asked £2 5s. per pair for the birds, but later Mr. Chas. Hallock succeeded in finding an animal dealer who agreed to furnish 100 pairs at £1 Ios. per pair. To-day many sportsmen would be glad to close with an offer such as this. Mr. Brewster, in the work already quoted, tells of Mr. Brewster, in the work already quoted, tells of TRANSPLANTING AMERICAN GAME BIRDS 531 an importation of these birds turned out in Massachu- setts. TRANSPLANTING AMERICAN GAME BIRDS. - He says: arly spring of 1884 or 1885, six pairs of “Tn the early spring of 1884 or 1885, six pairs of prairie hens brought from Iowa were liberated by Mr. Robert B. Nesbitt, of Cambridge, at various points along Concord Avenue, between Belmont and Concord. He tells me that he was afterwards informed—on somewhat questionable authority, however—that sev- eral of these birds reared broods of young that season. I can vouch for the fact that a year or two later an adult male spent most of the spring in a grain field near the village of Carlisle, Massachusetts, where it was seen by my friend, Mr. George H. Robbins, and several of his neighbors. “Another bird of the same sex was met with by Mr. Walter Faxon in the Fresh Pine Swamps (on the Ar- lington side of Little River) on May 14, 1892. The latter instance may be taken to indicate that at least a few of these grouse may have succeeded in maintain- ing themselves for a number of years, but there are no good reasons for believing that any of them are still living or have left living descendants. In short, the at- tempts to establish them permanently in the Cambridge region, as well as in other certain parts of Massachu- setts where they were liberated about the same time, have evidently proved a complete failure.” Efforts have been made to naturalize various Amer- Efforts have been made to naturalize various Amer- ican game birds in England, but so far as known, with no permanent success, except in the case of the turkey, and this only as a domestic bird. In 1861 Mr. Grant- AMERICAN GAME BIRD SHOOTING 532 ley F. Berkeley, a member of Parliament, wrote from Winkton House, in Hampshire, to his friend, Capt. Geo. D. Bayard, saying: “My friend in America up to this last week has been sending me over prairie grouse and quail for naturalization for our society for that purpose here, and I am charmed by being put in mind of the plains, by having a male prairie grouse walking about my garden, tamer than an English pheasant, coming to my whistle for food and making the devil’s own howls, with the skin blown out on either side of his neck, strutting and running around and calling for his mate, but I have no mate to give him. TRANSPLANTING AMERICAN GAME BIRDS. The four prairie grouse that came over are all males. A friend of mine, Lord Malmsbury, has imported some of your wood grouse, and they are doing well. “T have sent out to different places a great number of quails, some from Canada and some from New York,’ More than twenty years later Mr. Frank Sturgis, of Chicago, sent a large number of live pinnated grouse to a friend in England, who acknowledged them in a letter published in Forest and Stream in the following letter from Neath, under date of March 22, 1883, as follows: “T have unfortunately been upon the invalid list at the seaside for a few days, or I would have written at once to acknowledge Mr. Cheetham’s letter of the 3d inst., and to thank you most heartily and most sincerely for your princely present of prairie grouse. I am TRANSPLANTING AMERICAN GAME BIRDS 533 sure that I am quite right in stating you have altogether excelled all previous attempts in this direction, and the successful outcome of the shipment reflects the greatest credit upon your excellent arrangements. The birds being so rare, and the quantity so far in excess of any private requirements, I hope you will be pleased rather than offended at the distribution which has been arranged. Acting in concert with Mr. Henry Nash, who communicated with Mr. Lowell, we have pre- sented twenty to her Majesty the Queen, for Balmoral, and the Prince of Wales has been graciously pleased to accept twenty for Sandringham. We have sent ten to the Zodlogical Gardens in Regent’s Park, where they are very much prized and valued, and we have turned out sixteen to take their chances upon our Welsh hills—jointly upon Lord Jersey’s property and upon shooting land which is leased by the writer. Such of these last birds as have since been seen were all doing well, and I have a report to-day that those which were sent to the Zoological Gardens are also well and getting less shy than when first turned out. The birds which were sent to the Zoological Gardens are the only ones now in confinement, and it is understood that the so- ciety will reserve half of any young birds which may result in case those which are turned out should not in- crease their numbers. I shall hope to report further satisfactory progress very shortly. “PHILIP W. What became of these birds we do not know; that a TRANSPLANTING AMERICAN GAME BIRDS. FLOWER.” “PHILIP W. FLOWER.” What became of these birds we do not know; that a AMERICAN GAME BIRD SHOOTING 534 large number of them must have reached their destina- tion in good condition seems obvious, since no less than sixty-six are accounted for in the letter. Bobwhite was introduced into England in the earlier half of the last century, and is reported to have done well for a time. No serious attempt has been made to transplant No serious attempt has been made to transplant from one part of America to another any others of the grouse, except that one or more consignments of sharp- tailed grouse are said to have been shipped to Massa- chusetts, where they were turned out and disappeared. Sharp-tailed grouse were planted also on Grand Island in Lake Superior, but this is in or close to the native range of the species. What the fate of these Grand Island sharp-tail grouse may have been we do not know. Possibly they shared the fate of the exotic grouse there introduced, all of which, we are told, have disappeared. We read in the accounts of the early Spanish ex- plorers that the wild turkey had been domesticated by the Indians before the white man’s coming. A consid- erable portion of the sustenance of certain Florida In- dians was drawn from their turkeys, and we are told that the carnivorous animals in the great zoological gardens kept up by the Aztec, Montezuma, were fed on the flesh of turkeys. The domestic turkey is a descendant of the wild The domestic turkey is a descendant of the wild turkey of the Southwest. The bird was carried to Spain and thence spread over Europe, finally reaching England. That the common wild turkey of the East TRANSPLANTING AMERICAN GAME BIRDS 535 may also be domesticated has been shown by experi- ments carried on, on a plantation near Farmville, Va., by Prof. Robert Lee Blanton, an account of which was published in Forest and Stream, vol. 74, p. 450. When, if ever, game refuges shall be established in When, if ever, game refuges shall be established in the various States—game refuges properly protected, be it understood—they should be stocked with wild turkeys. DESTRUCTION: AND PROTECTION. Where game birds are left to look out for themselves, civilized man and the conditions he brings with him constitute their most destructive enemy. It is not so much the number of birds killed by man as the changes which he works in the land that affect the birds and often make life impossible for them. Of these changes, the most important is the clearing up and cultivation of woodland—the cutting down of forests and the drain- ing of swamps. Most wild creatures require cover to afford them shelter from their enemies, while the far- mer wishes the ground to be free from all cover except that which his crops afford, and these crops are standing for a few months only. Without cover, the quail, the wild turkey, the grouse and the woodcock cannot exist. Wild birds depend for safety largely on their protective coloring, which makes it easy for them to escape notice in their chosen haunts, and on their habit of flying—on the approach of danger—to refuges where they may hide. But if forest and brush are cut away the partridge and the quail have no places in which to seek safety ; if the tall grass of prairie sloughs and ravines is mowed and the soil ploughed up, the prairie chicken is without place of concealment; if the swamp is drained and the alders, birches and spice- 536 536 ‘aspoH “yo <a Aq peydeisojoyg Fen® jo [njpuey sfqnop y ‘a “yo <a Aq peydeisojoyg Fe jo [njpuey sfqnop y DESTRUCTION AND PROTECTION 537 wood that grow in it are cut down, the woodcock no longer has a home. While all these changes are going on, gunners are While all these changes are going on, gunners are becoming more numerous. With the settling up of the country there is an increasing demand for the flesh of game, and, between the attacks of man, the de- struction of their old homes and the ravages of pred- atory birds and mammals, in many regions the game birds have been almost exterminated. Among their natural enemies are all the carnivorous mammals, many of the hawks and owls, and not a few domestic animals. DESTRUCTION: AND PROTECTION. In the old times, when birds were less pursued by man and had a range far wider than at present, their annual increase in numbers more than made up the loss from the attacks of natural enemies, but over much of the territory of the United States that time has long passed. In thickly settled countries, the d In thickly settled countries, the domestic dog, self- hunting through the spring and summer, destroys great numbers of the nests and eggs of song and game birds, while the house cat, the pet of the kitchen or the house cat run wild, is very destructive. The harm done by the farmer’s dog in his travels through the fields is not generally understood. Two or three dogs from neigh- boring farms may start out in company and be gone all day—perhaps for several days—hunting. Among the prey that they capture may, perhaps, be a woodchuck, the remains of which the farmer finds later and speaks of with pride as having been killed by his dogs. He does not know that while hunting the dogs may have AMERICAN GAME BIRD SHOOTING 533 destroyed dozens of the nests of game birds and song- birds, which are the farmer’s best friends, since they are ever-working unpaid police officers, destroying all through the summer days the insect enemies which prey upon his crops. If the work of these insect-eating birds were to be stopped for a week, or for a month, the damage done to the crops of the United States would be incalculably great. In certain sections of the South an extremely de- structive enemy of ground-nesting birds of all sorts is the wild hog, which roams the forest, literally seeking what he may devour. The number of nests and eggs of turkeys, ruffled grouse and quail that these animals search out and destroy is very great. Some States have laws providing that hogs shall not be allowed to run at large, but such States are exceptions. On the other hand, it must be said that in States so thinly settled that hogs and cattle are permitted to run wild, there are comparatively few dogs and cats that roam the fields and woods. Mr. DESTRUCTION: AND PROTECTION. Herbert Brown reported a few years ago that previous to the introduction of ranch cattle the masked quail was quite common in southern Arizona, but that the cattle eating off and trampling down the tall grass had so destroyed the breeding and hiding places of this bird that it had practically disappeared from the United States side of the line, and at latest reports this was still the condition, as is seen by what he says on page 64. Under such adverse conditions, it is not strange that our stock of splendid game birds grows smaller Winter quarters of Dr. Hodge’s Quail. Photographed by C. F. Hodge. Winter quarters of Dr. Hodge’s Quail. Photographed by C. F. Hodge. DESTRUCTION AND PROTECTION 539 year by year. The problem as to what shall be done to keep this stock from wholly disappearing gives cause for much anxiety. For American sportsmen, American game birds are the best, and there is no reason why we should not have an abundance of these, provided only for a short time we practice a little self-control. The wild turkey was formerly found over the whole United States, east of the Missouri River, south of the Niobrara and east of the Rocky Mountains. Grouse of one sort and another formerly abounded over almost the whole United States and Canada, while in the southern portions of the United States there were great numbers of quail. These birds sportsmen may bring back, in large meas- ure, if they care to; but they cannot do this by killing them at every opportunity. All these birds are prolific and probably all can be hand-reared. If this can be done, they may be turned out in numbers on game refuges—if these shall be established—and if absolutely protected there, will soon spread themselves over the surrounding country. They must be protected, how- ever, alike against the gunner and against the attacks of their natural enemies. It is not yet fu It is not yet fully known what might be done with our grouse and quail, but the experiments of Prof. C. F. Hodge, of Worcester, with the ruffed grouse and bobwhite, justify the belief that all grouse may be reared in confinement. A short period of experimen- tation by men interested in the subject will probably result in the semi-domestication of practically all the AMERICAN GAME BIRD SHOOTING 540 American grouse. DESTRUCTION: AND PROTECTION. These birds, so reared in their natural ranges and then set free in refuges, would re- stock covers now barren. The process of restocking could be kept up indefinitely, and after a time at a very modest cost. If one-half as much time and money had been expended in attempts to domesticate or hand- rear some of our native species as has been devoted to the importation and rearing of exotic forms, the prob- lem of our game supply would now be much simpler than it is. With the Limicoline birds the case is somewhat dif- ferent. No experience has been had with them, and nothing is known about confining them or inducing them to breed in confinement. On the other hand, as migratory birds they are exposed to the gunner only for a short time, and then pass on to sections less thickly settled, and where, therefore, the dangers are less. In the United States most of the efforts to rear game birds have been devoted to foreign species, but to this rule the State of Massachusetts is a notable exception. On its public game farm it has for several years con- sistently tried to rear quail and ruffed grouse, and though not altogether successful, much experience has been gained. It was left to another Massachusetts man, Dr. Clif- ton F. Hodge, working from pure love of his task, to accomplish the greatest results. The work done by the Massachusetts Commissioners The work done by the Massachusetts Commissioners of Fisheries and Game, and by Dr. Hodge, is set forth ‘aspoH Oa ES) Aq poydeis0joyud *peyozey ysnf syoryo ssnoig poyny *peyozey ysnf syoryo ssnoig poyny DESTRUCTION AND PROTECTION 541 at some length in the Reports of the Massachusetts Commissioners for the years 1907 and 1908. In 1907 the efforts of the Massachusetts Commis- In 1907 the efforts of the Massachusetts Commis- sioners to rear ruffed grouse in confinement resulted in little more than the acquisition of some experience as to what not to do. It was learned, for one thing, that the males are extremely disposed to fight. During the winter, in a pen of three young birds one male killed another with which it had grown up. Even during the mating season there was need of great care lest the male kill the female. DESTRUCTION: AND PROTECTION. His attacks on the females caused them great alarm, since it was impossible for them to escape from the cage, and they hurt them- selves in their efforts to do so. In fact it was not safe to put the male in the coop for mating without closely watching him, and it was necessary to remove him almost at once. The birds in the coops of the commission soon be- came noticeably unafraid. They paid little attention to what was going on outside, and did not display the timidity and restlessness of the bobwhite. It was ob- served that they ate with appetite and appeared to enjoy their food. Nevertheless, the male grouse died late in the summer, evidently from an infectious dis- ease communicated to him probably through infected ground. No one has approached the success which has at- tended the work of Prof. C. F. Hodge, of Clark Uni- versity, who for some years, as a rest and recreation from his ordinary work, has been trying to rear ruffed AMERICAN GAME BIRD SHOOTING 542 grouse and quail in domestication. His experience is extraordinarily suggestive, and we quote much of his report to the Massachusetts Commissioners. His permits for the year allowed him to take 80 eggs of the ruffed grouse and to keep three pairs of bobwhite quail for purposes of propagation. He had 54 grouse eggs, from which 38 chicks were hatched, and of these he says: “As will be seen, the eggs did not hatch as well as in the previous years of experiment, possibly due to cold weather in May. The eggs were all hatched under Cochin bantam hens, as usual. Chicks are ac- counted for as follows: “Fifteen chicks, June 6th, left with hen mother, began dropping off after the first two weeks, and were all dead by August 15th. “Fifteen chicks, June 6th, transferred on morning of “Fifteen chicks, June 6th, transferred on morning of hatching to brooder; 2 escaped, 1 died, probably as result of accidental injury, 2 died from swallowing objects too large to pass into the gizzard (black cricket and large spider), 10 are healthy, well-grown birds at present. “Eight chicks, June roth, transferred to brooder, and all did well, until, on sudden change of weather, tem- perature ran too high and killed them all. DESTRUCTION: AND PROTECTION. “The experiment was conducted this season on my “The experiment was conducted this season on my new place, on uncontaminated ground, and I thought it desirable to try once more the simpler method of rearing with the hen. The result is decisive against this method. The fact that the hen carries parasites, DESTRUCTION AND PROTECTION 543 which are likely to prove fatal to young grouse, prob- ably explains why our native species have not long since been domesticated. All former attempts to domesticate them have been made in the poultry yard and its environment. The same is now being proved to explain why the turkey, another native American species, cannot be reared on ground contaminated by domestic fowls. This is the fifth year in which the experiment of rearing ruffed grouse with bantam hens has been tried, and although the second year 6 birds were reared out of a clutch of 12, for all the other years fatalities by this method have amounted to 100 per cent. This season is particularly decisive because the work was done on new ground under most favor- able conditions, with the benefit of past experience, and in clear competition with the brooder method.” On the other hand his experience led him to believe On the other hand his experience led him to believe that except for accidents, practically every ruffed grouse chick hatched may be reared to maturity by the brooder method. Brooders connected with yards in which the chicks may run during warm days offer a method which is not expensive. The young develop and feather out rapidly, and except for the two first weeks need no artificial heat, except during cold storms. Professor Hodge suggests the possibility of rearing Professor Hodge suggests the possibility of rearing a strain of hens to be hatched in incubators and reared on uncontaminated ground which possibly might be safe for mothers and brooders. Professor Hodge found the bobwhite quail much Professor Hodge found the bobwhite quail much easier to handle than the grouse, and experimented AMERICAN GAME BIRD SHOOTING 544 with a single pair kept as pets in the house. The hen laid 18 eggs in a nest made by the pair in a window cage, but since the birds did not seem inclined to in- cubate, the eggs were placed under a bantam hen. DESTRUCTION: AND PROTECTION. Of the 13 eggs first laid 6 hatched, but the chicks being left with the hen all died within ten days. Of the remaining 5, 3 hatched under a second hen and to these 5 eggs were added 11 obtained from the State Hatchery. Ten chicks were obtained from the 16 eggs. Of these, 3 died by accident and showed ne trace of the disease of liver and ceca which has proved fatal to so many of our young game birds as it has to turkeys. The remaining 7 birds did well from Sep- tember to the date of this report, November, 1907. Eleven quail chicks just out of the incubator were obtained August 19th from the State Farm, were reared in a brooder and to date are alive and well. One of the great difficulties attending these efforts One of the great difficulties attending these efforts to rear native game birds has been the persistent attacks of vermin, and this Professor Hodge was able to wholly guard against during the summer of 1907. Rats were troublesome, a skunk killed a hen quail, and a multi- tude of skunks were about. Traps were kept con- stantly set and the results were surprising—for it must be remembered that Professor Hodge lives in the large city of Worcester. These traps caught 17 skunks and a great number of rats, and all these before any dam- age had been done by the animals. Professor Hodge is carrying on this interesting experimental work purely as a labor of love, though in 1909 a grant of ‘aspoy “yO Aq paydeisojoyd “SHOTYO ayIYMQGog suIpsagq SIGS Ty ‘aspoy “yO Aq paydeisojoyd “SHOTYO ayIYMQGog suIpsagq DESTRUCTION AND PROTECTION 545 $500 from the Carnegie Institute paid a portion of the expenses. The The work of the Massachusetts State Farm started in the year of 1906 with a flock of breeding quail numbering 47. At the close of the season a partial count indicated 30, and at the beginning of winter 21 were left, while at the opening of the breeding season of 1907 there were but 12, or only about 25 per cent. of the original number. A nu A number of chicks were hatched in incubators, and this method seemed to Arthur Merrill, the superin- tendent, more successful than the use of hens. DESTRUCTION: AND PROTECTION. The loss from dead embryos was less than the usual break- age of eggs, and the chicks were stronger. The in- cubators were run in the same manner as for pheasants. The young quail were fed o pheasants. The young quail were fed on dried ants’ eggs and maggots, live ants’ eges and insects being added when practicable, with fruit, seeds and grain as the bird grew older. Insect food is unquestionably most de- sirable for young birds, but it is difficult to obtain. Berries, grasses and clover, or indeed almost any green food, are essential, but difficult. to secure. On the whole, up to the close of the year 1907 the experi- menters had learned chiefly what was to be avoided, and not what was to be done. In his report to the Commi In his report to the Commissioners on Fisheries and Game of Massachusetts for the year ending Decem- ber 31, 1908, Professor Hodge gives at length his experience for the previous season, writing under date of November 27th. Little could be done toward experi- AMERICAN GAME BIRD SHOOTING 546 menting with ruffed grouse, yet one clutch of 12 eggs was secured, 10 of these hatched and 3 reared to maturity. In the loss of 7 of these chicks a very im- portant discovery was made. Insect food being essen- tial to the young birds, a large number of striped plant bugs were obtained by sweeping the grass where they were abundant with insect nets. The young chicks ate them eagerly, and went to sleep and died as if they had been chloroformed. Professor Hodge points out that these bugs give out a strong odor similar to that of the squash bug, and Conradi found that when squash bugs were fed to toads, these died as though poisoned with chloroform. While Conradi found that 5 or 6 squash bugs might be sufficient to kill a toad, Miss Morse has fed as many as II to bobwhite at a single meal with no apparent ill effect. Toads ate the plant bugs in large quantities without injury, but it is ap- parent that strong-smelling bugs should not be fed to young ruffed grouse. The experiments with bobwhite proved very success- The experiments with bobwhite proved very success- ful. The season began with 5 pairs and several extra cocks. DESTRUCTION: AND PROTECTION. The first egg was laid by May 6th, and 4 of the hens had begun laying by May roth. Not less than 247 eggs were laid, an average of 49 to the pair, but Mr. Merrill, of the State Game Farm, had a hen quail which laid 100 eggs. Two of Professor Hodge's hens died during the summer, but the 3 that survived laid an average of 65 eggs each. In all 114 chicks were hatched and 75 reared. A most excellent showing, but one that will unquestionably be greatly Hodge. C.F. ite reared by Dr. Bobwh Hodge. C.F. ite reared by Dr. Bobwh DESTRUCTION AND PROTECTION 547 improved on as time goes on. Professor Hodge’s letter on his experiments shows extraordinary progress made in learning of the lives of these birds, There is a wide range of There is a wide range of difference in the actions of different pairs of bobwhites, especially in respect to breeding and care of young. One pair which occupied a large cage with the ruffed grouse got along with the larger birds without any trouble; the hen laid 58 eggs, making and filling several nests. On August 20th the cock began brooding a nest with 16 eggs, and on September 13th hatched 15 chicks. Both birds joined in the care of the brood. Another pair had occupied a cage 6 by 12 feet and produced in all 69 eggs, but neither bird showed any signs of brood- ing. The cock, however, took charge of and reared a brood of chicks, hatched under a bantam, but while he was doing this the hen quail refused to associate with him. A pair was kept in a cage 3 by 6 feet and produced 68 eggs, all laid in a single nest. The eggs were taken away from time to time, because neither bird showed signs of brooding. Nevertheless, on the 9 eggs last laid, the hen began to sit September 12th and brought out 9 chicks October 6th. The cock was attentive to his mate on the nest, but was seen sitting on it only once for a short time, while the hen was feeding. Both birds joined in the care of the young. DESTRUCTION: AND PROTECTION. Of the two unmated cocks one adopted and took care of a brood of chicks hatched under a hen, while the other refused to care for the chicks, pecked them savagely and would probably have killed any that had been left AMERICAN GAME BIRD SHOOTING 548 in his cage. It is evident, therefore, that a cage 3 by 6 feet is not too small for quail to do well in; a fact of interest, for it renders the work of experimenting in this matter practicable for almost anyone. Some temperatures of these birds were taken, and Some temperatures of these birds were taken, and it was found that the interior body temperature of a brooding male bobwhite was no less than 111 degrees— remarkably high. The brooding quail gives the eggs rather long periods of cooling once or twice a day. The records thus seem to suggest for incubators a temperature one or two degrees higher for quail than for domestic fowls. It has been noted also that the chicks of the ruffed grouse and the quail seem to thrive much better when the brooders are kept much warmer than is advised in the directions for domestic chickens. This is necessary, apparently, only for the first ten days or two weeks, but for that time it seems well to keep the temperature five degrees higher than re- quired for domestic fowl. The conclusion thus reached was confirmed by an experience in September when, of 68 chicks in brooders, 24 died in two days. Professor Hodge’s explanation of the ‘“brooder troubles,” which have caused the death of so many apparently healthy birds, seems wholly plausible. He regards these troubles as acute indigestion and sug- gests aremedy. He gives elaborate directions for feed- ing, lays an especial stress on lining brooders, which should have no chinks or cracks into which one of these tiny birds may creep, on keeping them free from in- fection from domestic fowls, and on giving the birds DESTRUCTION AND PROTECTION 549 sufficient water and a warm dust bath. These two things, he believes, attach the birds strongly to a hom- ing place. His birds were at liberty during the day. He says: “The little fellows have an affinity for brush patches, corn, asparagus beds, or any sort of natural cover, like that of ducks for water. DESTRUCTION: AND PROTECTION. Instead of proving the nuisance that I at first feared, this affinity may be turned to excellent account if properly indulged and arranged for. I always keep a little brush-pile, with an armful of hay or freshly cut weeds thrown over it, in the home cage. This also attracts them to the place. If they have located in a clump of bushes or a weed patch, they are safe for the day. Should a thunder storm come up, and the birds are as tame as they should be, they can be whistled home, or an insect net be put over the entire cluster and they can be car- ried into the brooder. When they began to fly in the second week, I expected to be obliged to clip their wings. I did not do this, however, and, as the sequel proved, their ability to fly is a great advantage in rearing them. Within the past few days a wind storm opened one of my cages containing adult and young birds. Though able to fly for miles, I drove them back into the cage as easily as if they had been a flock of little chickens. During the summer the flocks would range out afoot to feed and fly home. In feed- ing the birds I used from the first a low whistle, in imitation of the feeding note of the chicks themselves. On coming home at noon or night the flock might be AMERICAN GAME BIRD SHOOTING 550 nowhere in sight, but on giving the whistle I would be assured by a chorus of eager replies, and in a moment the air would be full of whirring wings, as the flock flew in with a rush and lighted at the entrance of the cage. This might be repeated as many as a dozen times a day. “Wintering the bobwhite offers no difficulty. I set “Wintering the bobwhite offers no difficulty. I set the cages with the shelter end open to the south, build a brush wood-pile in the middle of the cage, see that they are supplied with weed seed, grain mixture and water when snow is lacking, and keep a cabbage, a mangel wurzel or an apple where they can pick at it. DESTRUCTION: AND PROTECTION. It might be well to see that the cluster is not imprisoned under the ice after a sleet storm, but the brush-pile has afforded insurance against this so far. This brush wood-pile is made by placing a few stout branches on the ground in a sheltered sunny exposure, and on these pile about two feet of weeds, cut before the seeds fall—ragweed, lamb’s quarter, pigweed, smart-weed, wild buckwheat (chaff straw or loft sweepings would do if weeds are not at hand) ; then pile on a foot or two of stout brush which cannot be crushed down by heavy snows, and on top of this place a good thick covering of weeds. This will give the flock scratching mate- rial all winter, afford shelter from cold and storms, and protect from vermin, especially hawks, owls and cats. For bobwhite in the open I think this simple winter provision would insure against winter killing and ex- tend the range of the species at least several hundred miles to the north. The great value of the bird in Ruffed Grouse feeding from hand. Photographed by C. F. Hodge. Ruffed Grouse feeding from hand. Photographed by C. F. Hodge. DESTRUCTION AND PROTECTION 551 insect and weed seed destruction will amply ena farm- ers for their work. “As spring comes on the cocks fight a good deal; but still breeders generally advise keeping several pairs in the same cage. I tried both methods this season, keeping four pairs in the same cage (6 by 12 feet) through May and June. They laid well under these conditions, but there was so much disturbance and persecution that there was little hope of either hens or cocks beginning to brood. Each pair was then given a separate cage, and they did so much better that this plan will be generally adopted in future.” Professor Hodge gives various suggestions replying to many inquiries that have come to him, as to practical methods of rearing bobwhite. What he has said, how- ever, applies equally to the ruffed grouse and prob- ably to the prairie chicken as well. The results which he has obtained in six years of experiment will appear to most people very gratifying and very encouraging to further experimentation in this field. DESTRUCTION: AND PROTECTION. He very truly says: “We now see clearly that we must bid farewell to all our native game birds or work out methods of propagating them.” Besides the birds in question Professor Hodge ex- Besides the birds in question Professor Hodge ex- perimented with a pair of sharp-tail grouse from the Saskatchewan. The birds did very well and Professor Hodge discovered that they were exceedingly fond of clover. No doubt if he had given them alfalfa they would have eaten it with still greater relish, since, in AMERICAN GAME BIRD SHOOTING 552 their natural range, whenever the alfalfa is green, these birds resort to it, and for a part of the year it seems to constitute one of their chief foods. The hen prairie chicken laid ten eggs and then died, The hen prairie chicken laid ten eggs and then died, showing intense infection with coccidia, which appears to be the bane of so many of our native birds, including the domestic turkey. Five of the eggs were hatched under a bantam hen, but all died in Professor Hodge’s absence from home. He was promised eggs or breed- ing stock in prairie chickens for the following year, and hoped to experiment with them. In 1909, however, circumstances prevented his work on the problem. He reared three ruffed grouse and fourteen quail, and in 1910, owing to the pressure of affairs he turned over his breeding stock to the Massa- chusetts Commission. While Professor Hodge was doing this work at Worcester, the superintendent of the Sutton Hatchery, the Massachusetts Game Farm, was studying the same problems. The results obtained were not great in the number of birds, but much was learned as to the most serious disease likely to be met with, and the adapt- ability of quail and grouse to domestication. It was found that disease rather than the method of feeding is the controlling factor in rearing the young. One hundred and twenty-two ruffed grouse were hatched, but all except 2 birds were lost, though 11 lived to the age of seven weeks. Many of the deaths were due to coccidium, and others to what is called by the writer “brooder pneumonia,” and the inference drawn from DESTRUCTION AND PROTECTION 553 the report is that. DESTRUCTION: AND PROTECTION. many of these young birds received infection either from the hens which hatched them, or from the ground on which they were reared, this ground being a place where poultry had been kept. English pheasants appeared to be more resistant to dis- ease than the grouse and quail. During this season most of the eggs laid by the quail During this season most of the eggs laid by the quail were well fertilized, and no quail failed to lay. Four hundred and seventy eggs were laid and 222 hatched. There seemed no advantage in penning the birds with a greater number of females than males, and during the season of 1907, when the birds were paired, results were much more favorable than before. In a number of cases the males were active in caring for the chicks, and in one case a male adopted and reared a clutch of young ones. While in earlier attempts at hatching, much loss was experienced from breakage of eggs by the bantam hens, and while the next year incubators were used, hatching 80 per cent. of the eggs, during the season of 1908 incubators were unsatisfactory, while hens did much better than before, the greater number breaking no eggs at all. In other words, the result of each season shows the importance of more and more experiments on the part of those who have these matters in charge, and as they acquire this experi- ence their success will, of course, be greater. The food difficulty remains, but in this matter much The food difficulty remains, but in this matter much also is being learned. Like Professor Hodge, Mr. Merrill concludes that Like Professor Hodge, Mr. Merrill concludes that there appears to be no advantage of large pens over AMERICAN GAME BIRD SHOOTING 554 small ones. To be sure, the birds found some insect food, but this was soon exhausted. Small pens have given entire satisfaction for wintering quail and grouse, the only difficulty being wet and dampness when snow melts. The pen should be kept entirely dry, and should be open to the fresh air. A better comprehension of the mating and nesting A better comprehension of the mating and nesting processes of the quail and ruffed grouse is very nec- essary. The breeding of the grouse is complicated by the uncertain and quarrelsome temper of the male. DESTRUCTION: AND PROTECTION. The problem of hatching, like that of food, requires further study. Meantime, of course, the work of bacterio- logical investigation must be kept up, for one of the first problems in rearing these birds is to understand the diseases to which they are subject under these new and unnatural conditions. The success of the Sutton Farm with pheasants has The success of the Sutton Farm with pheasants has been better than with our native game, but even so has not been at all satisfactory. CONCLUSION. In view of all our experience it is clear that Amer- ican sportsmen cannot continue to kill off their game and still to have their covers full of birds. I believe that a time will come when there will be good shooting in many parts of the country, but this will not be until gunners have been taught the lesson of self-control. Methods must be devised for restocking our fields and woods, and the birds put out must have an opportunity to live and to reproduce their kind. This means better enforcement of law, shorter open-seasons and a bag limit. As conditions precedent to good shooting, must come also game refuges and the private preserve. If sportsmen long ago had been wise enough to demand from the legislatures the establishment of game refuges, we should now have public preserves which might have been large enough for anyone. Instead of demanding this, gunners often grumbled bitterly when land own- ers posted their fields and declined to allow everyone to run over their land, tear down their fences and shoot their domestic fowls, and even their animals. The private preserve has come to stay, and it is a good thing for gunners at large that it has come. What the general public should now demand is game refuges—public preserves—where the birds may in- crease and from which they will certainly scatter out 550 AMERICAN GAME BIRD SHOOTING 550 for the benefit of the general public. By the time such refuges have been generally established the problem of rearing our game birds will probably have been solved, and from that time on there will be little difficulty in our finding each autumn a few birds on which to work our dogs and exercise our skill in shooting. Only a few years ago the shooting seasons com- monly began on September Ist and closed Janu- ary Ist. Now in most States—above all in those States where birds are least abundant—the shooting season lasts for two months only and often closes Decem- ber 1st. Almost without our noticing it the length of the season has been reduced one-half. In many States bag limits have been established, and while too often these limits are disregarded, the sentiment that this law should be obyed is constantly growing stronger. CONCLUSION. Everywhere sportsmen might accomplish much more toward increasing the supply of game if they were organized and would work together for a common object. In some States organizations have been ef- fected, but sufficient interest has not yet been aroused to make them ready to work together for the common purpose. Sometitnes, too, the organization is brought together more for the purpose of making a showing or redounding to the credit of some one individual than it is for actual fish and game protective work, and when this takes place, the organization becomes a mere name, wholly inefficient for practical purposes. The State of Connecticut possesses an organization CONCLUSION Bez modeled on right lines. There are town gun clubs, which are members of the County Game Protective As- sociation, and these County Game Associations choose delegates who are members of the executive council of the central State Game Protective body. The town eun clubs are active and especially interested in their own especial territory. The occasional meetings held where all the towns of the country are represented, keep up this interest, and when the county delegates attend the meetings of the State Association they are able to report intelligently on what is taking place and on what should be done. If every State in the Union had such an organization, and if every State from time to time chose delegates to a central association which represented the English-speaking race on the conti- nent, matters would go forward much more rapidly, and much more would be accomplished than at present. Another thing that works against the sportsmen is Another thing that works against the sportsmen is that they have no method of communication with each other. The means are at their hand in the various sportsmen’s journals, but only a small proportion of the great number of gunners in thelandread these journals. If they were read by most sportsmen, who thus kept themselves informed as to what legislation is on foot and what other sportsmen are doing, intelligent action could be taken by a large body of men on the measures which should be taken to increase our stock of game. Sportsmen would then be acting together as one body, and many of the problems which now trouble us would have been solved. CONCLUSION. 558 AMERICAN GAME BIRD SHOOTING It is worth the while of all who are actively inter- ested in having our game supply increased to keep themselves informed of the legislation that is taking place, of the progress that is being made in the artificial rearing of fish and game, and above all of the views and opinions of other sportsmen. Unless those interested in this subject are willing to work together and to bring their united intelligence and influence to bear on the public, the reforms will come only slowly. The objects which sportsmen must work for are, the enforcement of the laws that we already have, and the passage and enforcement of better laws. Among the better laws needed are, first, statutes authorizing the establishment of game refuges; sec- ond, the prohibition of the sale of game; third, short shooting seasons and a reasonable bag limit. Many of the States now insist on a payment by gun- Many of the States now insist on a payment by gun- ners of a shooting license fee, and in many States the aggregate of these fees amounts to a great sum. It is proper that a large share of this money should be expended for the benefit of those who pay the tax, and a proportion of that expenditure should be for intel- ligent experimentation in rearing our native gamebirds. During the past twenty years great progress has During the past twenty years great progress has been made in game protection. To-day more interest is felt in this subject than ever before, and more money is available for it. If sportsmen will work together to a common end, the next ten or fifteen years should see in many parts of the United States better shooting than has been had there for a generation.
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Electroluminescent Organic and Quantum Dot LEDs: The State of the Art
Journal of display technology
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cc-by
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I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS T HE development of lighting solutions over the last 150 years has been driven by number of factors, namely lower cost, increased brightness, longer lifetime, and more efficient devices that use more environmentally-sustainable materials. T The tungsten light bulb was a revolutionary step up from candles and oil lamps. Owing to its effectiveness at filling rooms with white light at the flick of a switch, it quickly became the lighting solution of choice and became a fixture in almost all buildings with a source of electricity. But the tungsten lamp was not perfect; grossly inefficient it created significant excess heat. Nonetheless, its easy manufacturability and low cost meant it would be some time until its use began to be rivalled by the much more efficient compact fluorescent light bulb (CFL) (50–60 lm/W cf. 8–10 lm/W) that was introduced in the 1970s. Besides using less energy, CFLs also lasted much longer: in the region of 5000 hours cf. 800 hours. They have since become ubiquitous in households and commercial buildings as governments around the world introduced phase-out programs of traditional (so-called incandescent) bulbs in favor of newer generations of energy-efficient lighting. See Fig. 1. LED light bulbs make use of the principle of electrolumines- cence (EL), although it is first appropriate to consider the me- chanics of photoluminescence (PL) . PL is the term given to describe the radiative decay process in a material; it is a more general term than fluorescence or phosphorescence, which de- scribe emission from specific states: either excited singlet or triplet states, respectively. For organic molecules, fluorescence usually occurs from the lowest vibrational level of the excited singlet state. It is a so-called spin-allowed transition, so it oc- curs quickly, typically on the nanosecond time scale. Phospho- rescence, in contrast, occurs after intersystem crossing from an excited singlet state to an excited triplet state; and as this process is spin forbidden, it typically occurs over a longer period—in the microsecond to second time frame. The process is gener- ally described figuratively using a Jablonski diagram that shows the absorption and deactivation processes of organic molecules (Fig. 2). Many heavy metals, such as transition metals (e.g., Ir, Pt) and lanthanide/rare earths are “phosphorescent” owing to spin-orbit coupling, which weakens the rules around spin al- lowed and spin forbidden transitions. As can be seen in Fig. 480 480 JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 Manuscript received January 08, 2015; revised March 12, 2015; accepted March 19, 2015. Date of publication March 31, 2015; date of current version May 04, 2015. 1Minamata Convention on Mercury. [Online] Available: http://www.mer- curyconvention.org Poopathy Kathirgamanathan, Lisa M. Bushby, Muttulingam Kumaraverl, Seenivasagam Ravichandran, and Sivagnanasundram Surendrakumar Poopathy Kathirgamanathan, Lisa M. Bushby, Muttulingam Kumaraverl, Seenivasa Sivagnanasundram Surendrakumar Poopathy Kathirgamanathan, Lisa M. Bushby, Muttulingam Kumaraverl, Seenivasagam Ravichandran, and Sivagnanasundram Surendrakumar Fig. 1. Historic development of more efficient lighting solutions. Abstract—A detailed background into the reasons for the feverish interest and growing impact on commercial market share of organic light-emitting diode (OLED) technology for lighting and display applications is given. Following this, the increasing discussion over whether electroluminescent devices based on quantum dots will ever challenge that of organics in displays is considered. The unique physics and chemistry of quantum dots results in narrow emission bands and increased stability over organic dyes meaning the potential for this is significant. The current best efficiencies of quantum dot devices and associated device structures from the literature are presented followed by a comparison of devices fabricated with organic materials, in particular those of polymers, metal complexes (fluorescent and phosphorescent) and small molecules. Fig. 1. Historic development of more efficient lighting solutions. Although more energy efficient, the CFL bulb has its own environmental concerns, containing as it does the hazardous substance mercury, which means a number of controls must be instituted during manufacture and disposal to prevent re- lease into the environment and to protect human health. Parties and signatories to the UN's Minamata Convention on Mercury1 have agreed to ban the production, export, and import of cer- tain types of CFL by 2020 owing to their mercury content. As a result, the non-mercury-containing third generation of light bulb, the LED, which also provides further efficiency improve- ments (60–80 lm/W) over the CFL bulb has taken over signifi- cant swathes of market share and is continuing to do so. Index Terms—Inorganic light-emitting diodes, organic light-emitting diodes (OLEDs), quantum dots. The authors are with the Institute of Materials and Manufacturing, College of Engineering, Design and Physical Sciences, Wolfson Centre for Materials Processing, Brunel University London, Uxbridge, UB8 3PH, U.K. (e-mail: p.kathir@brunel.ac.uk). This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecommons.org/licenses/by/3.0/ This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecom I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS 2, the energy associated with emission is less than that of absorption (excitation) meaning that emitted photons have less energy than those that are absorbed and so Color versions of one or more of the figures are available online at http:// ieeexplore.ieee.org. Digital Object Identifier 10.1109/JDT.2015.2418279 Digital Object Identifier 10.1109/JDT.2015.2418279 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 481 Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro- cesses of organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 5. Mechanics of electroluminescence in organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro- cesses of organic molecules. Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro- cesses of organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro cesses of organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 5. Mechanics of electroluminescence in organic molecules. Fig. 3. Absorption and emission spectra of Zrq . Fig. 3. Absorption and emission spectra of Zrq . the spectra are shifted to longer wavelengths. In addition, as ab- sorption can occur from any one of several vibrational energy levels in the ground state to any one of various excited vibra- tional states, absorption and emission spectra of typical organic molecules, which include the vibrational levels of many atoms, are characteristically broad. See Fig. 3. Fig. 5. Mechanics of electroluminescence in organic molecules. Major breakthroughs in harnessing EL have been made pos- sible through the development of wide bandgap light emitting diodes (LEDs) in the 1960s, the development of evaporated or- ganic films in the 1980s and most recently through the develop- ment of solution-processable quantum dots. Where the excited triplet state is long lived and close in en- ergy to the excited singlet state, an additional mechanism for fluorescence may occur. Known as thermally activated delayed fluorescence (TADF), the singlet state is populated through: 1) excitation and 2) intersystem crossing then occurs to the triplet state. I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS In the case of LCDs, a white light source is used as the backlight and a liquid crystal, that can filter the white light to a variable degree, is placed in front of each subpixel in a device. The “filter” allows a range of wavelengths appropriate to its color through at any one time thus creating the multi-color capability. The brightness of the subpixel is controllable as the liquid crystal is energized or not to block or transmit light. It is the crystal filtering mechanism that causes poor viewing angles on LCD displays. The first observations of EL in organic materials proved problematic in terms of the potential for commercialization of the technology, however. For instance, in 1965, Helfrich and Schneider had to apply currents of 50–1000 V to anthracene crystals to observe fluorescence [4], and progress was further hampered by difficulties in crystal growth and short lifetimes. Around the same time, General Electric introduced the first commercial LEDs based on the inorganic semiconductor GaAsP [5]and over the next few decades, while research into organic EL waned, that related to inorganic LEDs flourished. It is possible to use LEDs as the white light source in LCD displays and these tend to produce better contrast and color co- ordinates than fluorescent backlights. Plasma displays are gen- erally not as efficient as so-called LED-LCDs. An inorganic (semiconductor) LED essentially consists of several layers including an n-type layer with a surplus of neg- ative electrons and a p-type layer with an insufficient amount of electrons (also described as a layer with a surplus of positive holes). An active layer is sandwiched between them. When an electric voltage is applied, the electrons and holes flow towards the layer they are attracted to. Their paths cross in the active layer where they recombine radiatively and light is emitted. See Fig. 6. In the first instance, LED technology would seem to tick many of the boxes required for display applications. ZnS and SrS are among the most common phosphor materials used in thin-film electroluminescent (TFEL) displays, although to ob- tain EL, the phosphor must be intentionally doped with either transition metal [7]or lanthanide luminescent impurities [8]. Ex- panding the applicability of semiconductor materials beyond lighting and backlighting solutions continues to pose significant challenges owing to the difficulties with the requirement to dis- play more than one color. I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS At this point, rather than the triplet relaxing back to the ground state: 3) a second intersystem crossing process back to the excited singlet occurs, from where: 4) fluorescence takes place, as shown in Fig. 4. One of the first observations of EL was in the 1930s by Des- triau [1], who found that certain zinc sulphide phosphors could be excited by low voltage, low frequency alternating current. Crucially, no heat was emitted during the process creating the potential for highly-efficient lighting solutions. Destriau also discovered that doping ZnS with “activators” such as Mn , Cu or Ag , could lead to enhanced emission, although charge compensation, i.e. the addition of “co-activators” such as Cl , Br or I is an important factor in this. EL is the phenomenon whereby light is emitted from a ma- terial following the application of an electric field to it. The process can be described by an analogous mechanism to that of PL where rather than exciting the molecule through the ab- sorption of radiation, the molecule is excited electrically. The molecule then relaxes to its ground state radiatively and EL re- sults (Fig. 5). Between 1953–1955, Bernanose published details of one of the first observations of EL of organic compounds, namely acri- dine derivatives [2], [3], in which brightness vs applied field followed the same emission laws to those found for inorganic phosphors. In particular, Bernanose studied acridine orange on cellulose films from which EL was observed following the ap- plication of high alternating voltages. His studies found that the emission spectrum was independent of the applied field with the The energy levels of organic molecules are often described as the lowest unoccupied molecular orbital (LUMO), or the excited state, and the highest occupied molecular orbital (HOMO), or the ground state, as can also be seen in Fig. 5, with the distance between the two levels the energy bandgap (Eg). 482 JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 Fig. 6. Principle of electroluminescence in semiconducting (inorganic) and organic LEDs. Fig. 6. Principle of electroluminescence in semiconducting (inorganic) and organic LEDs. proposed mechanism being either direct excitation of the dye molecules or excitation of electrons. another. CRTs, for example, have a much higher power usage than LCDs and require bulky devices. I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS A succession of LEDs based on III–V semiconductors, such as GaAs, GaP, AlGaAs, InGaP, AlInGaP, were all developed, but devices tended to be red, orange, green and yellow as the color of the emission is based on the energy gap of the semi- conducting material, and blue-colored devices remained elu- sive. It was some time before blue diodes based on materials such as ZnSe or GaN were developed and these still exhib- ited significantly lower efficiencies than other diodes [6]. It was for the blue emitting GaN-based device that The Nobel Prize in Physics 2014 was awarded. p-i-n junction inorganic LEDs offer long lasting and more efficient alternatives to older lighting technologies. Under active-matrix conditions, multi-color LEDs can be as- sembled into an EL display as demonstrated by Barrow and Tuenge of Planar Systems, Inc. [9]in a prototype multi-color dis- play using ZnS:Mn and ZnS:Tb phophor layers. EL subpixels can be arranged in two-dimensional arrays each with its own row and column address and associated data value. To make a full color display, one or more subpixels of different colors are grouped together to form a pixel. Thus each pixel on an EL dis- play includes one or more subpixels, e.g. red, green and blue. More than 30 years on, however, full-color TFEL displays based on semiconductors are generally only available in small format active matrix design as cost-effective full-color panels remain problematic to commercialize and the devices are still only used in backlighting applications. KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 483 Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 9. Selected commercial OLED displays. III. INTRODUCING OLED TECHNOLOGY TO THE MARKET: TWO ASPECTS—LIGHTING AND DISPLAYS Interest in organic EL was revived when Tang and VanSlyke fabricated devices using thin evaporated films ( 100 nm) of aluminum 8-hydroxyquinoline Alq and low voltages ( 10 V) [10]. This breakthrough development made it clear that, given improvements in efficiency and reliability, it would be possible to manufacture low-cost, flexible, multi-color and white displays based on organic EL. Further attention was drawn to this possibility with the advent of similar devices fabricated by Burroughes et al. [11] based on the conjugated polymer polyparaphenylenevinylene (PPV) which could be formed via the low-cost route of spin casting from solution and subsequent thermal treatment. The development of a solu- tion-processable precursor for PPV and poly(fluorenes) made this a potentially attractive approach for simple light-emitting diode fabrication. Fig. 9. Selected commercial OLED displays. OLEDs rely on a central layer of emissive organic material that is electroluminescent, that is, it will emit light when ex- cited by an electrical current. Electrons are injected from the cathode and holes from the anode. These travel inwards into the device where they recombine to form excitons in a singlet:triplet ratio of 1:3. The spin-allowed emission from fluorescent exci- tons may occur rapidly, otherwise the normally non-radiative triplet excitons may emit if the active layer is phosphorescent. As there are often difficulties in injecting electrons and holes (carriers) into the organic layer, the structure of an OLED usu- ally includes additional layers, such as electron injecting and transporting layers and hole injection and transport layers to facilitate the injection of charge carriers. The layers are de- posited on top of each other with the first deposited on a sub- strate (Fig. 7). The anode must be transparent to allow the pho- tons produced to be seen, and so it is usually transparent ITO, although it could equally be polyethyleneterephathalate (PET) coated with a transparent conductor for a flexible device or any other transparent material. involves the deposition of additional layers of emissive material onto the same substrate (Fig. 8). The inclusion of polymers and organic molecules as emissive layers in devices of various structures have been the subject of many thousand research papers and patent applications since the 1980s each demonstrating an improvement in efficiency, color coordinates, lifetime or brightness over the last, with the result that the efficiencies of the devices and thus the commercial vi- ability of the technology improved exponentially. II. MULTI-COLOR DISPLAY TECHNOLOGIES Traditional technologies for multi-color displays utilizing cathode ray tubes (CRT) or liquid crystal displays (LCD) have significant limitations that prohibit their use in one way or IV. QUANTUM DOTS Quantum dots (QDs) have become the subject of intense aca- demic and industrial (Nanoco, QD Vision, Nanosys, Samsung, Dow Chemicals) research over the past 15 years owing to their novel electronic, electrical, optical and catalytic properties with wide ranging applications in displays, lighting, lasers, solar cells and photoelectrochemical cells. Quantum dots are nanocrystals composed of III–V semicon- ductor (e.g. GaN, GaP, GaAs, InP and InAs) and II-VI semicon- ductor (e.g. ZnO, ZnS, CdS, CdSe, and CdTe) materials with all three dimensions in the approximately 1–10 nm size range. Among these, CdSe/ZnS systems have been studied most as far as the EL is concerned, although, Cd is environmentally re- stricted owing to its toxicological properties and its viability as a commercial material is consequently questionable. Fig. 11. White OLED vs white LED emission spectra. OLEDs can get much closer values of 90–95 to the ideal. The CIE chart showing the relative positions of warm and cool light is given in Fig. 10 and a typical emission spectra of a white OLED and white LED is given in Fig. 11 where the significant emission in the blue region of the spectrum can clearly be seen for the LED. It is the size of the QD that imparts unique physical properties on the material: in contrast to the bulk semiconductor, the elec- trons in a nanocrystal exhibit quantum mechanical effects. The so-called quantum confinement phenomenon occurs as the size of the semiconductor becomes comparable to or smaller than the exciton Bohr radius, and where the electron and hole are confined by the boundaries of the material. It leads to discrete energy levels, known as “confinement states”, as predicted by a particle in a box (Schrödinger's) equation [see (1)]. While technology into OLED lighting and research is some- what in its infancy, the same kind of improvements that were seen in OLED displays enabling commercial devices over the last few years are expected in lighting solutions in the next 3 years. For example, in 2009, Matsushita (National Panasonic) Electric Works demonstrated a WOLED with an efficiency of 37 lm/W with a lifetime in excess of 10000 hours at an initial luminance of 1000 cdm and improved it to 150 lm/W with a lifetime in excess of 20,000 hours at 3000 cdm in 2014 [12]. III. INTRODUCING OLED TECHNOLOGY TO THE MARKET: TWO ASPECTS—LIGHTING AND DISPLAYS OLEDs are now a commercial reality in many display appli- cations (Fig. 9) and it is worthy of note that as they can be de- posited onto virtually any substrate, e.g. glass, ceramic, metal or plastic. It is also possible to manufacture flexible or curved de- vices, which is not the case with semiconducting LEDs, LCDs or plasma technologies. In addition to use in displays, OLEDs have the potential to be used in lighting applications. A key advantage of using an OLED light over an LED light source is its favorable color rendering properties as given by its color rendering index (CRI). The CRI has been defined the International Commission of Illumination (CIE) as the effect of an illuminant on the color appearance of objects by conscious or subconscious comparison with their color appearance under a reference illuminant. Effectively, it is the quantitative measure of the ability of a light source to reveal the colors of objects in comparison with a natural light source. Devices are typically assessed through a number of character- ization measurements that include color coordinates (perceived color), current density (A cm ) versus voltage, luminance (a measure of brightness in cd m ) versus voltage, current efficiency (cd A) versus luminance, power efficiency (lm/W) versus luminance and lifetime (a measure of the stability of the device). Critically, the incremental cost and ease of producing multi- color devices over single color or white devices is negligible as the fabrication technique of making a multi-color device simply Semiconductor LED light bulbs have struggled to reach the more desirable warmer whites with values of 80–85 compared to the highest CRI achievable of 100 for a black body radiator. JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 484 Fig. 12. Projected market for OLED lighting, QDs and displays [IDtechEX, Santa Clara, November 2014]. Fig. 10. CIE color coordinate chart showing warm and cool warm light. Fig. 10. CIE color coordinate chart showing warm and cool warm light. Fig. 12. Projected market for OLED lighting, QDs and displays [IDtechEX, Santa Clara, November 2014]. Fig. 11. White OLED vs white LED emission spectra. IV. QUANTUM DOTS In addition to Matsushita, there are a number of large compa- nies heavily involved in OLED lighting, such as Samsung, LG, Pioneer, Hitachi, Osram, Philips, GE, and BOE. (1) (1) where is the quantum number; is Planck’s constant; is the electronic mass, and is the width of the box. These levels correspond to bonding (HOMO) and anti-bonding (LUMO) levels in the material (Fig. 13). While bulk semiconductors have a set bandgap (Eg) between the valence and conduction bands, an effect of quantum confine- ment means that the bandgap energy of a QD is inversely proportional to its size (smaller QDs emit higher energy than The market for both OLED lighting and displays has grown steeply since 2000, with the market, particularly for OLED lighting, predicted by IDTechEx to continue to show strong growth through 2018 and beyond (Fig. 12). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 485 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 485 Fig. 13. Energy bandgaps of bulk semiconductor and QD materials. Fig. 15. Cluster diameter versus energy gap. Fig. 15. Cluster diameter versus energy gap. Fig. 13. Energy bandgaps of bulk semiconductor and QD materials. QDs absorb all wavelengths higher in energy than their bandgap and convert them into a single color, i.e. they have broad absorption spectra, but narrow emission spectra. This feature gives them advantages over organic fluorophores be- cause the excitation wavelength can be anywhere within a broad range [15]. The narrow emission spectra of QDs and the tunability of such results in an extremely wide color gamut, and thus QD displays have the potential for improved color saturation over OLED displays. Fig. 14. Top image of PL of selected quantum dots in hexane [QD source: Sigma Aldrich]. Schematic of tunability of QDs—effect of size distribution on color of PL emission below. QLEDs are also solution processable (low cost) and have theoretical performance limits that meet or exceed that of all other display technologies [16]. As both the color coordinates and luminous efficiencies of QLEDs are good they are there- fore capable of being more power efficient. Furthermore, as with OLED displays, they can be deposited on any substrate enabling many exciting possibilities in terms of shape and design. It is well-known that the absorption spectrum shifts to red as the size of the nanocrystal increases. IV. QUANTUM DOTS Typical emission spectra show a full-width at half maximum (FWHM) of 30–40 nm depending on the degree of monodispersity achieved during synthesis [17]. QDs can be both electrically and optically excited, where in- ternal EL quantum efficiencies as high as 90% have been ob- tained for certain materials [18]. Fig. 14. Top image of PL of selected quantum dots in hexane [QD source: Sigma Aldrich]. Schematic of tunability of QDs—effect of size distribution on color of PL emission below. larger QDs), and this means that the emission from a QD is color tunable (Fig. 14). Owing to colloidal stability and the ability to make thin films without disrupting the physical integrity of the crystal, the use of QDs in flexible EL displays becomes a possibility. Further, solution processing enables low cost full color display man- ufacturing, e.g., using spin processing [19] or inkjet printing [20] techniques. Such techniques can be applied to additive pro- cessing, such as vacuum deposition [21], and hence are more affordable methods for display fabrication. The bandgap is also dependent on the cluster diameter, and this also affects the dis- tribution of spectral emission, i.e. the smaller the cluster, the narrower the emission band. See Fig. 15. Although radiative, a QD that comprises of a single semi- conducting material generally exhibits low quantum efficiencies owing to non-radiative electron-hole recombinations that occur at defects in the crystal and dangling bonds on the surface. For example, Xie et al. [13] found that after 12 hours in air, the ab- sorption spectrum of an InP QD sample shifted to the blue dra- matically, which was attributed to the fast shrinkage of the in- organic core by oxidation. As a result, shell materials, typically with wider bandgap energies, such as CdS or ZnS are grown on the surface of the core [14] in a process known as inorganic pas- sivation. Confining charge carriers within the luminescent core and away from the surface significantly reduces the effects of surface defects caused by atoms on the surface of the crystal. In this way, states that may function as centers for non-radiative recombination are protected and there is a consequent increase in photoluminescent quantum yield (PL QY). QDs are generally prepared by reacting inorganic precursors in the presence of organic ligands, which eventually form a molecular coating around the QD luminescent core and stabilise the nanoparticles against aggregation (Fig. 16). IV. QUANTUM DOTS Factors that are important to optimize during inorganic pas- sivation include the energy levels of the shell and core, the for- mation of a defect-free and uniform coating, coherence strain between with the shell and core lattice, and the thickness of the shell layer. The maximum PL efficiency of the core/shell QD is dependent on the thickness of the shell layer, which has Xie found that after growing a ZnS shell around the InP core, the absorption properties of the nanocrystal were unchanged fol- lowing exposure to air, indicating a much improved stability. Further, they found that the PL QY of the InP dot was low ( 1%), but that it reached 40% for the core/shell ones. 486 JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 Fig. 16. Example of a passivated core-shell quantum dot structure. Fig. 16. Example of a passivated core-shell quantum dot structure. Fig. 18. Typical synthetic route developed by Ludolph et al for the preparation of TOPO-capped CdSe using a simple precursor [26]. Fig. 17. Energy bandgaps in Type I, Reverse Type I and Type II QDs. Fig. 19. Schematic of capping ligand replacement mechanism. Fig. 17. Energy bandgaps in Type I, Reverse Type I and Type II QDs. been found to be less than two monolayers thickness for op- timum properties of a CdSe/CdS core/shell structured nanopar- ticle. Thicker capping layers lead to the formation of misfit dis- locations, which are also sites of non-radiative recombination, leading to a decrease in the PL QY [22]. Fig. 19. Schematic of capping ligand replacement mechanism. The surface of the core/shell QD may still possess highly re- active dangling bonds, and these can be used to further passivate the nanocrystal from its surrounding environment through the co-ordination of an organic ligand, such as tri-n-octylphosphine oxide (TOPO), trioctylphosphine (TOP), thiols (e.g. -mer- captoethanol), carboxylic acids (e.g. oleic acids), octadecene, oleylamine or 3-(aminopropyl)trimethoxysilane (APTMS). The ligand chelates to the surface of the QD by donating lone pair electrons to the surface metal atoms, resulting in a QD that demonstrates reduced particle agglomeration, reduced sensitivity to oxidation, improved electronic stability and may be soluble in relatively non-polar media [24]. It also stabilises the nanocrystals to the extent that it possible to extract them as free-standing powders [25]. IV. QUANTUM DOTS KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 487 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 487 Fig. 21. HOMO-LUMO levels of selected QD materials and hole and electron transporting materials. Fig. 20. Conventional OLED device structure. Fig. 20. Conventional OLED device structure. Further, QDs can be anchored by polymers (in particular light-emitting polymers) by producing a monomer with a phos- phonyl group, carrying out the QD synthesis in this medium and reacting the product with a polymerizable group (e.g., phenylvinylene) [27]. Fig. 21. HOMO-LUMO levels of selected QD materials and hole and electron transporting materials. This ability for solution-based synthesis enables effective control of the crystal size distribution. The phenomena of “Ostwald ripening” is utilized whereby the higher free energy of smaller QDs makes them lose mass to larger size QDs, eventually disappearing. Using a co-ordinating solvent, such as TOPO, stabilizes the QD dispersion, improves the passivation of the surface and provides a steric barrier to slow the growth of the QD. The final size of the QD is mainly controlled by the reaction time and the temperature. As crystal size is directly correlated to the HOMO-LUMO bandgap of the material, it is possible to take aliquots during the reaction process, and observe the corresponding absorption wavelength shift to stop the reaction when the appropriate size of crystal is achieved. This method has been used extensively to synthesize II-VI and III-V QDs [28]. The method provides sufficient thermal energy to anneal defects resulting a monodispersed solution of QD. A typical standard deviation of particle size of 5% is achieved. TABLE I HOMO-LUMO LEVELS OF TYPICAL QDS Fig. 22. Example OLED emissive layers with HTL/ETL energy levels. TABLE I HOMO-LUMO LEVELS OF TYPICAL QDS IV. QUANTUM DOTS The quantum efficiency of the QD will be further increased by a defect-free, and uniform shell coating to minimize the number of non-radiative recombination sites within the QD. Further, when the shell material coats the core surface the bonding lat- tice, lattice parameters, such as coherency strains result and can play an important role in the properties of these core/shell sys- tems. For instance, strain may cause the absorption and emission spectra of core/shell QDs to be red shifted [23]. Depending on the bandgaps and the relative position of the electronic levels of the involved semiconductors, the shell can have different functions in core/shell QDs. Fig. 17 gives an overview of the band alignment of the bulk materials where three types can be distinguished: Type I where the bandgap of the shell is larger than that of the core, Reverse Type I where the bandgap of the shell is smaller than that of the core and Type II where either the valence band edge or the conduction band edge of the shell material is located in the bandgap of the core. Ludolph et al. [26] has pioneered a route of synthesis for the preparation of a TOPO (tri-n-octylphosphine oxide) capped de- vice, as shown in Fig. 18. Organically-capped QDs may be photostable as the interface between the capping molecules and surface atoms is generally weak leading to the failure of passivation and the creation of new surface states under UV radiation. Surface modification by ligand replacement is possible to tailor the properties of the QD to the use for which it is required, for example it can be made hydrophobic or hyrophilic. Ligand replacement can be carried by reacting with an excess of another ligand or a ligand that has a higher complexing ability (high formation constant), for example a bidentate ligand (thiols), as shown in Fig. 19. It is crucial to match the energy levels of the shell and core. For Type I QDs, passivation of the QD surface should be with a material that has a larger bandgap energy to increase the ef- ficiency of charge injection into the radiative core. A wider bandgap shell material is desired to create a potential barrier around the QD core to confine the excitons. Confinement of charge carriers in the core region by the band offset potentials results in efficient and photostable luminescence from QDs. V. OLED AND QLED DEVICES Of the different types of emissive layer in OLEDs, typically small molecules, e.g., Alq have been shown to demonstrate good device lifetimes owing to the enhanced inherent stability of the molecules over phosphorescent materials, e.g. Ir , phosphors offer high efficiency along with high internal quantum efficiencies, and polymer materials, e.g., PPV, have the advantage of being solution processable, but have lower EL efficiency. y Owing to the multiplicity of the excited states formed when the charges meet in the active layer of an OLED, around 25% of excitons are generated in the singlet state, with three times more triplet excitons (approximately 75%) being formed. This means that OLEDs based on fluorescent molecules have a maximum internal efficiency of around 25%. The efficiency of OLEDs can be dramatically improved by using phosphors, such as organometallic complexes with iridium or platinum metal ions that enhance spin-orbit coupling and enable emission from the formally forbidden triplet state. Phosphorescent OLEDs consequently have theoretical maximum internal efficiencies of 100% as in addition to the 75% of triplet excitons generated, relaxation of spin-orbit coupling rules means that the singlets that are also generated may be converted to triplets through intersystem crossing. Fig. 23. Historic current efficiency data for OLED and QLED red [A], green [B], and blue [C] devices. Recent OLED activity utilizes thermally activated delayed fluorescence (TADF), or upconversion to achieve near unity in- ternal EL quantum yields from fluorphors by backfilling the sin- glet state with triplet excitons ( reverse intersystem crossing). The mechanism is described in Fig. 4, earlier. The need for more costly phosphorescent materials is eliminated, and the possibility of obtaining blue emitters that give very high total singlet yields [30], [31], is introduced. Indeed, Uoyama et al. [32] have reported OLEDs using EL metal-free molecules in which the energy gap between the singlet and triplet state is minimized by design allowing upconversion of excitons from the non-radiative triplet to the radiative singlet state thereby achieving efficiencies comparable to those in phosphorescence- based OLEDs. Green, orange and sky-blue OLEDs were fab- ricated with external EL quantum efficiencies of 19.3 1.5% (equivalent to 64.3%–96.5% internal efficiency), 11.2 1% and 8.0 1%, respectively. be wet processed into devices such as by inkjet printing or die coating much more cost effectively. Blue colored OLED devices remain a key challenge. V. OLED AND QLED DEVICES The basic structure of an OLED consists of one or more or- ganic films deposited between two electrodes, one of which is transparent (Fig. 20). Under electrical bias, electrons are in- jected from the cathode and holes from the anode into the or- ganic material where they travel in the applied field until they meet and form an excited state that leads to radiative emission. Fig. 22. Example OLED emissive layers with HTL/ETL energy levels. Devices fabricated using films that are deposited by subli- mation under vacuum are more costly than devices fabricated with films that are deposited from solution, e.g., by spin casting. Film thicknesses range from 10 to 100 nm. The emissive layer should have a high quantum yield and be an effective charge transporter. Additional layers are usually added between the anode/cathode and organic layer as hole or electron transporting aids. Considering the energy alignment of the ground and ex- cited electronic states of the emissive monolayers and the sur- rounding organic thin films is critical to the fabrication of an efficient device in order to reduce the energy level barriers be- tween layers, i.e. the ETL and HTL should be optimized as a well charge balanced device will have higher efficiency [29]. Fig. 21 shows energy levels of some common hole and electron transport materials used in OLEDs and HOMO-LUMO levels of typical QDs are shown in Table I. In addition to QLEDs, we consider three types of OLED (Fig. 22), i.e. those where the emissive layer is based on polymers (PLED), fluorescent small molecules (e.g., Alq , OLEDs) or organo–metallic phosphorescent small molecules (e.g., Ir ppy , Ir ppy acac , PHOLEDs). External quantum efficiency (EQE) can be expressed in anal- ogous manner to that of an OLED by a well understood multi- plication of four factors as described by (2) (2 (2) (2) JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 488 Fig. 23. Historic current efficiency data for OLED and QLED red [A], green [B], and blue [C] devices. where where recombination efficiency of holes and electrons; recombination efficiency of holes and electrons; fraction of charge carrier recombination in the emissive layer resulting in excitons with spin-allowed optical transitions; photoluminescent efficiency of the emitter; fraction of emitted photons that are coupled out of the device ; refractive index of the substrate (glass). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 489 TABLE II SELECTED TYPICAL PERFORMANCE DATA OF OLED AND QLEDS AT 1000 CDM Data given in the order: 1) CIE color coordinates (x,y); 2) lifetime; 3) current efficiency; 4) power efficiency TABLE II TABLE II SELECTED TYPICAL PERFORMANCE DATA OF OLED AND QLEDS AT 1000 CDM Data given in the order: 1) CIE color coordinates (x,y); 2) lifetime; 3) current efficiency; 4) power efficiency Fig. 24. Conventional [A] and inverted [B] QLED device structures. towards improved efficiencies for red, green and blue devices, both OLEDs and QLEDs. A comparison of typical device performance data based on fl t h h t l d QD i i l Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 26. Red device structure adapted from Kim and Jang [36]. Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 26. Red device structure adapted from Kim and Jang [36]. Fig. 24. Conventional [A] and inverted [B] QLED device structures. Fig. 24. Conventional [A] and inverted [B] QLED device structures. towards improved efficiencies for red, green and blue devices, both OLEDs and QLEDs. A comparison of typical device performance data based on fluorescent, phosphorescent, polymer and QD emissive layers at 1000 cdm is given in Table II. It demonstrates that the performance of QD devices still lag behind those of OLEDs and that progress on blue devices across the board is still required. QD device structures are largely similar to those of OLEDs (Fig. 24). A comparison of typical device performance data based on fluorescent, phosphorescent, polymer and QD emissive layers at 1000 cdm is given in Table II. It demonstrates that the performance of QD devices still lag behind those of OLEDs and that progress on blue devices across the board is still required. Fig. 26. Red device structure adapted from Kim and Jang [36]. has reported a red QD (CdSe/CdS) device that reaches a peak efficiency of 19 cd/A and 25 lm/W with an optimum thickness of 45 nm of QD and 0.68, 0.31 CIE coordinates. The lifetime was estimated to be 4 hours at 1000 cdm . The device config- uration is shown in Fig. 25. p g q QD device structures are largely similar to those of OLEDs (Fig. 24). V. OLED AND QLED DEVICES Com- pared to red (initial luminance 1000 cdm , 500,000 hours) and green (initial luminance 1000 cdm , 200,000 hours, according to Universal Displays at the OLED Summit 2013), the blue devices typically have lifetimes in the range 10,000–20,000 hours). Color coordinates of fluorescent blue OLEDs (0.15, 0.15) are considered acceptable with lifetimes of 50,000 hours at 1000 cdm reported by Idemitsu Kosan and Merck. Blue phosphorescent OLEDs have poorer color coordinates of (0.20, 0.35) with a reported lifetime of 8000 hours at initial 1000 cd/m . Printed PHOLEDs have even poorer lifetimes [33]. A further point of note is that OLEDs based on organic molecules have been found to degrade with time during prolonged excitation in a process known as photobleaching. Owing to the luminescent core being inorganic, which is then passivated by a protective inorganic shell, QDs are often much more resistant to photobleaching than organic dyes [34]. Strong intermolecular interactions usually result in the low solubility of organic molecules, further they tend to form crys- talline domains in the film state, which can act as carrier traps and raise the operational voltage of a device. As such, organic molecules in OLEDs are usually deposited onto a device by sub- limation under vacuum, a costly technique, but polymer OLED materials are readily soluble in ink solvents meaning they can Despite high theoretical performance levels of QLEDs in practice, thus far efficiencies still lag behind those for OLEDs, but as can be seen in Fig. 23, there is a marked general trend KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART It is frequently found that in the literature on QLED and OLED devices that not all the relevant data (EQE, power ef- ficiency, CIE co-ordinates and device lifetime) are reported for each device. While the PL efficiencies of QDs are high, the main reason for low EQEs in devices is largely attributed to poor charge carrier injection into the QD layers. Mashford et al.[35] Kim and Jang [14], [36] presented devices using CdSe/CdS/ZnS. Their red device achieved 6.5 cd/A and CIE (x,y)-coordinates of (0.70, 0.30), with a device structure shown in Fig. 26 and their green device an impressive 28 cd/A (0.16, 0.75), structure in Fig. 27. The lifetime was not reported. JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 490 Fig. 27. Green device structure adapted from Kim and Jang [36]. Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 30. Cd-free device structure (2013). (Lim et al. [38]) Fig. 27. Green device structure adapted from Kim and Jang [36]. Fig. 28. Yellow device structure adapted from Qasim et al. [37]. Fig. 30. Cd-free device structure (2013). (Lim et al. [38]) ITO, while holes are injected from Al. The HTL materials they used possessed different HOMO energy levels in the range of 5.1–6.0 e.V. ITO, while holes are injected from Al. The HTL materials they used possessed different HOMO energy levels in the range of 5.1–6.0 e.V. Fig. 28. Yellow device structure adapted from Qasim et al. [37]. HOMO-LUMO levels are easily determined from solution electrochemistry (viz. cyclic voltammetry) and the absorption edge from UV/VIS absorption spectroscopy of thin films [45]. Kathirgamanthan et al. [46] reported absorption, photolumines- cence and cyclic voltammetry studies on red and green QDs based on CdSe/ZnS provided by QD Vision. A yellow device has been reported by Qasim et al. [37] that achieves 1.6 cd/A with peak EL emission at 575 nm. Again, life- time data is not given. The device structure is given in Fig. 28. These devices show the potential of QDs in devices for dis- play applications, but their commercial viability is negligible owing to their reliance on the highly toxic and environmentally restricted Cd. VI. CONCLUSION AND FUTURE DEVELOPMENTS VI. CONCLUSION AND FUTURE DEVELOPMENTS One of the highest priorities for the commercialization of QLED technology will be to find suitable Cd-free quantum dots. InP-based dots and CuInS [44] are already showing much promise in this respect. We have also seen impressive results from devices that use ZnO cores with a MgO shell, indicating that ZnO is also a promising future Cd-free material. In terms of improving performance of QLEDs, there will certainly be much effort directed at optimizing the efficiency of devices. Research by Shen et al. [41], for example, has found that as the shell thickness of the quantum dot affects the charge confine- ment and charge injection in the QD. They recently reported a conventional QD device structure with a power efficiency of 19.7 lm/W for green emission. g As organic hole transporting materials with low HOMO levels (deep HOMO levels) are not readily available (for OLEDs, this is not so), the performance of QLEDs can be further improved by enhancing energy transfer from the hole transport layers into the QD layer through the careful selection of transport materials that can efficiently transfer excitons to QDs via Förster energy transfer [41]. Kim et al. [36] have described high-efficiency inverted QLED devices, which are useful for active matrix displays. They deposited two ETL layers, the first Al doped ZnO, followed by a second ETL. A maximum current efficiency of 28.29 cd/A and a power efficiency of 22.11 lm/W was achieved for their stacked ETL inverted green device; an approximately three times improve- ment on a QLED with a single ETL. Fig. 31. Absorption spectra of red and green QDs (upper chart) and PL of red and green QDs thin films (lower chart). Fig. 32. Cyclic voltammogram showing redox behavior of red QD in CH CN with CF SO Li as the supporting electrolyte. Using materials with high PLs in the red, green, and blue re- gions of the visible spectrum, Anikeeva et al. [43] were able to increase the efficiency of QLEDs four-fold for green devices and by 30% for orange devices. However, they found that there were still challenges in improving the efficiency of blue QLEDs, presumably owing to incomplete energy transfer from organics (weak spectral overlap between the blue QD and the electron and hole transporting materials used). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 491 Fig. 31. Absorption spectra of red and green QDs (upper chart) and PL of red and green QDs thin films (lower chart). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART As a result, efforts are now beginning to shift towards Cd-free device structures. While Lim et al. [38] pre- sented results from a Cd-free QD in 2011 using InP, the effi- ciency was poor at 0.006 cd/A (Fig. 29). Two years later, his group reported a 10.9 cd/A green device [39] that was also cad- mium free. (Fig. 30). The red QD’s absorption spectra (Fig. 31) of thin films spin coated from a hexane solution showed an edge, which we have attributed to the emissive energy gap in the nanocrystal at nm and which was at nm as a thin film. The green QDs showed clear absorption peaks at nm (in hexane); 509 nm (thin film). The PL spectra, also shown in Fig. 31, indicate the typical normal distribution type shape. The red QD showed PL emission peaks at (thin film); nm (in hexane) and the green at (thin film); nm (in hexane). 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They suggest this may be remedied by design and syn- thesis of wide bandgap hole and electron transporting organic materials for improved exciton energy transfer and direct charge injection into blue QDs. Along with continued improvements to luminance and lifetime results for OLEDs and QLEDs, we also expect to see the emergence of “hybrid” devices that incorpo- rate emissive layers using different types of emissive material, for instance a device could contain a blue emitting TADF layer, a green phosphorescent layer and a red QD layer. Fig. 32. Cyclic voltammogram showing redox behavior of red QD in CH CN with CF SO Li as the supporting electrolyte. determined the HOMO level to be set at 5.6 e.V. and the LUMO level at 3.4 e.V. for the red QD. Furthermore, we expect to see developments in the OLED lighting arena in the coming years owing to the appealing warm white light that can be created. The saturated colours from QDs, hitherto unattainable from OLEDs, give a technical advantage to QDs. However, the life-times of electroluminescent devices (RGB) are all very short (typically, 100 hours at 1000 cdm ) and need urgent attention. We have fabricated efficient red and green devices with organic electron injectors and transporters in inverted devices, the results of which will be published else- where. We anticipate a great future for quantum dots particularly A clear CV for the green QD could not be obtained as it did not form a stable colloid in the acetonitrile. This is supported by Zeta potential measurements that we carried out. A value of 66.32 mV was obtained for the red QD in acetonitrile indicating excellent stability in solution, but a value of 39.68 mV was ob- tained for the green QD indicating very poor colloidal stability. We estimated a HOMO level of 5.72 e.V. for the green QD from the CV and a value of 2.41 e.V for its LUMO level from the absorption edge [46]. KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 493 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART Poopathy Kathirgamanathan received the Ph.D. degree from Exeter University, Devon, U.K., in 1980. Poopathy Kathirgamanathan received the Ph.D. degree from Exeter University, Devon, U.K., in 1980. Poopathy Kathirgamanathan received the Ph.D. degree from Exeter University, Devon, U.K., in 1980. in recognition of his achievements in developing electroactive and light emit- ting materials and winner of awards for the best R&D in OLED's in 2006 and 2007 at the Organic Semiconductor Conference (Frankfurt, Germany). He is an Associate Editor of the IEEE/OSA JOURNAL OF DISPLAY TECHNOLOGY since 2012. He was recently awarded GB £4 million by EC to develop flexible OLED lighting. He has been a Fellow of the Royal Society of Chemistry since 1992, and of the Institute of Physics since 1994 in recognition of his achievements in developing electroactive and light emit- ting materials and winner of awards for the best R&D in OLED's in 2006 and 2007 at the Organic Semiconductor Conference (Frankfurt, Germany). He is an Associate Editor of the IEEE/OSA JOURNAL OF DISPLAY TECHNOLOGY since 2012. He was recently awarded GB £4 million by EC to develop flexible OLED lighting. He has been a Fellow of the Royal Society of Chemistry since 1992, and of the Institute of Physics since 1994 1980. He is a Professor of Electronic Materials Engi- neering, Wolfson Centre, Brunel University, U.K., and is one of the worlds leading experts in Organic Electronics. Following the Ph.D. degree from Exeter (Sir Arthur Reed Scholar), he pioneered research and development in the area of Organic Electronics at Newcastle University, Cookson Group plc., Uni- versity College London, and London South Bank University (Chair in Electronic Materials Engineering since 1993). In 2000, he founded OLED-T, based on the technology that he developed at London South Bank University and served as the CTO of OLED-T until the company and most of the I.P. (48 patents) of OLED-T was sold to Merck Chemicals, Germany, in August 2008. He moved to Brunel University, Uxbridge, U.K., in March 2009, where he continues to develop new materials for OLEDs, QLEDs, OPV, and OTFT, in addition to electrochemistry and nanotechnology. He is passionate about the commercialization of OLEDs for displays and lighting and OPV (Organic PV). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART He has received a total of GB £2.1 million pounds from Royal Society, TSB, EU, and industrial funding over the last three years. He has over 200 publications and patents, and over 150 conference papers. Prof. Kathirgamanathan is currently an Executive Committee Member of the Materials Chemistry Group, Society of Chemical Industries, London, U.K., and also served as the Chairman of the Group from 1999 to 2000. He is also a Com- mittee Member of the Materials Chemistry Division (RSC) since July 2013, and the Regional Vice President, Europe, Society for Information Displays, since May 2013. He was a recipient of prestigious Sir Monty Finniston Award REFERENCES Surendrakumar, “Red and green quantum dot based LEDs demon- strating excellent color coordinates,” in SID Conf. Proc., 2015. Lisa M. Bushby, photograph and biography not available at time of publication. Lisa M. Bushby, photograph and biography not available at time of publication. Muttulingam Kumaraverl, photograph and biography not available at time of publication. Seenivasagam Ravichandran, photograph and biography not available at time of publication. Prof. Kathirgamanathan is currently an Executive Committee Member of the Materials Chemistry Group, Society of Chemical Industries, London, U.K., and also served as the Chairman of the Group from 1999 to 2000. He is also a Com- mittee Member of the Materials Chemistry Division (RSC) since July 2013, and the Regional Vice President, Europe, Society for Information Displays, since May 2013. He was a recipient of prestigious Sir Monty Finniston Award Prof. Kathirgamanathan is currently an Executive Committee Member of the Materials Chemistry Group, Society of Chemical Industries, London, U.K., and also served as the Chairman of the Group from 1999 to 2000. He is also a Com- mittee Member of the Materials Chemistry Division (RSC) since July 2013, and the Regional Vice President, Europe, Society for Information Displays, since May 2013. He was a recipient of prestigious Sir Monty Finniston Award Sivagnanasundram Surendrakumar, photograph and biography not available at time of publication.
https://openalex.org/W2325344227
https://www.epj-conferences.org/10.1051/epjconf/201611402139/pdf
English
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Numerical experiment about the arrangement determination of multiple bluff cylinders which obtain the most suitable new type stir
EPJ web of conferences
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3,689
1 Introduction demerit in which carbon dioxide falls off from the liquid by the centrifugal force produced in a motion of the muddler will occur. The good method of raising the efficiency of stirring should just make a muddler reciprocating movement within the glass horizontally. However, the method is not efficiently mixed over the overall fluid in the glass. In the restaurant in Japan, various alcoholic beverages are offered like the countries of Europe. Beer, sake, white distilled liquor, wine, whiskey, etc. are prepared there. Consumption of plum wine with soda is increasing in such an alcoholic beverage. This is because plum wine with soda passes along a throat smoothly. In women or the younger age group, it is reported that the amount of consumption of plum wine with soda is increasing [1]. In response to this matter, alcoholic beverage brewing industry companies vied in production increase-in- quantity sale of a "soda cocktail" drink mutually, and planned it. In order to attain differentiation on sale, the improvement idea of packaging, addition of a sales promotion article, etc. were planned. Development of a new type "muddler" was desired as one of the plans of differentiation on sale. Here, although the "muddler" is "cocktail stir" in use, it is the popular name currently called in usually in the food-and-drink industry of Japan. The popular name was used in this study. Generally, the "muddler" of the stirring use of the cocktail drink is a long and slender bar configuration. The method for use agitates by making it circle horizontally in the liquid in a glass. Since the liquid in a glass also rotates synchronizing with a muddler, in the shape of an ordinary muddler, stirring efficiency is low. Therefore, in order to obtain enough stirring effect, it is necessary to continue rotating a muddler over a long time. In a "soda cocktail", if it continues rotating a muddler over a long time, the The author is observing that mix as the symmetrical vortex street discharged from two circular cylinders which are oscillating in the direction of a flow flows and goes down through study of a lock-in phenomenon until now, and rearrangement occurs as a vortex of another kind [2, 3]. By using the lock-in phenomenon and phenomenon of vortical rearrangement, the author conjectured what can promote liquid stirring. 1 Introduction Then, the author performed creation development of the muddler (type shaken longitudinal) of the new shape based on interference stirring by vortices. And the muddler was used as the product and sent out to the market [1]. This paper reports the details which are the numerical simulations performed in the early stage of the creation development. Numerical experiment about the arrangement determination of multiple bluff cylinders which obtain the most suitable new type stir Yoshifumi Yokoi1,2,a 1National Defense Academy, Department of Mechanical Engineering, 1-10-20 Hashirimizu, Yokosuka 239-8686, Japan 2Techniche Universität Berlin, Müller-Breslau-Str. 8, 10623 Berlin, Germany Yoshifumi Yokoi1,2,a Abstract. When developing the stirring rod for mixing two liquid with which the densities into which gas melted differ in little stirring operation, verification of the stirring effect was performed in the numerical simulation using a vortex method. Since it is calculation for specification examination of the preceding paragraph which creates a producing model, it becomes important to obtain a calculation result early and to feed back to the design. Two dimension calculation of the double file circular cylinder group which carried out staggered arrangement of the nine circular cylinders was performed. The aspects of the flow which strayed off from the design point or the design point were shown. The mixed degree was judged from the degree of the complexity of the flow pattern. In a design point, the complicated flow by a vortex is formed with oscillating 1 period, and interference stirring occurs. Adaptation ranges other than a design point are that the ranges of the value of P/L are 0.8-1.2. DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 201 / 0 1 (201 ) 201 epjconf EPJ Web of Conferences , 1 61 6 6 1 1 0 4 4 2 213 39 9 DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 201 / 0 1 (201 ) 201 epjconf EPJ Web of Conferences , 1 61 6 6 1 1 0 4 4 2 213 39 9 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits distribution, and reproduction in any medium, provided the original work is properly cited.    2.1 Test design of the muddler for numerical simulations Mixture of ume liquor and carbonated water was assumed with the cylinder glass with a capacity of 460 ml (the a Corresponding author: yokoi@nda.ac.jp a Corresponding author: yokoi@nda.ac.jp This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits distribution, and reproduction in any medium, provided the original work is properly cited.    EPJ Web of Conferences this thing, the design of the coil centre distance L and the design of a coil pitch P were performed. The value of P/d was set to 3.5 in order to secure the space which has been rolled before the circular cylinder by the side of downstream by the separating shear layer discharged from the circular cylinder by the side of the upstream. The value of space ratio P/L which is the ratio of a coil pitch P and the coil centre distance L was decided to be 1.0 (design point). As a result, it was decided that it would be P = L = 14 mm. The length of the coil section was made into the 4 times roll (4P = 56 mm) in consideration of the appearance as the muddler. The conditions of oscillation were decided as follows. Half oscillating amplitude a was set to 30 mm so that oscillating amplitude might become equivalent to the length of the coil section. Frequency f of oscillation was set to 1 Hz in consideration of the cardiac beats rate of the human body at the time of drinking. diameter of 70 mm, the depth of 120 mm) containing ice. In order to make it mix moderately in a little operation, a motion of the muddler makes it reciprocating movement in the perpendicular direction of the glass. In order to enlarge the contact surface product when moving in one operation in the inside of a liquid, the tip section of a stick made shape of a muddler the shape rolled spirally. It was considered as the shape of a coil. The shape is shown in figure 1. The circular cylinder which became this coil shape plans making a vortex, discharging and agitating surrounding fluid by the mutual interference of the discharged vortices. The muddler is inconvenient with the metal which rusts on a health concept target in order to touch a drink. It becomes a cost overrun although what is necessary is just to use stainless steel for the material. a Corresponding author: yokoi@nda.ac.jp The cast made of resin which can be mass-produced at a low price also from the field of manufacture expense was assumed. In consideration of intensity and productivity, the diameter d of a material round bar was determined as 4 mm. 2.2 The calculation method and apparatus As for a vortex shedding occurring independently mutually, it is known for parallel two circular cylinder that the ratio (G/d = (L - d)/d) of the circular cylinder interval G and the circular cylinder diameter d is 1.0 or more. In tandem two circular cylinders, it is known that the separating shear layer discharged from the upstream side circular cylinder at the time of about P/d (ratio of circular cylinder center interval P and circular cylinder The numerical simulation was performed by commercial fluid analysis tools and the commercial note type computer. The fluid analysis tools of the marketing used "Uzu Cruise 2D Ver.1.1.3 Rev.H" by College Master Hands, Inc. The numerical computation technique of the used fluid analysis tools is a vortex method which introduced the surface of wall vortex element method which is a type the vortex method application level 3 which used together the boundary element method and the vortex method developed. The details as the calculation technique of the vortex method are written to reference [5] and reference [6]. The computer used the commercial note type personal computer (NEC LaVie LL850/L). Figure 1. Muddler tip section shape 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 P/L 1.0 0.9 0.8 0.7 0.6 0.5 2.0 1.6 1.2 1.4 L/d P/d Independent vortex shedding in parallel case Independent vortex shedding in tandem case 0.4 0.3 0.2 Figure 2. The diagram of the calculation points diameter d) = 2.5 will carry out a reattachment to the downstream side circular cylinder [4]. In consideration of which is a type the vortex method application level 3 which used together the boundary element method and the vortex method developed. The details as the calculation technique of the vortex method are written to reference [5] and reference [6]. The computer used the commercial note type personal computer (NEC LaVie LL850/L). Moving direction Figure 1. Muddler tip section shape Moving direction Figure 1. Muddler tip section shape Movin directio Moving direction 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 P/L 1.0 0.9 0.8 0.7 0.6 0.5 2.0 1.6 1.2 1.4 L/d P/d Independent vortex shedding in parallel case Independent vortex shedding in tandem case 0.4 0.3 0.2 Figure 2. 3.1 Aspect of the flow in a design point The aspect of the flow for every time progress in a design point (P/L = 1.0, L/d = 3.5, P/d = 3.5) is shown in figure 3. Here, "Origin" is in the state of the calculation start. "T.D.C." and "B.D.C." mean the top dead center of oscillation, and the bottom dead center of oscillation, respectively. The "cycle" expresses one period and the numeric character in a parenthesis shows the number of calculation steps. Since preservation of calculated data was performed every ten steps, the flow pattern of each period, the top dead center, and the bottom dead center used the thing of the step near it. It is imagined that mixture of flow is promoted at the time of the turn to the bottom dead center from the top dead center and the turn to the top dead center from the bottom dead center. When it sees through oscillating one period, it turns out that the complicated flow by vortices was formed and interference stirring has occurred. Furthermore, the flow increased complexity by continuing oscillation. After In such the state, in order to investigate the applicability of space ratio (P/L), the value of space ratio (P/L) was varied and the simulation was performed. The value of space ratio (P/L) was changed with 0.25, 0.5, 0.8, 1.0, 1.2, and 2.0. The total number of times of repetition calculation is 1000 times, and this corresponds by 2.5 times the oscillating period. Figure 2 shows a design Figure 3. The time history of the flow pattern in a design point P/L = 1.0, L/d = 3.5, P/d = 3.5 (a) Origin (0) (i) 2 cycle (750) (b) T.D.C. (80) (c) 0.5 cycle (170) (d) B.D.C. (270) (e) 1 cycle (360) (f) T.D.C. (460) (g) 1.5 cycle (560) (h) B.D.C. (650) (j) B.D.C. (850) (k) 2.5 cycle (950) (l) Final calculation (1000) (c) 0.5 cycle (170) (d) B.D.C. (270) (a) Origin (0) (b) T.D.C. (80) (d) B.D.C. (270) (c) 0.5 cycle (170) (a) Origin (0) (b) T.D.C. (80) (c) 0.5 cycle (170) (g) 1.5 cycle (560) (a) Origin (0) (e) 1 cycle (360) ( ) ( ) (f) T.D.C. (460) (h) B D C (650) (i) 2 cycle (750) (e) 1 cycle (360) (e) 1 cycle (360) (h) B.D.C. (650) (f) T.D.C. (460) (g) 1.5 cycle (560) (h) B.D.C. (650) (l) Final calculation (1000) (g) 1.5 cycle (560) (k) 2.5 cycle (950) (f) T.D.C. 2.3 Calculation conditions point and each calculating point in each space ratio with the aspect of the flow of parallel two circular cylinders, and the aspect of the flow of tandem two circular cylinders. An abscissa is the distance ratio in the case of parallel two circular cylinders, and an ordinate is the distance ratio in the case of tandem two circular cylinders. The straight lines in the figure mean the space ratio (P/L). This calculation is calculation for specification examination of the preceding paragraph which creates a producing model. So, it is important to obtain the calculation result early and to feed back to a design. In this study, two dimensional calculation of the circular cylinder group which treated the section including the main axis of the coil, then carried out staggered arrangement of the nine circular cylinders was performed. The mixed degree was judged from the degree of the complexity of the flow pattern. Two kinds of liquids with which density differs from specific gravity in fact are mixed. However, in order to verify the process of mixture by the vortex, it was considered as single fluid and the physical-properties value of water was used for calculation here. Here, the spatial restriction supposing a glass, ice, etc. is not provided. The state of early fluid is stationary. Half oscillating amplitude was set to a = 30 mm, and the oscillation frequency was set to f = 1 Hz. The Reynolds number (Re = umaxd/) based on the maximum movement speed of body (umax = 2 af) was about 660. If the Strouhal number was calculated using the body oscillation frequency and amplitude, the value of the Strouhal number was about 0.02. 2.2 The calculation method and apparatus The diagram of the calculation points 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 P/L 1.0 0.9 0.8 0.7 0.6 0.5 2.0 1.6 1.2 1.4 L/d P/d Independent vortex shedding in parallel case Independent vortex shedding in tandem case 0.4 0.3 0.2 Figure 2. The diagram of the calculation points Figure 1. Muddler tip section shape diameter d) = 2.5 will carry out a reattachment to the downstream side circular cylinder [4]. In consideration of diameter d) = 2.5 will carry out a reattachment to the downstream side circular cylinder [4]. In consideration of diameter d) = 2.5 will carry out a reattachment to the downstream side circular cylinder [4]. In consideration of diameter d) = 2.5 will carry out a reattachment to the downstream side circular cylinder [4]. In consideration of 02139-p.2 02139-p.2 EFM 2015 3.1 Aspect of the flow in a design point (460) (j) B.D.C. (850) (k) 2.5 cycle (950) (l) Final calculation (1000) (j) B.D.C. (850) (i) 2 cycle (750) Figure 3. The time history of the flow pattern in a design point P/L = 1.0, L/d = 3.5, P/d = 3.5 02139-p.3 EPJ Web of Conferences Figure 4. The time history of the flow pattern when straying off ±20% from a design point (P/L = 0.8 and 1.2) Origin T.D.C. 0.5 cycle B.D.C. 1 cycle P/L = 1.0, L/ d= 3.5, P/d = 3.5 P/L = 0.8, L/d = 3.5, P/d = 2.8 P/L = 0.8, L/d = 4.375, P/d = 3.5 P/L = 1.2, L/d = 2.917, P/d = 3.5 P/L = 1.2, L/d = 3.5, P/d = 4.2 Origin T.D.C. 0.5 cycle B.D.C. 1 cycle P/L = 1.0, L/ d= 3.5, P/d = 3.5 P/L = 1.0, L/ d= 3.5, P/d = 3.5 P/L = 0.8, L/d = 3.5, P/d = 2.8 P/L = 0.8, L/d = 4.375, P/d = 3.5 P/L = 1.2, L/d = 2.917, P/d = 3.5 Figure 4. The time history of the flow pattern when straying off ±20% from a design point (P/L = 0.8 and 1.2) e history of the flow pattern when straying off ±20% from a design point (P/L = 0.8 and 1.2) independent of each circular cylinder are discharged. Figure 4 shows the flow patterns of the conditions used as P/L = 0.8 and P/L = 1.2 which strayed off from the space ratio in a design point ±20 %. On every condition, the complicated flow by a vortex was formed with oscillating one period, and interference stirring has occurred, and it can grasp so that it may scarcely change to the aspect of the flow in a design point. On the other hand, figure 5 shows the flow pattern in the case of the space ratio which strayed off from the design point further. When a result is seen, it turns out that enough on any conditions stirring is not obtained. These matters show that the ranges of P/L of space ratio suitable for stirring are 0.8- 1.2. Here, when it is on the line of the same space ratio, the one where the value of L/d or P/d is smaller is considered to be a desirable thing on the size of the product. passing over oscillating two periods, mixture by large- scale vortices can be seen. 3.1 Aspect of the flow in a design point This thing suggests that "good stirring" is obtained by little movement. EFM 2015 EFM 2015 Figure 5. The time history of the flow pattern when straying off further from a design point (P/L = 0.25, 0.5 and 2.0) Origin T.D.C. 0.5 cycle B.D.C. 1 cycle P/L = 1.0, L/d = 3.5, P/d = 3.5 P/L = 0.25, L/d = 7.0, P/d = 1.75 P/L = 0.5, L/d = 3.5, P/d = 1.75 P/L = 0.5, L/d = 7.0, P/d = 3.5 P/L = 2.0, L/d = 3.5, P/d = 7.0 Origin T.D.C. 0.5 cycle B.D.C. 1 cycle P/L = 1.0, L/d = 3.5, P/d = 3.5 T.D.C. P/L = 1.0, L/d = 3.5, P/d = 3.5 P/L = 0.25, L/d = 7.0, P/d = 1.75 P/L = 0.5, L/d = 3.5, P/d = 1.75 P/L = 0.5, L/d = 7.0, P/d = 3.5 P/L = 2.0, L/d = 3.5, P/d = 7.0 P/L = 2.0, L/d = 3.5, P/d = 7.0 Figure 5. The time history of the flow pattern when straying off further from a design point (P/L = 0.25, 0.5 and 2.0 3.2 Examination of the space ratio suitable for stirring The aspects of the flow of space ratios other than a design point are shown in figure 4 and figure 5. The aspect of the flow of the design point is also displayed on both figures for comparison. In the design point, the complicated developed flow is formed oscillating one period. Therefore, the flow patterns of the "initial state", the "top dead center", the "half cycle", the "bottom dead center", and the "one period" are shown here, respectively. The conditions currently colored by yellow all over the figure show the case where the vortices which became 02139-p.4 References 1. T. Monna, Y. Yokoi, Proc. 50th all Japan packing technical research convention, 265-268 (2012) The "Kanpai-muddler" was the schedule of 20,000 piece creation in the first plan. However, since it was reputable, additional creation of the " Kanpai-muddler " was performed, and 80,000 totals were created. 2. Y. Yokoi, K. Hirao, Trans. Jpn Soc. Mech. Eng. 74- 748, 2466-2475 (2008) 3. Y. Yokoi, K. Hirao, Trans. Jpn Soc. Mech. Eng. 77- 775, 590-601 (2011) Figure 8. The surface of the covering letter attached to the muddler e covering letter attached to the 4. JSME ed., JSME Standard S012, B78(1998) 5. K. Kamemoto, Computational Fluid Dynamics, 2-1, 20-29 (1993) 6. K. Kamemoto, Computational Fluid Dynamics, 2-2, 28-39 (1994) Acknowledgement In order to develop the stirring rod for mixing two liquid with which densities called sodium carbonate and ume liquor differ in little stirring operation, verification of the stirring effect was performed in the numerical simulation using the vortex method. The following conclusions were obtained. An author expresses gratitude to Sapporo Breweries, Ltd., Daiko Advertising, Inc., and Avalanche, Inc. The opportunity to which an author carries out research from the staff of those companies was obtained. And they sent out the product borne by this research to the market. An author expresses deep gratitude to the staff members anew. (1) In a design point, a complicated flow is made from oscillation of the one period by vortices. And interference stirring occurs. (2) Adaptation ranges other than the design point are that the ranges of the value of P/L are 0.8-1.2. (3) The calculation using the vortex method is the simple technique whose product development is possible while making it feed back to the design. 02139-p.5 EPJ Web of Conferences is written. Here, the busy condition of the muddler was also shown. Appendix The data of "the new type muddler" which the author developed are attached. Figure 6 shows the blueprint and its finished goods. The material of parts is plastic resin. This article is the goods developed for advertisement sales promotion of ume liquor. Figure 7 shows the muddler attached to the package of ume liquor. The sale was performed by such packaging. Figure 8 shows the covering letter attached to the muddler. Since the campaign was performed in Japan, it is written in Japanese. The muddler is shown in the beer jug which a model woman (right-hand side) has in a hand. The author (left-hand side) is also introduced as a designer. Since "the cheers" was called "Kanpai" in Japan, this muddler was named the "Kanpai-muddler." Figure 9 is the back of the covering letter, and the method for use of the muddler Figure 6. The blueprint and finished goods of the muddler Figure 8. The surface of the covering letter attached to the muddler Figure 9. The back of the covering letter attached to the muddler and the busy condition of the muddler Figure 6. The blueprint and finished goods of the muddler Figure 7. Packaging of goods Figure 9. The back of the covering letter attached to the muddler and the busy condition of the muddler Figure 7. Packaging of goods Figure 7. Packaging of goods 02139-p.6
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https://europepmc.org/articles/pmc6433726?pdf=render
English
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Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β‐cells
Journal of Cellular and Molecular Medicine
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Correspondence Correspondence Dusica Maysinger, Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada. Email: dusica.maysinger@mcgill.ca Funding information Canadian Diabetes Society; Canadian Institutes of Health Research; McGill University K E Y W O R D S autophagy, islets, LAMP2, PLIN2, TFEB Received: 27 July 2018  |  Revised: 14 December 2018  |  Accepted: 29 December 2018 DOI: 10.1111/jcmm.14172 Received: 27 July 2018  |  Revised: 14 December 2018  |  Accepted: 29 December 2018 DOI: 10.1111/jcmm.14172 Received: 27 July 2018  |  Revised: 14 December 2018  |  Accepted: 29 December 2018 DOI: 10.1111/jcmm.14172 Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β‐cells Jeff Ji1 | Maria Petropavlovskaia2 | Armen Khatchadourian1 | Jason Patapas2 |  Julia Makhlin2 | Lawrence Rosenberg2 | Dusica Maysinger1 Jeff Ji1 | Maria Petropavlovskaia2 | Armen Khatchadourian1 | Jason Patapas2 |  Julia Makhlin2 | Lawrence Rosenberg2 | Dusica Maysinger1 1Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada 1Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada Abstract Abstract Both type 2 diabetes (T2D) and obesity are characterized by excessive hyperlipidae‐ mia and subsequent lipid droplet (LD) accumulation in adipose tissue. To investigate whether LDs also accumulate in β‐cells of T2D patients, we assessed the expression of PLIN2, a LD‐associated protein, in non‐diabetic (ND) and T2D pancreata. We ob‐ served an up‐regulation of PLIN2 mRNA and protein in β‐cells of T2D patients, along with significant changes in the expression of lipid metabolism, apoptosis and oxidative stress genes. The increased LD buildup in T2D β‐cells was accompanied by inhibition of nuclear translocation of TFEB, a master regulator of autophagy and by down‐regu‐ lation of lysosomal biomarker LAMP2. To investigate whether LD accumulation and autophagy were influenced by diabetic conditions, we used rat INS‐1 cells to model the effects of hyperglycaemia and hyperlipidaemia on autophagy and metabolic gene expression. Consistent with human tissue, both LD formation and PLIN2 expression were enhanced in INS‐1 cells under hyperglycaemia, whereas TFEB activation and autophagy gene expression were significantly reduced. Collectively, these results sug‐ gest that lipid clearance and overall homeostasis is markedly disrupted in β‐cells under hyperglycaemic conditions and interventions ameliorating lipid clearance could be beneficial in reducing functional impairments in islets caused by glucolipotoxicity. 2Department of Surgery, McGill University, Montreal, QC, Canada 2.5 | qRT‐PCR For RNA extraction, human pancreatic samples stored at −80°C in RNAlater were homogenized in RLT buffer and processed in QiacubeTM (Qiagen, Toronto, ON, Canada) using RNEasy mini kit according to manufacturer protocol. Quality and integrity of RNA was assessed by 1.5% agarose gel electrophoresis. For RNA ex‐ traction in cultured cells, INS‐1 were seeded at 2 000 000 cells in 150 mm plates (Sigma) and 48 hours after seeding were exposed to 5 mmol/L or 30 mmol/L [GLU] with or without 500 μmol/L OA, PA or 250 μmol/L OA + 250 μmol/L PA for 24 hours. Cells were lysed in RLT buffer and processed as above. Equal amounts of RNA, based on OD260, were reverse transcribed using oligo‐dT primers and Omniscript RT kit (Qiagen). One microlitre of cDNA was used for a 20 μL qPCR reaction performed with IQTM SYBR® Green Supermix (Bio‐Rad, Mississauga, ON, Canada) in CFX96TM Real‐Time System 2.3 | Stable EGFP‐TFEB transfection of INS‐1 cells INS‐1 cells were seeded in 6‐well plates (Starstedt, Montreal, QC, Canada) and transfected with pEGFP‐N1‐TFEB (CMV promoter, neomycin resistance) using Lipofectamine 2000 (FischerScientific) in culture medium for 48 hours. The medium was supplemented with 400 μg/mL geneticin (Sigma, Oakville, ON, Canada) to select for resistant cells and subsequently for single colonies by reseeding into 96‐well plates. EGFP‐positive clones showing functional TFEB translocation when starved in HBSS for 1 hour at 37°C were cul‐ tured with 200 μg/mL geneticin in the medium. Targeted inactivation or repression of autophagy in β‐cells has been shown to cause diabetes in animal models. β‐cell–specific Atg7 knockout led to islet degeneration in mice, accumulation of protein aggregates and decreased insulin production.20 Similarly, β‐cell–spe‐ cific Tsc‐2 knockout, which caused mTORC1 hyperactivation and repression of autophagy, increased mitochondrial oxidation and ER stress, resulting in β‐cell failure.21 mTORC1 is a central kinase re‐ sponsible for regulating many aspects of metabolism, energy utili‐ zation and cell growth in response to nutrient abundance within the cell. A direct effect of mTORC1 activity on LD formation in rat islet cells has been previously reported.22 mTORC1 inhibits autophagy partly through phosphorylation of transcription factor EB (TFEB) which prevents its nuclear translocation. During starvation, mTORC1 is suppressed and TFEB translocates to the nucleus and up‐regulates genes involved in autophagic and lysosomal production.23 TFEB is necessary for lipid degradation in the liver24 but its role in human pancreatic islets in the context of T2D has not been reported. 2.2 | Cell culture INS‐1 rat insulinoma cells (AddexBio, San Diego, CA, USA) were cul‐ tured in RPMI‐1640 media containing 11.1 mmol/L [GLU], 2 mmol/L L‐glutamine, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 2 g/L sodium bicarbonate, 10% FBS, 50 μmol/L 2‐mercaptoethanol, 1% penicillin‐streptomycin (Invitrogen, Waltham, MA, USA) and main‐ tained at 37°C with 5% CO2. 2.4 | FA/BSA complex preparation Oleic acid (OA) (Sigma) and palmitic acid (PA) (Sigma) were dis‐ solved in Krebs‐Ringer bicarbonate buffer complexed with 5% fatty‐acid free BSA (Sigma) under gentle heating and stirring and sterile‐filtered through a 0.22 μm filter. FA concentration was quan‐ tified using Wako HR series NEFA‐HR(2) according to manufacturer instructions. The goal of this study was to investigate the impact of T2D on LDs, autophagy and islet metabolism by assessing the expression and localization of PLIN2, TFEB, lysosome‐associated membrane protein‐2 (LAMP2) and genes associated with metabolism, oxida‐ tive stress, apoptosis and mitochondrial function in human pancre‐ atic tissue from normal and T2D subjects. We have suggested that nutrient overload in diabetes causes LD accumulation due to de‐ creased TFEB activation and suppression of autophagy and tested this hypothesis in vitro, using the rat insulinoma β‐cell line INS‐1. and as a platform for the recruitment of signalling molecules.6,7 Perilipin associated proteins (PAT) coat and stabilize the surface of LD by reducing lipases access to the lipid core. Perilipin‐2 (PLIN2) is the main housekeeping LD protein that is ubiquitously expressed and used as a marker for LDs in many human and animal tissues.8 PLIN5 is highly expressed in oxidative tissues and plays multiple metabolic roles such as fatty acid (FA) mobilization and has been shown to regulate post‐prandial insulin secretion in β‐cells.9,10 High cellular lipid uptake (eg, post‐prandial period) increases PLIN2 (and other PAT proteins) expression and triggers their recruitment to the LD surface.11 These events temporarily reduce the cytosolic FA concentration. However, chronic lipid overload can lead to the for‐ mation of abnormally large and unstable LDs prone to FA spillover, which could lead to endoplasmic reticulum (ER) stress, induction of apoptosis and insulin resistance in β‐cells.12,13 The timely removal of excessively ‘leaky’ LDs is therefore necessary to prevent lipotoxicity in β‐cells. Aside from cytosolic lipase activity, lipophagy is emerging as an important mechanism for LD degradation using the autophagic pathway14,15 and is suggested as a general mechanism for degrading LD in diverse cell populations.16-19 RNAlaterTM (Qiagen, Toronto, ON, Canada) for RNA extraction or fixed in 10% formalin (Fisher Scientific, Ottawa, ON, Canada) and paraffin‐embedded for immunolabelling (Pathology Unit, Montreal General Hospital, Montreal, Quebec, Canada). Donor information is summarized in Table S1. The study consisted of 22 ND and 17 type 2 diabetic patients. 1 | INTRODUCTION that excessive lipids and the ensuing lipotoxicity play a major role in these processes by promoting insulin resistance and β‐cell dysfunc‐ tion.3 However, the exact impact of lipid overload on β‐cell dysfunc‐ tion remains poorly understood. T2D is characterized by β‐cell failure, insulin resistance, hypergly‐ caemia, and hyperlipidaemia. β‐cells are nutrient sensors that reg‐ ulate insulin secretion in response to elevated levels of glucose and lipids.1 Nutrient overload is viewed as the main cause of insulin resis‐ tance that increases the demand on β‐cells to secrete more insulin. If β‐cells fail to adapt to insulin resistance and become dysfunctional, glucose intolerance and overt diabetes develops.2 Evidence suggests LDs are dynamic organelles implicated in metabolic disorders including obesity and T2D, which are marked by abnormal LD ac‐ cumulation.4,5 Structurally, LDs are natural micelles composed of a hydrophobic core containing triglycerides, cholesterol esters and a corona made of phospholipids that can serve as lipid storage sites J Cell Mol Med. 2019;23:2890–2900. J Cell Mol Med. 2019;23:2890–2900. JI et al. 2891 2.11 Cells were seeded into 24‐well plates (Starstedt) at 150 000 cells per well for 24 hours treatment or 75 000 cells per well for 48 hours treatment, 24 hours post‐seeding, cells were incubated in treatment media for 24 hours/48 hours. Cell count analysis: Cells were incu‐ bated with 10 μmol/L Hoechst 33342 for 10 minutes followed by imaging using a fluorescence microscope with a 4X objective. Four images per well were taken to represent the average number of cell in the well. The number of nuclei per field were counted using ImageJ. MTT assay 500 ug/mL MTT (Sigma) was added to cells and incubated for 40 minutes at 37°C. After incubation, the media were aspirated and DMSO was added to each well to dissolve formazan crystals. The absorbance was read at 595 nm using a plate reader. 2.12 | Microscopy Confocal imaging was performed with Zeiss LSM 510 NLO inverted confocal microscope using a Plan Achromat 63X/1.4 Oil DIC objec‐ tive (Carl Zeiss Canada Ltd., Toronto, ON, Canada). All images were acquired at a resolution of 1024 × 1024 pixels (x,y). Fluorescence mi‐ croscopy was performed with a Leica DMI4000 inverted microscope (Leica Microsystems Inc, Concord, ON, Canada) using a 4×, 10× or 63× oil objective captured using a Leica DFC345 FX camera (Leica Microsystems Inc). 2.7 | Lipid droplet staining and quantification INS‐1 cells were seeded onto poly‐d‐lysine coated coverslips and in‐ cubated for 48 hours before treatment with 500 μmol/L OA in nor‐ moglycaemic (5 mmol/L [GLU]) or hyperglycaemic (30 mmol/L [GLU]) medium for 24 hours. Torin‐1 (Tocris, Bristol, UK) was added 3 hours before and with the 24 hours treatment. Cells were fixed in 2% PFA in PBS+ (with 0.5 mmol/L MgCl2, 1 mmol/L CaCl2) for 10 minutes at room temperature (RT). After washing with PBS+ cells were labelled with 20 μmol/L BODIPY 493/503 and 10 μmol/L Hoechst 33342 for 10 minutes at RT and washed with PBS+. Coverslips were mounted onto microscope slides with non‐hardening EverBrite Mounting me‐ dium (Biotium, Fremont, CA, USA) and the edges sealed with nail polish. 2.10 After treatment, cells were fixed in 2% PFA in PBS+ (with 0.5 mmol/L MgCl2, 1 mmol/L CaCl2) for 10 minutes at RT and permeabilized with 0.1% Triton X‐100 in PBS+. Cells were blocked with 10% goat serum and incubated in primary antibody overnight at 4°C and incubated in secondary antibody for 1 hour at RT. Coverslips were mounted onto microscope slides using Aqua Poly/Mount. 2.1 | Human pancreatic tissue Adult human pancreata were obtained from Quebec Transplant with prior consent for research use. Pancreatic tails were preserved in JI et al. JI et al. 2892 2.6 Tissue sections (5 μm) of pancreatic samples were deparaffinized in xylene and rehydrated in ethanol‐water mixtures. Antigen retrieval was performed in 95°C citrate buffer (10 mmol/L, 0.05% Tween‐20, pH 6.0) for 40 minutes and was followed by blocking in 2% horse serum/10% goat serum/1 mmol/L HEPES/0.1% sodium azide/0.3% Triton X‐100 in HBSS for 2 hours. Incubation with primary an‐ tibodies (Table S3) at 4°C overnight was followed by secondary antibodies, staining of nuclei in Hoechst 33342 or DAPI and mount‐ ing using Aqua‐Poly/Mount (Polysciences, Warrington, PA, USA). Quantification was performed in ImageJ by assessing the average fluorescence intensity normalized to the area and expressed as ar‐ bitrary units. 2.8 | TFEB translocation test INS‐1‐TFEB‐EGFP cells seeded in 24‐well plates (Starstedt) were treated in 5 mmol/L or 30 mmol/L [GLU] in the presence or ab‐ sence of 500 μmol/L OA, PA or 250 μmol/L OA + 250 μmol/L PA for 48 hours. After treatment, cells were starved for 1 hour in HBSS, fixed in 2% PFA for 10 minutes at RT and washed twice with PBS+. After labelling nuclei with 10 μmol/L Hoechst 33342, cells were imaged by fluorescence microscopy and the images were analysed using ImageJ. Four fields were randomly chosen per well and EGFP‐ positive cells were randomly chosen per field. TFEB‐EGFP nuclear translocation was assessed as the ratio of the area of normalized EGFP fluorescence intensity in the nucleus over the cytosol. A higher ratio indicates greater nuclear TFEB‐EGFP translocation. 2.9 | Western blot After treatment, total cellular protein was extracted using RIPA buffer containing protease inhibitors. Protein concentration was quantified using the Pierce BCA Protein Assay (ThermoFisher, Waltham, MA, USA). Proteins were subjected to SDS‐PAGE and transferred onto PVDF membrane. Membranes were blocked with 5% milk/TBST and incubated with primary antibodies followed by horseradish peroxidase‐conjugated secondary antibodies. Blots were developed using ClarityTM Western ECL Substrate (BIO‐RAD, Hercules, CA, USA) and imaged using Amersham Imager 600 (GE Healthcare, Saint‐Laurent, QC, Canada). (Bio‐Rad) and primer pairs shown in Table S2. Multiple plates of ex‐ perimental data, run with an inter‐plate calibrator, were combined into gene studies using glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), β‐actin and succinate dehydrogenase complex flavopro‐ tein (SDHA) as reference genes in human samples and β‐actin and β‐tubulin in INS‐1 samples (all M < 0.5). Fold change in gene expres‐ sion (∆∆Ct) were obtained using the data analysis software CFX Manager3.1. 2.13 Statistical significance was considered at P < 0.05. Statistical analysis was done using R v3.2.2. 2.13 All data are expressed as means ± standard error of the mean (SEM). Statistical differences were analysed by either student's t test or two‐way analysis of variance (ANOVA) followed by post‐hoc Tukey's JI et al. JI et al. 2893 at P < 0.05. Statistical y increased in (Figure 1C). This abnormal accumulation of PLIN2 and PLIN5 and, by extension, of LDs in β‐cells is similar to that in hepatocytes in fatty liver disease or macrophages in atherosclerosis, where it is usually linked to the abnormal lipid catabolism.27 This prompted us to investigate whether the observed LD accumulation in T2D is accompanied and perhaps caused by an impaired processing of lipids in β‐cells. FI G U R E 1 Expression of Lipid Droplet markers in the human pancreas. (A) Gene expression analysis of lipid droplet regulation genes from pancreatic tails (CAV1 = caveolin 1, PLIN2 = perilipin 2, FSP27 = fat specific protein 27). ND = 11 patients, T2D = 10 patients, graph represents average fold change ± standard error of the mean (SEM) in gene expression over ND. (B) Pictomicrograph of sections double immunolabelled with PLIN2 (red), C‐peptide (green) and stained with Hoechst 33342 (blue). Images are representative from four different donors per group. White arrows point to acinar tissue (exocrine tissue) which did not up‐ regulate PLIN2. (C) Photomicrograph and fluorescence quantification of sections labelled with PLIN5 (red), C‐peptide (green) and stained with Hoechst 33342 (blue). Quantified from >21 islets from two different donors per group. Statistical analysis was evaluated by Student's t test and significance is indicated by * (compared to ND), *P < 0.05 (Figure 1C). This abnormal accumulation of PLIN2 and PLIN5 and, by extension, of LDs in β‐cells is similar to that in hepatocytes in fatty liver disease or macrophages in atherosclerosis, where it is usually linked to the abnormal lipid catabolism.27 This prompted us to investigate whether the observed LD accumulation in T2D is accompanied and perhaps caused by an impaired processing of lipids in β‐cells. (Figure 1C). This abnormal accumulation of PLIN2 and PLIN5 and, by extension, of LDs in β‐cells is similar to that in hepatocytes in fatty liver disease or macrophages in atherosclerosis, where it is usually linked to the abnormal lipid catabolism.27 This prompted us to investigate whether the observed LD accumulation in T2D is accompanied and perhaps caused by an impaired processing of lipids in β‐cells. test. 3.2 | TFEB activation is suppressed and LAMP2 abundance is reduced in T2D (A) Representative images of TFEB immunolabelling in ND, and T2D patients and quantification of TFEB nuclear fluorescence of cells within islets. Islets regions (encircled) were traced from bright‐field images based on morphology and nuclei were stained with Hoechst 33342. Quantifications are from ND = 8 patients (471 cells measured), T2D = 6 patients (416 cells measured). (B) Representative images of LAMP2 immunolabelling in ND and T2D patients and quantification of LAMP2 fluorescence within the islet. Quantifications are from ND = 6 patients (48 islets measured), T2D = 6 patients (61 islets measured). Statistical analysis was evaluated by Student's t test and significance is indicated by * (compared to ND), *P < 0.05, error bars = SEM receptor‐alpha (PPAR‐α, 2.1 ± 0.3 ↑, P < 0.01) (Figure 3E), while PPAR‐γ expression did not differ between T2D and ND. Together, these changes in mRNA level in mitochondria, anti‐ROS defense and apoptosis‐associated genes in the pancreata of T2D subjects, sug‐ gest the activation of compensatory mechanisms to withstand meta‐ bolic stress and that the effects of diabetes were likely still within the adaptable range and have not reached the stage of irrevocable damage. To assess the morphology and architecture of the islets, we immunostained for glucagon (α‐cells), insulin (β‐cells) and somato‐ statin (δ‐cells) using sections from several patients in both ND and T2D groups. Both ND and T2D islets contained similar distribution of α‐, β‐ and δ‐cells (Figure S2), suggesting that the altered metabolic profiles in T2D subjects were not caused by gross morphological changes. 3.3 | Expression of islet metabolism, redox and apoptosis related genes is altered in T2D Since T2D has a major impact on islet metabolism, we examined the expression of pancreatic genes regulating mitochondrial func‐ tion, apoptosis and oxidative stress, as illustrated in Figure 3. Among mitochondrial genes, there was a significant up‐regulation in the expression of Carnitine palmitoyltransferase 1A (CPT1A), a crucial FA transporter on the surface of mitochondria (3 ± 0.5 ↑, P < 0.01, Figure 3B). This suggests an increase in β‐oxidation to combat the increased lipid load. Elevated β‐oxidation would increase the trans‐ membrane potential of the mitochondria. Accordingly, Uncoupling protein 2 (UCP2) was also up‐regulated in T2D (1.7 ± 0.2 ↑, P < 0.05) suggesting an increase in non‐ATP coupled proton leak (Figure 3B). 3.2 | TFEB activation is suppressed and LAMP2 abundance is reduced in T2D We assessed whether T2D leads to changes in LD‐associated pro‐ teins: PLIN2, caveolin (CAV1) and fat specific protein 27 (FSP27) by qRT‐PCR (Figure 1A). PLIN2 mRNA was significantly up‐regulated in T2D (2.5 ± 0.3 ↑ fold increase, P < 0.001) (Figure 1A). However, there was no significant difference in gene expression of other LD‐ associated proteins – CAV1, which plays an important role in cho‐ lesterol transport, lipogenesis and LD biogenesis,25 or FSP27, which promotes LD growth by lipid exchange and fusion.26 We evaluated the homeostatic clearance of lipids by autophagy by focusing on the activation of TFEB. TFEB nuclear transloca‐ tion gauged by immunofluorescence was significantly decreased in islets from T2D donors compared to ND (3 ± 0.01 ↓, P < 0.001) (Figure 2A), which suggested down‐regulation of autophagic genes. Since TFEB controls lysosome‐related genes, we quantified LAMP2 protein, a structural marker for lysosomes and found that it was sig‐ nificantly decreased in T2D compared to ND (3.3 ± 0.2 ↓, P < 0.001, Figure 2B). These data support our hypothesis that T2D‐associated reduction in islet TFEB and subsequently in lysosomal activity led to suppression of autophagy. Immunostaining of pancreatic sections for the LD‐associated pro‐ teins showed a marked increase in PLIN2 protein in T2D islets, and demonstrated its predominant localization in β‐cells based on co‐stain‐ ing with C‐peptide (Figure 1B), whereas it was undetectable in the acinar tissue. PLIN5 was also up‐regulated in β‐cells from T2D donors JI et al. JI et al. 2894 2894  |     JI et al. FI G U R E 2 Autophagy markers in human islets. (A) Representative images of TFEB immunolabelling in ND, and T2D patients and quantification of TFEB nuclear fluorescence of cells within islets. Islets regions (encircled) were traced from bright‐field images based on morphology and nuclei were stained with Hoechst 33342. Quantifications are from ND = 8 patients (471 cells measured), T2D = 6 patients (416 cells measured). (B) Representative images of LAMP2 immunolabelling in ND and T2D patients and quantification of LAMP2 fluorescence within the islet. Quantifications are from ND = 6 patients (48 islets measured), T2D = 6 patients (61 islets measured). Statistical analysis was evaluated by Student's t test and significance is indicated by * (compared to ND), *P < 0.05, error bars = SEM 2894  | FI G U R E 2 Autophagy markers in human islets. 3.2 | TFEB activation is suppressed and LAMP2 abundance is reduced in T2D UCP2 up‐regulation was previously reported in response to oxi‐ dative stress in β‐cells.28 We next evaluated expression of several genes related to anti‐oxidant defence and apoptosis and found sig‐ nificant up‐regulation of glutathione peroxidase 1 (GPX1, 3.8 ± 0.3 ↑, P < 0.001) and heme oxygenase 1 (HMOX1, 2.2 ± 0.3 ↑, P < 0.01) in T2D pancreata (Figure 3C), which correlated with an increased immunofluorescence for Nrf2 (Figure S1), a transcription factor regulating GPX1, HMOX1 and other genes involved in oxidant de‐ fense and redox signalling.29 One of these genes, p62 (SQSTM1), is directly involved in autophagy and is used as a measure of au‐ tophagy flux,30 was also up‐regulated in T2D (Figure 3). Expression of anti‐apoptotic gene BCL2 was strongly increased (11.3 ± 4.6 ↑, P < 0.05) (Figure 3D), however pro‐apoptotic gene BAX was also up‐ regulated (2.3 ± 0.3 ↑, P < 0.001). Finally, we examined transcription factors linked to metabolism. T2D leads to significant changes in the expression of transcription factors forkhead box protein 1 (FOXO1, 1.7 ± 0.3 ↑, P < 0.05), and peroxisome proliferator‐activated 3.4 | Hyperglycaemia increases LD and Plin2 in INS‐1 CPT1A = carnitine palmitoyltransferase 1A couples carnitine to acyl groups which enables fatty acids to enter the mitochondria and undergo β‐oxidation. UCP2 = uncoupling protein 2 enables protons to flow back into the mitochondria matrix without oxidative phosphorylation which generates heat instead of ATP, p62 = sequestome 1, delivers cargo to autophagosomes for degradation, regulates Nrf2 activity by binding with Keap1. HMOX1 = heme oxygenase 1, converts denatured heme, oxygen and NADPH to Billiverdin, carbon monoxide and iron. It is an anti‐oxidant gene up‐regulated by oxidative stress. GPX1 = glutathione peroxidase 1 is an anti‐oxidant enzyme which scavenges hydrogen peroxide. BAX = BCL2 associated X, BCL2 = BCL2 are respectively pro‐apoptotic and anti‐apoptotic regulators. FOXO1 = forkhead box protein 1, PPAR‐α = peroxisome proliferator activated receptor‐α, PPAR‐γ = peroxisome proliferator activated receptor‐γ are transcription factors which regulates many facets of metabolism. Gene expression analysis of (B) mitochondria, (C) oxidative stress/oxidant defense, (D) apoptosis‐related, (E) metabolism regulation related genes from pancreatic tails. Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups over ND. N = 11 patients for ND, N = 10 patients for T2D. Statistical analysis was evaluated by Student's t test and significance is indicated by * (compared to ND), *P < 0.05 translational level and that Plin2 alone is not sufficient for LD for‐ mation and growth. (Figure S3C,D). INS‐1 cells treated with OA under hyperglycae‐ mia showed a considerable increase in the number of LD (five‐ fold increase in LD area compared to normoglycaemia, P < 0.01, Figure 4A,B). Furthermore, two‐way ANOVA analysis revealed sig‐ nificant synergistic effect (P < 0.05) between hyperglycaemia and OA treatment on LD area per cell. The increased LD storage due to hyperglycaemia was partially abolished by pre‐treatment with the mTOR inhibitor/autophagy inducer Torin‐1, which suggests that activation of autophagy could reduce lipid accumulation. In addition, we show that hyperglycaemia alone was the main factor leading to significant up‐regulation of Plin2 (P < 0.001, Figure 4C), while FA alone (normoglycaemic conditions) only slightly elevated Plin2 mRNA. Interestingly, FA treatment under hyperglycaemia lowered the effect of hyperglycaemia on Plin2 expression. This discrepancy between Plin2 mRNA and the LD number under FA+30 mM [GLU] may indicate that Plin2 is also regulated at the 3.4 | Hyperglycaemia increases LD and Plin2 in INS‐1 Although pancreatic dysfunction in T2D and obesity is multifac‐ torial and involves a host of metabolic and hormonal changes, two major contributors are hyperglycaemia and hyperlipidaemia. To better understand the observed changes in LDs and in gene expression in T2D pancreata, we examined the direct effects of hyperglycaemia and hyperlipidaemia on LD and autophagy in vitro using INS‐1 cells. 5 mmol/L glucose was taken as normoglycaemia and 30 mmol/L glucose as hyperglycaemia. Hyperlipidaemia was induced by 500 μmol/L OA, 500 μmol/L PA or 250 μmol/L oleic acid +250 μmol/L palmitic acid (OA+PA), as used by others.22,31 This was the maximal dose which did not significantly reduce cell number or induce apoptosis (Figure S3A,B,E) yet it did increase mi‐ tochondrial metabolic activity relative to 5 mmol/L [GLU] control 2895 JI et al. FI G U R E 3 Metabolism, redox and apoptosis related gene expression in human pancreata. (A) Schematic of the genes assayed below. CPT1A = carnitine palmitoyltransferase 1A couples carnitine to acyl groups which enables fatty acids to enter the mitochondria and undergo β‐oxidation. UCP2 = uncoupling protein 2 enables protons to flow back into the mitochondria matrix without oxidative phosphorylation which generates heat instead of ATP, p62 = sequestome 1, delivers cargo to autophagosomes for degradation, regulates Nrf2 activity by binding with Keap1. HMOX1 = heme oxygenase 1, converts denatured heme, oxygen and NADPH to Billiverdin, carbon monoxide and iron. It is an anti‐oxidant gene up‐regulated by oxidative stress. GPX1 = glutathione peroxidase 1 is an anti‐oxidant enzyme which scavenges hydrogen peroxide. BAX = BCL2 associated X, BCL2 = BCL2 are respectively pro‐apoptotic and anti‐apoptotic regulators. FOXO1 = forkhead box protein 1, PPAR‐α = peroxisome proliferator activated receptor‐α, PPAR‐γ = peroxisome proliferator activated receptor‐γ are transcription factors which regulates many facets of metabolism. Gene expression analysis of (B) mitochondria, (C) oxidative stress/oxidant defense, (D) apoptosis‐related, (E) metabolism regulation related genes from pancreatic tails. Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups over ND. N = 11 patients for ND, N = 10 patients for T2D. Statistical analysis was evaluated by Student's t test and significance is indicated by * (compared to ND), *P < 0.05 FI G U R E 3 Metabolism, redox and apoptosis related gene expression in human pancreata. (A) Schematic of the genes assayed below. 3.5 | Hyperglycaemia inhibits TFEB nuclear translocation and down‐regulates autophagy Given that TFEB nuclear translocation and LAMP2 expression are suppressed in the islets of T2D, we hypothesized that hyperglycae‐ mia and hyperlipidaemia may inhibit TFEB nuclear translocation and autophagy. To test our hypothesis, INS‐1 cells stably transfected with TFEB‐EGFP were exposed to 5 or 30 mmol/L [GLU] in the presence and absence of FAs for 48 hours and then subjected to starvation to measure TFEB nuclear translocation. Treatment with OA and OA+PA under hyperglycaemic conditions significantly re‐ duced TFEB nuclear translocation (Figure 5A,B). These results show that the combination of high glucose concentration with lipid 2896  | | 2896 JI et al. FI G U R E 4 Effect of hyperglycaemia and hyperlipidaemia on LD and LD‐associated proteins in INS‐1. (A) Representative image and (B) quantification of INS‐1 pre‐treated with 1 μmol/L Torin‐1 for 3 h before treatment with 5 mM (normoglycaemic) [GLU] or 30 mM [GLU] (hyperglycaemic) medium ±500 μM oleic acid (OA) for 24 h and stained for LD with BODIPY 493/503 (green) and Hoechst 33342 (blue). LD was quantified as the total area of LD per cell and represents the mean from three independent experiments. (C) Gene expression analysis of INS‐1 Plin2 under 5 mmol/L [GLU], 30 mmol/L [GLU]), treated with 500 μmol/LM OA, 500 μmol/L palmitic acid (PA) or 250 μmol/L OA + 250 μmol/L PA. Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups from three independent experiments. Statistical analysis was evaluated by two‐way ANOVA followed by Tukey's test. Dual arrows * indicate a significant main effect due to hyperglycaemia (P < 0.05), * beside each point indicate significant difference (P < 0.05) compared to 5 mmol/L [GLU] Ctrl FI G U R E 4 Effect of hyperglycaemia and hyperlipidaemia on LD and LD‐associated proteins in INS‐1. (A) Representative image and (B) quantification of INS‐1 pre‐treated with 1 μmol/L Torin‐1 for 3 h before treatment with 5 mM (normoglycaemic) [GLU] or 30 mM [GLU] (hyperglycaemic) medium ±500 μM oleic acid (OA) for 24 h and stained for LD with BODIPY 493/503 (green) and Hoechst 33342 (blue). LD was quantified as the total area of LD per cell and represents the mean from three independent experiments. (C) Gene expression analysis of INS‐1 Plin2 under 5 mmol/L [GLU], 30 mmol/L [GLU]), treated with 500 μmol/LM OA, 500 μmol/L palmitic acid (PA) or 250 μmol/L OA + 250 μmol/L PA. 3.5 | Hyperglycaemia inhibits TFEB nuclear translocation and down‐regulates autophagy Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups from three independent experiments. Statistical analysis was evaluated by two‐way ANOVA followed by Tukey's test. Dual arrows * indicate a significant main effect due to hyperglycaemia (P < 0.05), * beside each point indicate significant difference (P < 0.05) compared to 5 mmol/L [GLU] Ctrl overload inhibits TFEB activation. Hyperglycaemia lowered the expression of Tfeb, Lamp1, and Lc3 (Figure 5C), meaning that au‐ tophagy was down‐regulated at the transcriptional (TFEB), lysoso‐ mal (Lamp1) and autophagosome assembly (LC3) levels. Expression of p62, a scaffold protein known to bind ubiquitin and LC3 and degrade by autophagy,30 followed the same pattern as LC3 except for PA+30 mmol/L [GLU] when no down‐regulation by high glucose was observed. Since p62 protein accumulates when autophagy is inhibited, we used p62 accumulation (seen as aggregated puncta) as a measure of autophagic blockage. Chloroquine (inhibitor of autophagy) was used as a positive control for autophagy blockage (Figure S4A). PA+30 mmol/L [GLU] significantly increased p62 lev‐ els and aggregation (Figure S4A,B), while OA+PA+30 mmol/L [GLU] had a lesser effect. Inhibition of autophagy was further confirmed by a significant decrease in LC3BII/I ratio under hyperglycaemia (Figure S4C). Given that down‐regulation of autophagy is known to dysregulate insulin production and islet metabolism, we performed qRT‐PCR for Ins1 and Ins2, Ppar‐α, Ppar‐γ, Ucp2 in INS‐1 cells ex‐ posed to 5 or 30 mmol/L [GLU] with or without OA, PA, OA+PA (Figure S5). Under normoglycaemia, FA treatments stimulated a significant increase in insulin expression, whereas under hypergly‐ caemia FA had a smaller effect, suggesting that prolonged hypergly‐ caemia can reduce insulin production. This corroborates with the recently reported significant reduction in insulin expression in INS‐1 cells after 14‐day exposure to 22.4 mmol/L [GLU] due to methyla‐ tion silencing of the insulin promoter.32 4 | DISCUSSION (C) Gene expression analysis of INS‐1 Tfeb, Lamp1, Sqstm1 and Lc3 under 5 mmol/L [GLU]/30 mmol/L [GLU]), treated with 500 μmol/L OA, 500 μmol/L palmitic acid (PA) or 250 μmol/L OA +250 μmol/L PA. Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups from three independent experiments. Statistical analysis was evaluated by two‐ way ANOVA followed by Tukey's test. Dual arrows * indicate significant main effect due to hyperglycaemia (P < 0.05), * beside each point indicate significant difference (P < 0.05) compared to 5 mmol/L [GLU] Ctrl up‐regulated PLIN2 and substantially increased OA‐induced LD for‐ mation. The primary function of LDs is to store lipids for energy but also to prevent acute lipotoxicity by sequestering otherwise toxic FA.4 In isolated rat β‐cells, the accumulation of LD was inversely pro‐ portional to the cytotoxicity for a given FA, for example palmitate lead to lower triglyceride accumulation and greater cell toxicity com‐ pared to oleate.34 However, nutrient‐induced LD accumulation is also associated with β‐cell dysfunction.22 Reduction in LD by PLIN2 knockdown in β‐cells lead to decreased ER stress, enhanced autoph‐ agy and decreased β‐cell apoptosis in diabetic animal models.35This is consistent with our observations of the changes in autophagy‐re‐ lated proteins TFEB and LAMP2 and in the gene expression in T2D pancreata suggesting the activation of compensatory mechanisms to withstand metabolic stress. For instance, to reduce the ATP/ADP ratio under hyperglycaemic conditions, mitochondria can increase proton leakage through uncoupling proteins. We report an increase in both UCP2 and CPT1A expression in the pancreas of T2D patients which suggests an increase in both FA mitochondrial import and in β‐oxidation. We also observed a significant increase in mitochondrial metabolic activity in INS‐1 exposed to hyperglycaemic conditions after 24 and 48 hours. In this study, PLIN2 protein was used as a LD marker to gauge the size of the LD pool. 4 | DISCUSSION Results from this study show that hyperglycaemia and hyperlipi‐ daemia in humans and rat insulin‐producing cells (INS‐1) lead to: (1) an up‐regulation of LD‐associated protein PLIN2, (2) a significant decrease in TFEB activity and in lysosomal biomarker LAMP2, con‐ sistent with inhibition of autophagy and (3) dysregulation of genes implicated in lipid metabolism, mitochondrial function and cell survival. A schematic of the proposed mechanisms is illustrated in Figure 6. Under physiological conditions, β‐cells preferentially use FA over glucose through β‐oxidation.33 We showed that hyperglycaemia JI et al. JI et al. 2897 OA‐induced LD for‐ which suggests an increase in both FA mitochondrial import and in FI G U R E 5 TFEB translocation and autophagy‐associated gene expression in INS‐1. (A) INS‐1‐TFEB‐EGFP was cultured for 48 h in normoglycaemia (5 mmol/L [GLU]), hyperglycaemia (30 mmol/L [GLU]) and treated with 500 μmol/L OA, 500 μmol/L PA, or 250 μmol/L OA + 250 μmol/L PA and starved for 1 h in HBSS to induce TFEB nuclear translocation. (A) Representative images, (B) quantification of nuclear/ cytosolic TFEB fluorescence ratio. n > 50 cells from three independent experiments. White arrows point to TFEB nuclear translocation. (C) Gene expression analysis of INS‐1 Tfeb, Lamp1, Sqstm1 and Lc3 under 5 mmol/L [GLU]/30 mmol/L [GLU]), treated with 500 μmol/L OA, 500 μmol/L palmitic acid (PA) or 250 μmol/L OA +250 μmol/L PA. Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups from three independent experiments. Statistical analysis was evaluated by two‐ way ANOVA followed by Tukey's test. Dual arrows * indicate significant main effect due to hyperglycaemia (P < 0.05), * beside each point indicate significant difference (P < 0.05) compared to 5 mmol/L [GLU] Ctrl FI G U R E 5 TFEB translocation and autophagy‐associated gene expression in INS‐1. (A) INS‐1‐TFEB‐EGFP was cultured for 48 h in normoglycaemia (5 mmol/L [GLU]), hyperglycaemia (30 mmol/L [GLU]) and treated with 500 μmol/L OA, 500 μmol/L PA, or 250 μmol/L OA + 250 μmol/L PA and starved for 1 h in HBSS to induce TFEB nuclear translocation. (A) Representative images, (B) quantification of nuclear/ cytosolic TFEB fluorescence ratio. n > 50 cells from three independent experiments. White arrows point to TFEB nuclear translocation. 4 | DISCUSSION PLIN2 is actively degraded in the cytosol when it is not on the surface of LD.36 Interestingly, a number of studies re‐ port that PLIN2 has additional functions not directly related to lipid accumulation, such as mediating Wnt/LiCl signalling,37 activation of ER and unfolded protein response in β‐cells (UPR),35 and inhibition of glucose uptake through interactions with SNAP23.38 Expression of PLIN2 is up‐regulated by lipids,39 ROS,40 ER stress and UPR.35 We found that 30 mmol/L [GLU] alone was the strongest inducer of PLIN2 expression in INS‐1, whereas FA alone did not have an ef‐ fect (Figure 4). We were surprised to see no effect of FA on PLIN2 mRNA in low glucose, contrary to the notion that PLIN2 expression is up‐regulated by lipids.39 However, the differing culturing glucose 2898  | 2898 JI et al. FI G U R E 6 Schematic for dysregulated TFEB and lipid droplet accumulation during hyperglycaemia and hyperlipidaemia. Elevated glucose and FFA increase mTORC1 activity which leads to greater suppression of TFEB through inhibitory phosphorylation. As a consequence, LDs accumulate inside of β‐cells due to increased lipid load as well as decreased lipophagy due to the down‐regulation of autophagy and lysosomal biogenesis FI G U R E 6 Schematic for dysregulated TFEB and lipid droplet accumulation during hyperglycaemia and hyperlipidaemia. Elevated glucose and FFA increase mTORC1 activity which leads to greater suppression of TFEB through inhibitory phosphorylation. As a consequence, LDs accumulate inside of β‐cells due to increased lipid load as well as decreased lipophagy due to the down‐regulation of autophagy and lysosomal biogenesis both a mouse model of type 1 diabetes (Akita mice) and in obese patients.47 concentration and different treatment times complicates the com‐ parison between our study and that of Faleck et al. 39 Active mTORC1 up‐regulates anabolic processes and suppresses autophagy. Vernier et al have previously demonstrated that hyper‐ glycaemia and hyperlipidaemia lead to increased mTORC1 activ‐ ity.22 Our data on inhibition of LDs in 30 mmol/L [GLU]+OA treated cells by Torin‐1 are consistent with those findings. As an extension, we found a decreased rate of starvation‐induced TFEB nuclear translocation in β‐cells pre‐treated with elevated glucose and FA. Hyperactivation of mTORC1 may be a general metabolic dysregu‐ lation due to nutrient overload. 4 | DISCUSSION Rapamycin inhibition of mTORC1 reversed insulin resistance in adipose tissue, skeletal muscle and liver in hyperinsulinaemic rats.48 Importantly, rapamycin inhibition of mTORC1 greatly diminished LD accumulation in rodent β‐cells.22 Although abnormally active mTORC1 leads to deleterious diabetic‐ like symptoms, normal mTORC1 function is still critical for β‐cell pro‐ liferation and metabolic function. Long‐term rapamycin treatment leads to worsened glucose intolerance, lipid accumulation in the liver and β‐cell loss in diabetic animals.49,50 Whether increased TFEB ac‐ tivation can rescue β‐cell loss and improve insulin sensitivity has yet to be explored. In both genetic and diet‐induced models of obesity, TFEB overexpressing mice had lower blood glucose levels, improved glucose tolerance and decreased fat gain compared to WT, whereas TFEB‐knockout mice experienced increased fat gain.24 Interestingly, TFEB activity is increased during physical exercise and is necessary for the beneficial effects of exercise such as increased FA oxidation, insulin response and improved mitochondrial function in skeletal muscle.51 Up‐regulation of PLIN2 in T2D patients is likely caused by a com‐ bination of hyperglycaemia, hyperlipidaemia, inflammation and oxi‐ dative stress, among others. As a chronic phenomenon, prolonged accumulation of PLIN2 and LD can contribute to ER stress and β‐cell loss. Consistent with this, we show an increase in pro‐apoptotic gene BAX in T2D as well as up‐regulation of anti‐apoptotic gene BCL2 and oxidant defense genes (GPX1, HMOX1, NRF2) to counteract ROS induced by ER stress. The PLIN family of proteins are also known to regulate the activity of a multitude of LD associated proteins,41 including lipases such as ATGL42 and HSL.43 Future studies should examine the expression and activity of LD associated proteins such as lipid synthesis enzymes, lipases and trafficking proteins to com‐ pletely characterize the regulation of lipid storage and breakdown in T2D. Our data, from T2D samples and INS‐1 cells, are consistent with inhibition of autophagy by hyperglycaemia and hyperlipidae‐ mia. Autophagy (and lipophagy) plays multiple roles in maintaining cellular homeostasis.44 TFEB is a master regulator of autophagic genes and lysosome biogenesis. A previous study has reported that mice fed a high‐fat diet have reduced TFEB mRNA levels in pancreatic islets.45 Our observation of reduced TFEB nuclear translocation and LAMP2 protein levels in T2D islets is consistent with previous findings of reduced LAMP2 and cathepsin‐B/D gene expression in islets from T2D patients.46 Reduction in TFEB activ‐ ity as a consequence of diabetes is not limited to β‐cells. REFERENCES 1. Oropeza D, Jouvet N, Bouyakdan K, et al. PGC‐1 coactivators in beta‐cells regulate lipid metabolism and are essential for insulin se‐ cretion coupled to fatty acids. Mol Metab. 2015;4:811‐822. cretion coupled to fatty acids. Mol Metab. 2015;4:811‐8 2. Alejandro EU, Gregg B, Blandino‐Rosano M, et al. Natural history of β‐cell adaptation and failure in type 2 diabetes. Mol Aspects Med. 2015;42:19‐41. 3. Yazici D, Sezer H. Insulin resistance, obesity and lipotoxicity. Adv Exp Med Biol. 2017;960:277‐304. 4. Welte MA. Expanding roles for lipid droplets. Curr Biol. 2015;25:R470‐R481. 5. Onal G, Kutlu O, Gozuacik D, et al. Lipid droplets in health and dis‐ ease. Lipids Health Dis. 2017;16. Combined therapeutic approaches targeting TFEB and mTORC1 with intermittent fasting could be a rewarding direction in the inter‐ vention of T2D and other metabolic disorders. A limitation of our current study is the scarcity of human pancreatic tissue and the dif‐ ficulty of obtaining freshly isolated islets. Instead, we used the well‐ studied rat β‐cell line (INS‐1) to model the effects of hyperglycaemia on LD and autophagy in vitro. We used supraphysiological levels of glucose (30 mmol/L) to model hyperglycaemia in INS‐1 cells for two reasons: (1) rat diabetic models have two‐ to threefold higher circu‐ lating glucose compared to humans56,57; (2) previous in vitro studies using INS‐1 cells employed ≥25 mmol/L glucose to model hypergly‐ caemia.58,59 Having established an important connection between autophagy dysregulation and LD accumulation in our current study, future experiments should explore whether TFEB overexpression in β‐cells can rescue islet dysfunction in vivo. Complementary ex‐ periments with human islets could provide new directions for the therapeutic interventions utilizing lipophagy to normalize lipid homeostasis. 6. Arrese EL, Saudale FZ, Soulages JL. Lipid droplets as signaling platforms linking metabolic and cellular functions. Lipid Insights. 2014;7:7‐16. 7. Walther TC, Farese RV. Lipid droplets and cellular lipid metabolism. Annu Rev Biochem. 2012;81:687‐714. 8. 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These studies were supported by Canadian Institute for Health Research (DM), Canadian Diabetes Society (LR) and McGill University for con‐ tributing to the internal studentship of JJ, and AK. JJ, MP and AK performed all of the experiments with technical assistance from JM. JJ, MP and DM contributed to the preparation of the drafted manuscript. JP participated in tissue collection and human islet isolation. DM initiated the study, revised multiple drafts and sug‐ gested analyses of the data. LR read the final draft and provided human pancreatic tissue. 16. Ouimet M, Franklin V, Mak E, et al. Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase. Cell Metab. 2011;13:655‐667. 17. Hubbard VM, Valdor R, Patel B, et al. Macroautophagy regulates energy metabolism during effector T cell activation. J Immunol. 2010;185:7349‐7357. 18. Kaushik S, Rodriguez‐Navarro JA, Arias E, et al. 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The role of ER stress in lipid metabolism and lipotoxicity. J Lipid Res. 2016;57:1329‐1338. 13. Olofsson SO, Bostrom P, Andersson L, et al. Lipid droplets and their role in the development of insulin resistance and diabetic dyslipid‐ emia. Clin Lipidol. 2009;4:611‐622. 14. Liu K, Czaja MJ. Regulation of lipid stores and metabolism by li‐ pophagy. Cell Death Differ. 2013;20:3‐11. 15. Ward C, Martinez‐Lopez N, Otten EG, et al. Autophagy, lipoph‐ agy and lysosomal lipid storage disorders. Biochem Biophys Acta. 2016;1861:269‐284. Nutrient overload is a new phenomenon at the evolutionary timescale and in this context Neel proposed a thrifty gene hypothe‐ sis52 to explain the adaptation of β‐cells in oscillating periods of low and high energy load. Periods of fasting would enable β‐cells to up‐ regulate autophagy through TFEB nuclear translocation and recycle worn‐out organelles and accumulating lipids. A recent study on islet regeneration has shown fasting in diabetic mice lowers mTOR activ‐ ity, promotes β‐cell neogenesis and reverses diabetic symptoms.53 Intermittent fasting proved beneficial in other diseases associated with impaired autophagy including Alzheimer's disease, excitotoxic‐ ity and in aging.53-55 CONFLICT OF INTEREST The authors declare that they have no conflicts of interests. 23. Settembre C, Di Malta C, Polito VA, et al. TFEB links autophagy to lysosomal biogenesis. Science. 2011;332:1429‐1433. 24. Settembre C, De Cegli R, Mansueto G, et al. TFEB controls cellular lipid metabolism through a starvation‐induced autoregulatory loop. Nat Cell Biol. 2013;15:647‐658. 4 | DISCUSSION A recent study in cardiomyocytes has found reduced TFEB expression in JI et al. 2899 Fasting‐mimicking diet pro‐ motes Ngn3‐driven beta‐cell regeneration to reverse diabetes. Cell. 2017;168:775‐788 e12. 33. Malaisse WJ, Best L, Kawazu S, et al. 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Scientific reports. 2017;7:40855. 56. Sudre B, Broqua P, White RB, et al. Chronic inhibition of circu‐ lating dipeptidyl peptidase IV by FE 999011 delays the occur‐ rence of diabetes in male Zucker diabetic fatty rats. Diabetes. 2002;51:1461‐1469. 36. Takahashi Y, Shinoda A, Kamada H, et al. Perilipin2 plays a positive role in adipocytes during lipolysis by escaping proteasomal degra‐ dation. Sci Rep. 2016;6:20975. 37. Liu X, Lu X, Song K, et al. Natural functions of PLIN2 mediating Wnt/LiCl signaling and Glycogen Synthase Kinase 3 (GSK3)/GSK3 substrate‐related effects are modulated by lipid. Mol Cell Biol. 2016;36:421‐437. 57. Qian C, Zhu CY, Yu WQ, et al. Diet/low‐dose streptozotocin‐in‐ duced type 2 diabetes in rats impacts osteogenesis and Wnt signal‐ ing in bone marrow stromal cells. PLoS ONE. 2015;10:e0136390. 58. Hanchang W, Semprasert N, Limjindaporn T, et al. Testosterone protects against glucotoxicity‐induced apoptosis of pancreatic beta‐cells (INS‐1) and male mouse pancreatic islets. Endocrinology. 2013;154:4058‐4067. 38. Senthivinayagam S, McIntosh AL, Moon KC, et al. Plin2 inhibits cel‐ lular glucose uptake through interactions with SNAP23, a SNARE complex protein. PLoS ONE. 2013;8:e73696. 39. Faleck DM, Ali K, Roat R, et al. Adipose differentiation‐related protein regulates lipids and insulin in pancreatic islets. Am J Physiol Endocrinol Metab. 2010;299:E249‐E257. 59. Brereton MF, Rohm M, Shimomura K, et al. Hyperglycaemia induces metabolic dysfunction and glycogen accumulation in pancreatic beta‐cells. Nat Commun. 2016;7:13496. 40. Jin Y, Tan Y, Chen L, et al. 45. Liu HY, Javaheri A, Godar RJ, et al. Intermittent fasting preserves beta‐cell mass in obesity‐induced diabetes via the autophagy‐lyso‐ some pathway. Autophagy. 2017;13:1952‐1968. 25. Cohen AW, Razani B, Schubert W, et al. Role of caveolin‐1 in the modulation of lipolysis and lipid droplet formation. Diabetes. 2004;53:1261‐1270. 26. Gong J, Sun Z, Wu L, et al. Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites. J Cell Biol. 2011;195:953‐963. 46. Masini M, Bugliani M, Lupi R, et al. Autophagy in human type 2 dia‐ betes pancreatic beta cells. Diabetologia. 2009;52:1083‐1086. 47. Trivedi PC, Bartlett JJ, Perez LJ, et al. Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obe‐ sity and diabetes. BBA‐Mol Cell Biol L. 2016;1861:1893‐1910. 27. Onal G, Kutlu O, Gozuacik D, et al. Lipid droplets in health and disease. Lipids Health Dis. 2017;16. https://doi.org/10.1186/ S12944-017-0521-7 48. Ueno M, Carvalheira J, Tambascia RC, et al. Regulation of insulin sig‐ nalling by hyperinsulinaemia: role of IRS‐1/2 serine phosphorylation and the mTOR/p70 S6K pathway. Diabetologia. 2005;48:506‐518. 28. Li LX, Skorpen F, Egeberg K, et al. Uncoupling protein‐2 partici‐ pates in cellular defense against oxidative stress in clonal beta‐cells. Biochem Biophys Res Comm. 2001;282:273‐277. 49. Wang Y, He Z, Li X, et al. Chronic rapamycin treatment exacer‐ bates metabolism and does not down‐regulate mTORC2/Akt sig‐ naling in diabetic mice induced by high‐fat diet and streptozotocin. Pharmazie. 2015;70:604‐609. 29. Ma Q. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. 2013;53:401‐426. 30. Bjorkoy G, Lamark T, Pankiv S, et al. Monitoring autophagic degra‐ dation of p62/SQSTM1. Methods Enzymol. 2009;452:181‐197. 50. Schindler CE, Partap U, Patchen BK, et al. Chronic rapamy‐ cin treatment causes diabetes in male mice. Am J Physiol‐Reg. 2014;307:R434‐R443. 31. Zummo FP, Cullen KS, Honkanen‐Scott M, et al. Glucagon‐like peptide 1 protects pancreatic beta‐cells from death by increas‐ ing autophagic flux and restoring lysosomal function. Diabetes. 2017;66:1272‐1285. 51. Mansueto G, Armani A, Viscomi C, et al. Transcription factor EB controls metabolic flexibility during exercise. Cell Metab. 2017;25:182‐196. 32. Ishikawa K, Tsunekawa S, Ikeniwa M, et al. Long‐term pancreatic beta cell exposure to high levels of glucose but not palmitate in‐ duces DNA methylation within the insulin gene promoter and re‐ presses transcriptional activity. PLoS ONE. 2015;10:e0115350. 52. Neel JV. The "thrifty genotype" in 1998. Nutr Rev. 1999;57:S2‐S9. 53. Cheng CW, Villani V, Buono R, et al. Reactive oxygen species induces lipid droplet accumulation in HepG2 cells by increasing perilipin 2 ex‐ pression. Int J Mol Sci. 2018;19:3445. 2900  | 2900 JI et al. JI et al. ORCID Dusica Maysinger  https://orcid.org/0000-0002-0017-5201 SUPPORTING INFORMATION 41. Sztalryd C, Brasaemle DL. The perilipin family of lipid droplet proteins: gatekeepers of intracellular lipolysis. Bba‐Mol Cell Biol L. 2017;1862:1221‐1232. Additional supporting information may be found online in the Additional supporting information may be found online in the Supporting Information section at the end of the article. Supporting Information section at the end of the article. 42. Wang H, Bell M, Sreenivasan U, et al. Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet‐associated protein. J Biol Chem. 2013;288:10952. How to cite this article: Ji J, Petropavlovskaia M, Khatchadourian A, et al. Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β‐cells. J Cell Mol Med. 2019;23:2890–2900. https://doi.org/10.1111/ jcmm.14172 How to cite this article: Ji J, Petropavlovskaia M, Khatchadourian A, et al. Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β‐cells. J Cell Mol Med. 2019;23:2890–2900. https://doi.org/10.1111/ jcmm.14172 43. Whytock KL, Shepherd SO, Wagenmakers A, et al. Hormone‐sen‐ sitive lipase preferentially redistributes to lipid droplets associated with perilipin‐5 in human skeletal muscle during moderate‐intensity exercise. J Physiol. 2018;596:2077‐2090. 44. Wang CW. Lipid droplets, lipophagy, and beyond. Biochem Biophys Acta. 2016;1861:793‐805.
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&amp;lt;i&amp;gt;Letter to the Editor:&amp;lt;/i&amp;gt; A comparison of F-region ion velocity observations from the EISCAT Svalbard and VHF radars with irregularity drift velocity measurements from the CUTLASS Finland HF radar
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A comparison of F-region ion velocity observations from the EISCAT Svalbard and VHF radars with irregularity drift velocity measurements from the CUTLASS Finland HF radar J. A. Davies, T. K. Yeoman, M. Lester, S. E. Milan Radio and Space Plasma Physics Group, Department of Physics and Astronomy, University of Leicester, University Road, Leicester, LE1 7RH, UK e-mail: jaq@ion.le.ac.uk Received: 24 September 1999 / Revised: 21 February 2000 / Accepted: 24 February 2000 Received: 24 September 1999 / Revised: 21 February 2000 / Accepted: 24 February 2000 and Ossakow, 1983) and observational (Villain et al., 1985; Ruohoniemi et al., 1987; Baker et al., 1990; Davies et al., 1999), suggests that the motion of F-region irregularities measured by HF coherent scatter radars, which, unlike their VHF or UHF counterparts, are not limited to E-region observation at high-latitude due to the refractive nature of radio propagation at HF, is determined by the ambient plasma ¯ow. This has also been found to be true of irregularities generated arti®cially in the F-region by high-power heating facilities (e.g. Eglitis et al., 1998). Abstract. During August 1998, the UK EISCAT special programme SP-UK-CSUB, which combines operation of both the mainland VHF and Svalbard UHF inco- herent scatter radars, was run for several hours around magnetic midnight on four consecutive days. The CUTLASS Finland HF coherent scatter radar was, at these times, operating in a discretionary mode, sounding on all 16 beams, one at high-time resolution. This study presents a comparison of the velocities measured by coherent and incoherent techniques during the SP-UK- CSUB experiments. Agreement, particularly between the ion velocities measured by the EISCAT Svalbard radar and irregularity drift measurements by the Fin- land radar, is remarkable, thereby validating the scien- ti®c integrity of both data sets. This work highlights the substantive contribution to our understanding of the solar-terrestrial environment which can be made by use in concert of incoherent and HF coherent scatter radars. The studies by Villain et al. (1985), Ruohoniemi et al. (1987) and Baker et al. (1990) were based on data taken over a restricted time interval and, consequently, on a very limited number of velocity pairs. In contrast, Davies et al. (1999) compared EISCAT UHF radar observations from over 1000 hours of common pro- gramme 1 (CP-1) and common programme 2 (CP-2) experiments to the drift velocity of natural irregularities, the latter measured by the CUTLASS Finland HF radar. Despite their extensive database, Davies et al. (1999) still found themselves limited to a relatively small number of velocity pairs, the EISCAT mainland site being by no means optimally located within the Finland radar ®eld-of-view from which to receive F-region returns. A comparison of F-region ion velocity observations from the EISCAT Svalbard and VHF radars with irregularity drift velocity measurements from the CUTLASS Finland HF radar J. A. Davies, T. K. Yeoman, M. Lester, S. E. Milan Radio and Space Plasma Physics Group, Department of Physics and Astronomy, University of Leicester, University Road, Leicester, LE1 7RH, UK e-mail: jaq@ion.le.ac.uk Here, irregularity drift speeds measured by the CUTLASS Finland radar are compared to ion velocity observations by the EISCAT mainland VHF radar and the EISCAT Svalbard radar (ESR). Key words: Ionosphere (ionospheric irregularities; plasma convection; instruments and techniques) 1 Introduction Irregularity drift velocity measurements by coherent scatter radars have been compared to bulk plasma velocity measurements by other instrumentation, in- cluding satellite borne probes and incoherent scatter radars. Haldoupis and Schlegel (1990), and authors referenced therein, con®rmed experimentally previous theoretical evidence that the phase velocity of E-region irregularities, measured with the STARE VHF coherent scatter radar, is limited in magnitude to near the ion- acoustic speed. Evidence, both theoretical (e.g. Keskinen Ann. Geophysicae 18, 589±594 (2000) Ó EGS ± Springer-Verlag 2000 Ann. Geophysicae 18, 589±594 (2000) Ó EGS ± Springer-Verlag 2000 LETTER Correspondence to: J. A. Davies 2 Experimental arrangement In SP-UK-CSUB, which is identical to the VHF common programme CP-4, long pulse and power pro®le codes are transmitted. The long pulse, to which data this study is limited, provides line- of-sight observations, on each beam, over 20 gates of range resolution 65.3 km with the ®rst gate centred at a range of 533.0 km. Like those from Svalbard, VHF observations were analysed at 1 min temporal resolu- tion. Beam 1 of the VHF radar is aligned in azimuth along beam 7 of the Finland radar whereas beam 2 is less convenient with regard the present study in this respect, crossing as it does beams 8, 9 and 10 of the Finland radar. Fig. 1. The geographic location of the ®eld-of-view of the CUTLASS Finland radar, the ESR beam (solid line) and both the VHF radar beams (dashed lines). Beams 7 and 9 of the CUTLASS radar are highlighted explicitly. The locations of the radars are represented by large solid circles. The smaller solid circle indicates the position of the geomagnetic pole while the position of the geographic pole is represented as a cross within a circle During the third run, ionospheric scatter is observed by the Finland radar within the common viewing area only between 2100 and 2200 UT on August 22. Over the other two runs, there is a preponderance of coherent scatter identi®ed as being of ionospheric origin within that portion of the Finland ®eld-of-view accessible to the incoherent scatter radars. During the third run, ionospheric scatter is observed by the Finland radar within the common viewing area only between 2100 and 2200 UT on August 22. Over the other two runs, there is a preponderance of coherent scatter identi®ed as being of ionospheric origin within that portion of the Finland ®eld-of-view accessible to the incoherent scatter radars. Figure 2 presents long pulse observations of line-of- sight ion velocity from 2100 UT on 21 August to 0100 UT on 22 August 1998 from the EISCAT Svalbard radar (Fig. 2a), and beams 1 and 2 of the mainland VHF radar (Fig. 2b, c, respectively). Figure 2d, e depicts line-of-sight irregularity drift velocity measure- ments along beam 7 and beam 9, respectively, of the CUTLASS Finland radar. 2 Experimental arrangement The UK EISCAT special programme SP-UK-CSUB, which combines operation of both the VHF radar and the ESR, was run over four four-hour intervals com- mencing at 21 UT on 20, 21, 22 and 23 August 1998. During these times, the CUTLASS radar at Hankasalmi in Finland (e.g. Davies et al., 1999 and references therein) was operating in a discretionary mode in which all 16 beams (numbered clockwise 0 to 15) were sounded at a dwell time of 2 s, but with the radar returning to 590 J. A. Davies et al.: A Comparison of F-region ion velocity observations LETTER Fig. 1. The geographic location of the ®eld-of-view of the CUTLASS Finland radar, the ESR beam (solid line) and both the VHF radar beams (dashed lines). Beams 7 and 9 of the CUTLASS radar are highlighted explicitly. The locations of the radars are represented by large solid circles. The smaller solid circle indicates the position of the geomagnetic pole while the position of the geographic pole is represented as a cross within a circle sound on beam 9 between each; the scan is said to camp on beam 9. In total 75 range gates were sampled along each beam with a gate length of 45 km, and a range to the ®rst gate of 180 km. The ESR, located at Longyearbyen (e.g. Wannberg et al., 1997) was, during SP-UK-CSUB, directed south- ward, with a geographic azimuth of 161.6° and an elevation of 31.0°. The ESR beam is aligned, at least in azimuth, along beam 9 of the CUTLASS Finland radar ®eld-of-view. The ESR was transmitting the GUP0 radar code, a multi-frequency long pulse. Received signals were integrated over 10 s and the data subse- quently analysed at a temporal resolution of 60 s. Analysis of ESR data is such that the user can vary the range resolution, in this case with gating of 12 km below a range of 330 km, which corresponds to an altitude of around 175 km, and 36 km range gates above 330 km. In SP-UK-CSUB, the EISCAT VHF radar at Tromsù operated in a split beam mode with one beam (beam 2) directed along the boresight which corresponds to a geographic azimuth of 359.5° and the other (beam 1) phased 14.5° west to an azimuth of 345.0°; both beams are at a elevation of 30°. 2 Experimental arrangement Coherent returns identi®ed as arising from ground scatter are excluded as indeed are all Finland radar data with a line-of-sight irregularity drift velocity of magnitude less than 50 m s)1; the latter is an attempt to remove potentially misidenti®ed ground scatter (e.g. Davies et al., 1999). Velocities are plotted as a function of geomagnetic latitude. Velocities are de®ned as being positive towards the radar, except in the case of the Svalbard radar where positive velocities represent ¯ow away from the radar in order to retain a consistency between the general orientations of the observations. An in-depth assessment of the geophysical processes arising during this interval is not attempted here, that being the subject of a forthcoming study. Figure 1 presents a map on which is depicted the ®eld-of-view of the CUTLASS Finland radar, beams 7 and 9 of which are illustrated explicitly. Moreover, coverage by the ESR is represented by a solid line and the VHF radar beams are indicated by dashed lines. The large ®lled circles represent the locations of the radars themselves and the cross within a circle, the geographic pole; the magnetic pole is indicated by the small ®lled circle. 3 Results at altitudes below some 140 km, where ion-neutral collisions are signi®cant, will not be representative of the component of the E ´ B velocity along the radar line-of-sight. ments, being of F-region origin. The lowest range gate for VHF observations corresponds to an altitude of some 280 km while the lowest altitude at which the Svalbard radar observes is 108 km. As such, it should be borne in mind that ion velocities in the ®rst six or so ESR range gates, i.e. at altitudes below some 140 km, where ion-neutral collisions are signi®cant, will not be representative of the component of the E ´ B velocity along the radar line-of-sight. ignored. Although deviations from free-space propaga- tion have been found to be small, less than the length of a single range cell (e.g. Ruohoniemi et al., 1987), they are, nevertheless, not insigni®cant, particularly in stud- ies of this type. Despite these possible sources of discrepancy, many small-scale ¯ow features observed by the Finland radar appear to be mirrored in the incoherent scatter obser- vations, for example the equatorward velocity burst observed at around 2300 UT both in beam 9 of the CUTLASS ®eld-of-view and by the Svalbard radar. The relationship between F-region ion velocity and irregu- larity drift velocity is investigated on a more quantita- tive basis by employing appropriate observations from all but the ®rst SP-UK-CSUB experiment. Even a cursory glance reveals good general agree- ment between the observations from the coherent and incoherent systems, and, indeed, between the two incoherent scatter radars themselves. Exact agreement is not anticipated due to disparities between the sampling volumes of the systems (the beam width of the incoherent scatter radars being almost an order of magnitude smaller than that of the HF radar) and the integration periods of the data sets; velocities from both the ESR and VHF radars are integrated over 60 s, whereas beam 9 of the Finland radar is integrated for 2 out of every 4 s and beam 7 for only 2 s out of a total scan time of 64 s. Further discrepancies will be intro- duced due to geometrical factors. Finland velocities can be assumed orthogonal to the local magnetic ®eld, this being the condition necessary for coherent scatter. This need not be so for incoherent scatter, and, indeed, is not so here. 3 Results It should also be noted that in the calculation of the range from which coherent returns are attributed assumes straight line propagation of HF waves, i.e. refraction e€ects are neglected and group retardation is Ion velocities measured by the ESR are compared statistically to the irregularity drift velocities from beam 9 of the Finland radar in Fig. 3a while velocities from beam 1 of the VHF radar are compared to drift speeds from Finland Beam 7 in Fig. 3b; the sign convention of the previous ®gure is maintained, with positive velocities directed towards the Finland radar. As with the previ- ous ®gure, all Finland radar data with a line-of-sight irregularity drift speed less than 50 m s)1, in addition to those returns conventionally identi®ed as arising from ground scatter, are excluded. Moreover, a 3 dB thresh- old has been applied to the CUTLASS data and a 2% signal-to-noise ratio level to the EISCAT VHF obser- vations. In the situation where there are multiple estimates of irregularity drift velocity within a single Fig. 3a±c. Scattergram of irregularity drift velocity a measured by the Finland radar versus F-region ion velocity measured by the ESR and b along beam 1 of the VHF radar, during the SP-UK-CSUB experiments. Both ion velocity and irregularity drift speed are binned with a width of 50 m s)1, with the number of observations in each bin represented by the colour scale to the right. c Reproduces the data from the upper panel of Fig. 5 of Davies et al. (1999), in which ion velocity from the UHF radar is plotted against F-region irregularity drift velocity from the Finland radar, in an identical format to a and b. The solid lines indicate a hypothetical equality of irregularity drift and ion velocity from the upper panel of Fig. 5 of Davies et al. (1999), in which ion velocity from the UHF radar is plotted against F-region irregularity drift velocity from the Finland radar, in an identical format to a and b. The solid lines indicate a hypothetical equality of irregularity drift and ion velocity from the upper panel of Fig. 5 of Davies et al. (1999), in which ion velocity from the UHF radar is plotted against F-region irregularity drift velocity from the Finland radar, in an identical format to a and b. The solid lines indicate a hypothetical equality of irregularity drift and ion velocity Fig. 3a±c. 3 Results The four runs of SP-UK-CSUB are not equally useful with regard a comparison of irregularity drift velocity and F-region ion velocity. During the ®rst run, 20 August 2100 UT±21 August 0100 UT, there are a limited number of coherent echoes observed by the Finland radar within the viewing range of the ESR and VHF radars, mainly in the lower CUTLASS range gates of the common ®eld-of-view. The vast majority of these returns are, however, characterised by very narrow spectral widths and low velocities and, as such, have been identi®ed as ground scatter. For the purpose of the present study this interval is not considered further. The vagaries of propagation in the HF band is such that the altitude from which scatter originates is not well determined. Elevation angle-of-arrival measurements by the Finland system are consistent with the ionospheric scatter observed in the common viewing area of the three instruments during the SP-UK-CSUB experi- 591 J. A. Davies et al.: A Comparison of F-region ion velocity observations Fig. 2a±e. Line-of-sight velocity measurements during the SP-UK- CSUB experiment run from 2100 UT on 21 August to 0100 UT on 22 August, 1998. Successive panels illustrate data from a the EISCAT Svalbard radar, b beam 1 of the VHF radar, c be radar, d beam 7 of the CUTLASS Finland radar an Finland radar, each as a function of geomagnetic l J. A. Davies et al.: A Comparison of F-region ion velocity observations LETTER Svalbard radar, b beam 1 of the VHF radar, c beam 2 of the VHF radar, d beam 7 of the CUTLASS Finland radar and e beam 9 of the Finland radar, each as a function of geomagnetic latitude Fig. 2a±e. Line-of-sight velocity measurements during the SP-UK- CSUB experiment run from 2100 UT on 21 August to 0100 UT on 22 August, 1998. Successive panels illustrate data from a the EISCAT J. A. Davies et al.: A Comparison of F-region ion velocity observations 592 LETTER ments, being of F-region origin. The lowest range gate for VHF observations corresponds to an altitude of some 280 km while the lowest altitude at which the Svalbard radar observes is 108 km. As such, it should be borne in mind that ion velocities in the ®rst six or so ESR range gates, i.e. 3 Results Scattergram of irregularity drift velocity a measured by the Finland radar versus F-region ion velocity measured by the ESR and b along beam 1 of the VHF radar, during the SP-UK-CSUB experiments. Both ion velocity and irregularity drift speed are binned with a width of 50 m s)1, with the number of observations in each bin represented by the colour scale to the right. c Reproduces the data 593 J. A. Davies et al.: A Comparison of F-region ion velocity observations LETTER radar, in an identical format to Fig. 3a, b. In this case the ion velocity plotted is the component of the vector velocity (the UHF being a tristatic system) resolved along the direction of the appropriate Finland beam. Davies et al. (1999) di€erentiated between E- and F-region drift velocities on the basis of interferometric evidence. Figure 3c, in common with Fig. 3a, b, reveals a number of points corresponding to anomolously high values of irregularity drift velocity, which are attributed to uncorrelated spikes sometimes observed in CUT- LASS data (Davies et al., 1999). EISCAT integration period, these have been averaged together. Moreover, where there are more than one ion velocity observation within a single CUTLASS range gate, as is the case with the ESR, these too have been averaged together. Although coherent returns of veloc- ity less than 50 m s)1 have been excluded, the average of the irregularity drift velocity over an EISCAT integra- tion period may still be less than that value. ESR range gates centred below 140 km altitude are excluded from the analysis, the velocities therein not being representa- tive of magnetised ¯ow. EISCAT integration period, these have been averaged together. Moreover, where there are more than one ion velocity observation within a single CUTLASS range gate, as is the case with the ESR, these too have been averaged together. Although coherent returns of veloc- ity less than 50 m s)1 have been excluded, the average of the irregularity drift velocity over an EISCAT integra- tion period may still be less than that value. ESR range gates centred below 140 km altitude are excluded from the analysis, the velocities therein not being representa- tive of magnetised ¯ow. g The line-of-sight ion velocities measured by the ESR and along beam 1 of the VHF radar must be corrected for geometrical e€ects in order that direct comparison be made with the Finland drift observations. 3 Results As noted previously, coherent scatter requires the radar wave vector to be near orthogonal to the magnetic ®eld. Although at approximately the same azimuths as beams 7 and 9 of the Finland radar, the ion velocities measured by the incoherent scatter systems are not perpendicular to the magnetic ®eld, rather at an angle determined by the radar beam elevation and direction of the local geomagnetic ®eld. The ion velocities are corrected such that they represent the ®eld-perpendicular ¯ow, albeit assuming that there is no ®eld-parallel motion of the ion population, an assumption the validity of which is discussed in more detail in the subsequent section, and that the radar beam lies in the same plane as the magnetic ®eld, the latter of which is reasonable under these circumstances of a mainly northward directed beam. The solid lines on Fig. 3 indicate a hypothetical equality of irregularity drift and ion velocity for comparative purposes. ( ) The correspondence, not inconsistent with the equiv- alence of irregularity drift and bulk plasma motion in the F-region, noted by Davies et al. (1999) is surpassed in the present observations, especially with regard the EISCAT Svalbard radar, corrected velocities from which compare remarkably well with the line-of-sight drift velocity measurements from the CUTLASS Fin- land radar. There is signi®cant spread, however, which may be ascribed to a number of potentially deleterious e€ects such as discrepant temporal and spatial sampling. There does appear, however, to be a small yet systematic o€set, of some 50 to 100 m s)1, between the irregularity drift measurements and the ion velocities determined by the Svalbard radar. It is suggested that this is attribut- able, at least in part, to the assumption that there is no motion of the ion population along the magnetic ®eld direction, used in correcting the incoherent scatter velocity measurements from the Svalbard and VHF radars such that they represent ®eld-perpendicular ¯ows. On the nightside, the general antisunward hori- zontal motion of the neutral atmosphere, in response to pressure gradients set up by solar heating, will drive ions up the magnetic ®eld with a velocity of some several tens of m s)1 (e.g. Rodger et al., 1992). Invoking an upward ®eld-aligned velocity of this order does indeed signi®- cantly reduce the o€set observed between the HF drift and ESR velocity measurements. 4 Discussion These observations, made over a four day interval, provide an exceptional opportunity for the comparison of F-region ion velocity with the drift speed of F-region ®eld-aligned irregularities. This is especially noteworthy given that in the work of Davies et al. (1999) coherent returns from the Finland radar were observed in the vicinity of the EISCAT site for a time totalling less than 4 h out of an entire database comprising more than 1000 h of measurements from the two most frequently run UHF common programmes, taken over a three year interval. Due to the geometrical arrangement of the SP- UK-CSUB experiment, the present VHF and ESR observations have the advantage over the UHF obser- vations employed in the velocity comparison of Davies et al. (1999) of being simultaneous over a number of CUTLASS Finland range gates. Moreover, the present study concentrates on latitudes poleward of those considered by Davies et al. (1999) where the nature of HF propagation limits Finland returns from the F-region. y Perhaps one of the most noteworthy features of this work is the number of velocity pairs available for comparison, over 3300 for the ESR and nearly 1500 for the VHF radar compared to a mere 525 simultaneous estimates of UHF ion velocity and F-region irregularity drift speed in the study of Davies et al. (1999); this is even more remarkable given that the latter was derived from a data set more than 50 times as long as the combined lengths of the four runs of SP-UK-CSUB. In addition to the superior geometrical arrangement of the SP-UK-CSUB experiment for a study of this type, with regard the location and extended latitudinal range of the common viewing area, this is possibly also attributable to the conditions associated with the more active part of the solar cycle. References operation of the CUTLASS Finland HF radar. Line-of- sight velocity measurements from the three instruments exhibit a consistency even with regard to small-scale features in the convective ¯ow pattern. The geometrical arrangement of the experiment permits, moreover, the direct statistical comparison of line-of-sight F-region ion velocities measured by the ESR and along one beam of the VHF radar with irregularity drift velocity measure- ments from the Finland radar. Baker, K. B., R. A. Greenwald, J. M. Ruohoniemi, J. R. Dudeney, M. Pinnock, P. T. Newell, M. E. Greenspan, and C. I. Meng, Simultaneous HF radar and DMSP observations of the cusp, Geophys. Res. Lett., 17, 1869±1872, 1990. Davies, J. A., M. Lester, S. E. Milan, and T. K. Yeoman, A comparison of velocity measurements from the CUTLASS Finland radar and the EISCAT UHF system, Ann. Geophysicae, 17, 892±902, 1999. Eglitis, P., T. R. Robinson, M. T. Rietveld, D. M. Wright, and G. E. Bond, The phase speed of arti®cial ®eld-aligned irregu- larities observed by CUTLASS during HF modi®cation of the auroral ionosphere, J. Geophys. Res., 103, 2253±2259, 1998. Statistical comparison of ion velocities measured by the ESR and irregularity drift measurements by the Finland radar is particularly satisfying, despite a num- ber of potential sources of discrepancy, demonstrating once again that the CUTLASS system provides an invaluable spatial context in which to interpret small- scale features in the high-altitude ionosphere identi®ed by incoherent scatter techniques and, indeed, how signi®cant a contribution to our understanding of solar-terrestrial physics can be made by collaborative use of observations from incoherent and HF coherent scatter radars. Haldoupis, C., and K. Schlegel, Direct comparison of 1-m irregularity phase velocities and ion acoustic speeds in the auroral E-region ionosphere, J. Geophys. Res., 95, 18989±19000, 1990. Keskinen, M. J., and S. L. Ossakow, Theories of high-latitude ionospheric irregularities ± A review, Radio Sci., 18, 1077±1091, 1983. Rodger, A. S., R. J. Mo€ett, and S. Quegan, The role of ion drift in the formation of ionisation troughs in the mid- and high-latitude ionosphere ± A review, J. Atmos. Terr. Phys., 54, 1±30, 1992. Ruohoniemi, J. M., R. A. Greenwald, K. B. Baker, J.-P. Villain, and M. A. McCready, Drift motions of small-scale irregularities in the high-latitude F-region: an experimental comparison with plasma drift motions, J. Geophys. Res., 92, 4553±4564, 1987. Acknowledgements. 5 Summary and conclusions During August 1998, the UK EISCAT special pro- gramme SP-UK-CSUB, combining operation of both the mainland VHF and Svalbard incoherent scatter radars, was run in conjunction with discretionary mode g Figure 3c reproduces the data from the upper panel of Fig. 5 of Davies et al. (1999), in which ion velocity from the UHF radar is plotted against the drift velocity of F-region irregularities measured by the Finland J. A. Davies et al.: A Comparison of F-region ion velocity observations 594 LETTER The Editor-in-chief thanks C. Senior for her help in evaluating this paper. Acknowledgements. The authors wish to acknowledge support from the EISCAT group at the Rutherford Appleton Laboratory and other members of the UK campaign team. Additional thanks are due to N. M. Wade. EISCAT is funded collaboratively by the research councils of Finland, France, the Federal Republic of Germany, Japan, Norway, Sweden and the UK funding body, PPARC. CUTLASS is funded jointly by PPARC, the Swedish Institute for Space Physics and the Finnish Meteorological Institute. References The authors wish to acknowledge support from the EISCAT group at the Rutherford Appleton Laboratory and other members of the UK campaign team. Additional thanks are due to N. M. Wade. EISCAT is funded collaboratively by the research councils of Finland, France, the Federal Republic of Germany, Japan, Norway, Sweden and the UK funding body, PPARC. CUTLASS is funded jointly by PPARC, the Swedish Institute for Space Physics and the Finnish Meteorological Institute. p p y Villain, J.-P., G. Caudal, and C. Hanuise, A SAFARI-EISCAT comparison between the velocity of F-region small-scale irreg- ularities and the ion drift, J. Geophys. Res., 90, 8433±8443, 1985. Wannberg G., L. Wolf, L.-G. Vanhainen, K. Koskenniemi, J. RoÈ ttger, M. Postila, J. Markkanen, R. Jacobsen, A. Stenberg, R. Larsen, S. Eliassen, S. Heck, and A. Huuskonen, The EISCAT Svalbard radar: A case study in modern incoherent scatter radar system design, Radio Sci., 32, 2283±2307, 1997. The Editor-in-chief thanks C. Senior for her help in evaluating this paper.
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Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection
Virology journal
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BioMed Central BioMed Central Received: 13 June 2009 Accepted: 15 July 2009 Received: 13 June 2009 Accepted: 15 July 2009 © 2009 Sainz 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 Sainz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection Bruno Sainz Jr†1, Veronica TenCate†1 and Susan L Uprichard*1,2 ddress: 1Department of Medicine, The University of Illinois at Chicago, Chicago, IL 60612, USA and 2Department o mmunology, The University of Illinois at Chicago, Chicago, IL 60612, USA Published: 15 July 2009 Virology Journal 2009, 6:103 doi:10.1186/1743-422X-6-103 Received: 13 June 2009 Accepted: 15 July 2009 This article is available from: http://www.virologyj.com/content/6/1/103 © 2009 Sainz 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: In order to elucidate how Hepatitis C Virus (HCV) interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV- associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells. Results: When cultured in the RWV, Huh7 cells became morphologically and transcriptionally distinct from more standard Huh7 two-dimensional (2-D) monolayers. Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4α, Albumin, TTR and α1AT), were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, β-Catenin and E- Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization. Conclusion: These findings show that when cultured in 3-D, Huh7 cells acquire a more differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system. anese Fulminant Hepatitis, JFH-1) that can replicate and produce infectious HCV in vitro in the Huh7 human hepatoma-derived cell line [2-4], allowing for the study of the entire viral life cycle. This system, however, is limited in that it makes use of a non-differentiated cell line that does not recapitulate the cellular conditions encountered by HCV in vivo [5,6]. In particular, hepatocyte polarity is likely relevant to HCV entry as growing evidence suggests interplay between HCV and tight junction (TJ) proteins Virology Journal Open Access Page 1 of 8 (page number not for citation purposes) http://www.virologyj.com/content/6/1/103 http://www.virologyj.com/content/6/1/103 claudin-1 (CLDN1) [7] and occludin [8,9] is essential for viral uptake. In fact, recent reports surprisingly suggests that hepatocyte polarity may restricts HCV entry [10,11]. While an inverse relationship between viral entry and hepatocyte polarity would potentially represent a unique determinant of HCV entry, to date attempts to dissect this relationship have been difficult and inconclusive due to the inability of cell culture grown hepatocyte-derived cell lines, such as Huh7 cells, to mimic the complex polarized phenotype of hepatocytes in vivo. To circumvent these restriction, studies investigating HCV entry into Caco-2 cells [10] and HepG2 cells [11] have been performed as these cells can polarize to differing degrees in vitro, how- ever, neither Caco-2 or HepG2 cells supports efficient HCV infection limiting their utility. As such, a more phys- iologically relevant hepatocyte tissue culture model is still needed to assess if cell polarity negatively affects HCV infection. lished using previously described techniques [13,14], with minor modifications. Briefly, 5 × 106 Huh7 cells were trypisinized, incubated with 250 mg Cytodex-3 microcarrier beads (Sigma, St. Louis, MO) for 30 minutes at room temperature in a total volume of 30 ml complete DMEM. Cell-bead complexes were introduced into the RWV bioreactor (Synthecon, Inc., Houston, TX) at a ratio of 20 cells/bead, transferred to 37°C, and vessel rotation was initiated at 20 rotations per minute. Medium was replenished every 24 h and rotation speed was increased as aggregates developed to maintain cells in free-falling suspension. lished using previously described techniques [13,14], with minor modifications. Briefly, 5 × 106 Huh7 cells were trypisinized, incubated with 250 mg Cytodex-3 microcarrier beads (Sigma, St. Louis, MO) for 30 minutes at room temperature in a total volume of 30 ml complete DMEM. Cell-bead complexes were introduced into the RWV bioreactor (Synthecon, Inc., Houston, TX) at a ratio of 20 cells/bead, transferred to 37°C, and vessel rotation was initiated at 20 rotations per minute. Medium was replenished every 24 h and rotation speed was increased as aggregates developed to maintain cells in free-falling suspension. Protocols for JFH-1 in vitro transcription and HCV RNA electroporation have been described elsewhere [28]. RNA isolation and RTqPCR Total cellular RNA was isolated by the guanidine thiocy- anate method using standard protocols [29]. One μg of RNA was used for cDNA synthesis using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA), followed by SYBR green real-time quantitative PCR analysis (RTqPCR) using an Applied Biosystems 7300 real-time thermocycler as previously described [30]. Rela- tive expression levels of hepatocyte-specific genes and Phase I and Phase II metabolic genes were assessed using the primers described in [30] and normalized to β-actin levels. HCV JFH-1 and GAPDH transcript levels were determined relative to a standard curve of serially diluted plasmid containing the JFH-1 cDNA or the human GAPDH gene, respectively, using primers described in [28]. In the current study we demonstrate that RWV-cultured Huh7 cells formed complex, multilayered, 3-D aggregates that exhibited up-regulation of metabolic and hepatocyte- specific transcripts as well as increased expression and re- localization of tight junction, cell adhesion, and polarity markers. Importantly, these aggregates remained highly permissive for HCV infection suggesting that hepatic polarity does not limit HCV entry in 3-D-cultured Huh7 cells. As such, RWV-cultured Huh7 cells may represent a more appropriate physiologically relevant system for fur- ther in vitro studies of HCV entry and infection dynamics. Immunofluorescence f Immunofluorescence analysis of aggregates was per- formed as previously described [14]. Briefly, Huh7 3-D aggregates were fixed with 4% (v/v) paraformaldehyde (Sigma, St. Louis, MO), free aldehydes quenched with 50 mM NH4Cl in DPBS for 10 minutes at room temperature and cells permeabilized with 0.1% Triton-X 100 (Fisher). In parallel, Huh7 2-D monolayers were seeded in 8-well chamber slides at 80% confluence and fixed 48 hours post seeding. 3-D aggregates and 2-D monolayer cells were stained with antibodies specific for scavenger receptor class B member 1 (SR-BI) (BD Biosciences, Franklin Lakes, NJ), CD81 (AbD Serotec, Raleigh, NC), CLDN1 (Abnova, Taipei, Taiwan), CD26 (Abcam, Cambridge, UK), β-Cat- enin (Zymed, San Francisco, CA), E-cadherin (Zymed), zona occludens 1 (ZO-1) (Zymed), Occludin (Zymed) or HCV E2 (C1 [31]) overnight at 4°C, followed by incuba- http://www.virologyj.com/content/6/1/103 JFH- 1 cell culture-propagated HCV (HCVcc) viral stocks were obtained by infection of naïve Huh7-1 cells at a multiplic- ity of infection (MOI) of 0.01 focus forming units (FFU)/ cell, using medium collected from Huh7 cells on day 18 post transfection with in vitro transcribed pJFH-1 RNA as previously described [2]. The NASA-engineered RWV is a horizontally rotating cylindrical culture vessel which reduces shear and turbu- lence associated with conventional stirred bioreactors; therefore, it simulates aspects of microgravity similar to the environment encountered during fetal development [12-14]. In contrast to conventional static tissue culture systems, cells grown in the RWV are cultured in "sus- pended animation" where they are continuously free-fall- ing [12,15]. Thus, while the 2-D environment of plastic substrates may alter gene expression and prevent cellular differentiation [12,16-21], the fluid dynamics of the RWV culture system allow cells to co-localize into three-dimen- sional (3-D) aggregates, promoting in vivo-like exchange of growth factors and efficient cell-to-cell interactions [12- 14,20,21]. This in vivo-like environment thus can pro- mote transformed and primary cell lines to become more structurally and functionally similar to their in vivo coun- terparts [13,15,20-24]. Page 2 of 8 (page number not for citation purposes) Background g Hepatitis C virus (HCV), a liver tropic positive-stranded RNA flavivirus, infects ~170 million people worldwide, causing acute and chronic hepatitis and hepatocellular carcinoma [1]. However, since its discovery in 1989, a major obstacle impeding HCV research has been the lack of robust cell culture and small animal infection models. Notably significant advancement has been made with the identification of a genotype 2a HCV consensus clone (Jap- Page 1 of 8 (page number not for citation purposes) Page 1 of 8 (page number not for citation purposes) Virology Journal 2009, 6:103 HCV infection kinetics Huh7 3-D aggregates were infected with JFH-1 HCVcc at an MOI of 0.01 FFU/cell at day 1, 7 or 14 post RWV seed- ing by injection of the viral inoculum directly into the RWV. At indicated times post infection (p.i.), medium was harvested for titration analysis and RNA was isolated from ~0.5 ml of aggregates for reverse transcription fol- lowed by RTqPCR as described above. High-fidelity 3-D Huh7 RWV aggregates Figure 1 High-fidelity 3-D Huh7 RWV aggregates. (A) Phase contrast micrograph of Huh7 3-D aggregates cultured in the RWV for 14 days (400×). (B) Fluorescence micrograph of Hoechst-stained OCT sections of 3-D Huh7 aggregates (400×). (C-D) Light micrographs of H&E-stained paraffin sec- tions of 3-D Huh7 aggregates [200× (C), 600× (D)]. (*) = 100 μm microcarrier bead. Cell culture and viruses Huh7 cells (also known as Huh7/scr cells [25,26] and Huh7-1 cells [27]) were obtained from Dr. Chisari (The Scripps Research Institute, La Jolla, CA) [2] and cultured as previously described [2]. 3-D Huh7 cultures were estab- Page 2 of 8 (page number not for citation purposes) Page 2 of 8 (page number not for citation purposes) Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 High-fidelity 3-D Huh7 RWV aggregates Figure 1 High-fidelity 3-D Huh7 RWV aggregates. (A) Phase contrast micrograph of Huh7 3-D aggregates cultured in the RWV for 14 days (400×). (B) Fluorescence micrograph of Hoechst-stained OCT sections of 3-D Huh7 aggregates (400×). (C-D) Light micrographs of H&E-stained paraffin sec- tions of 3-D Huh7 aggregates [200× (C), 600× (D)]. (*) = 100 μm microcarrier bead. tion with a 1:1,000 dilution of an appropriate Alexa555- conjugated secondary antibody (Molecular Probes, Carlsbad, CA) for 1 h at room temperature. Cell nuclei were stained by Hoechst dye. Bound antibodies were vis- ualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Images were analyzed using Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss), and brightness and contrast were adjusted using Adobe® Photoshop® (San Jose, CA). Alternately, 3-D aggregates were embedded in OCT freezing medium (TissueTek, Fisher) or paraffin, sec- tioned and stained with Hoechst dye or Hematoxylin and Eosin (H&E), respectively. Establishment of Huh7 3-D Aggregates gg g To assess the utility of the RWV as a culture method for Huh7 cells, Huh7 cells were cultured on Cytodex-3 micro- carrier beads in the RWV for 26 days. Morphological and cytological examination of cultures demonstrated that Huh7 cells efficiently adhered to the collagen-coated microcarrier beads and that these individual beads then assembled to form 3-D tissue-like aggregates containing ~10–20 beads per aggregate (Fig. 1A). To determine if these aggregates consisted of multilayered cells, aggregates were embedded in OCT freezing medium or paraffin, sec- tioned, stained with either Hoechst stain (Fig. 1B) or H&E (Fig. 1C–D) and examined by fluorescence or light micro- scopy, respectively. Panels C and D highlight the multilay- ered cellular infrastructure of the Huh7 3-D aggregates, while Hoechst's staining in Panel B illustrates similar infrastructure and confirms that the aggregates are devoid of necrotic cores. Expression and Organization of Cellular Tight Junction and Polarity Markers in 3-D Huh7 Aggregates While the effect of HCV on cell polarity and TJs (and vice- versa) cannot be accurately studied in 2-D monolayer Huh7 cultures [10], these interactions are of particular Infectivity titration assay Culture supernatants were serially diluted 10-fold and used to infect triplicate Huh7 cultures in 96-well plates. At 24 h p.i., cultures were overlayed with complete DMEM containing 0.4% methylcellulose (Fluka BioChemika, Switzerland) to give a final concentration of 0.25% meth- ylcellulose. Seventy-two hours p.i., cells were fixed in 4% paraformaldehyde (Sigma), and immunohistochemically stained for HCV E2 using the anti-HCV E2 antibody C1 [31]. Viral titers are expressed as FFU/ml, determined by the average E2-positive foci number detected at the high- est HCV-positive dilution. Gene Expression within Huh7 3-D Aggregates p gg g One measure of hepatocyte differentiation is up-regula- tion of expression of transcription factors such as hepato- cyte nuclear factors (HNF) [32,33], which regulate the expression of liver secretory proteins [33] such as albumin [34], alpha-1-antitrypsin (α1AT; [35]), and transthyretin (TTR; [36]). Likewise, induction of enzymes and trans- porters involved in Phase I and II xenobiotic metabolism [37,38], which include cytochrome P450s (CYPs) and UDP-glucuronosyltransferase (UGTs) enzymes, respec- tively, is another hallmark of hepatocyte differentiation. Hence, to determine whether culturing Huh7 cells in the RWV allows for cellular differentiation at the transcrip- tional level, expression of hepatocyte-specific genes, CYPs, and UGTs were analyzed. At indicated times post seeding, total cellular RNA was extracted from 0.5 ml of 3-D Huh7 aggregates or 2-D Huh7 monolayers grown to confluence, and relative gene expression was assessed by RTqPCR analysis. As illustrated in Fig. 2, mRNA levels for the hepa- tocyte-specific genes and the CYP and UGT enzymes were significantly induced in 3-D Huh7 aggregates (relative to 2-D Huh7 monolayers) and increased in a time-depend- ent manner while cultured in the RVW. Expression and Organization of Cellular Tight Junction and Polarity Markers in 3-D Huh7 Aggregates Expression and Organization of Cellular Tight Junction and Polarity Markers in 3-D Huh7 Aggregates While the effect of HCV on cell polarity and TJs (and vice- versa) cannot be accurately studied in 2-D monolayer Huh7 cultures [10], these interactions are of particular Page 3 of 8 (page number not for citation purposes) http://www.virologyj.com/content/6/1/103 Virology Journal 2009, 6:103 Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates Figure 2 Increased differentiation-specific gene expression 3-D Huh7 RWV aggregates. At indicated time points post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 104 cells) were removed from the RWV, pelleted at 1400 RPM for 5 minutes and total RNA extracted. Expression (A) hepatocyte-specific genes, (B) Phase I (CYP) and (C) Phase II (UGT) metabolic genes in Huh7 3-D aggregates w assessed by RTqPCR. Expression of each transcript, relat interest as TJ proteins are involved in the entry of numer- ous viruses [39-41] and the TJ proteins CLDN1 [7] and occludin [8,9] have recently been shown to be involved in HCV entry. Therefore, we assessed the expression and organization of the HCV receptors (CD81 and SR-B1), cell adhesion molecules (E-Cadherin and β-Catenin), cellular TJ proteins (CLDN1, ZO-1 and Occuldin-1) and the polarity marker (CD26) in 3-D Huh7 aggregates and their 2-D monolayer counterparts (Fig. 3). The expression of known HCV receptors and polarity markers were increased in 3-D Huh7 aggregates as compared 2-D Huh7 monolayers, similar to that observed by Mee et al in polar- ized Caco-2 cells [10]. This was not a consequence of increased mRNA levels, as normalized transcript levels for all markers examined were similar between 3-D and 2-D Huh7 cultures, as determined by RTqPCR (data not shown). Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates Figure 2 Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates. At indicated time points post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 × 104 cells) were removed from the RWV, pelleted at 1400 RPM for 5 minutes and total RNA extracted. Expression of (A) hepatocyte-specific genes, (B) Phase I (CYP) and (C) Phase II (UGT) metabolic genes in Huh7 3-D aggregates was assessed by RTqPCR. Expression of each transcript, relative to 2-D Huh7 monolayer cultures, was determined using the method [50], by normalizing to β-actin expression and is graphed as fold induction compared to 2-D monolay- ers. Increased d Huh7 RWV Figure 2 Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates. At indicated time points post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 × 104 cells) were removed from the RWV, pelleted at 1400 RPM for 5 minutes and total RNA extracted. Expression of (A) hepatocyte-specific genes, (B) Phase I (CYP) and (C) Phase II (UGT) metabolic genes in Huh7 3-D aggregates was assessed by RTqPCR. Expression of each transcript, relative to 2-D Huh7 monolayer cultures, was determined using the method [50], by normalizing to β-actin expression and is graphed as fold induction compared to 2-D monolay- ers. 2-DDC T Page 4 of 8 (page number not for citation purposes) Expression and Organization of Cellular Tight Junction and Polarity Markers in 3-D Huh7 Aggregates 2-DDC T As expected, the cell adhesion molecules E-Cadherin and β-Catenin were membrane localized both in 2-D and 3-D Huh7 cultures; however, there was a profound decrease in the accumulation of nuclear β-Catenin-containing com- plexes in the 3-D Huh7 aggregates. Because atypical nuclear localization of β-Catenin in transformed cells has been well documented [42], the loss of this cancer-specific phenotype in the 3-D cultured Huh7 aggregates is consist- ent with the loss of cancer-specific markers observed in other continuous cell lines when cultured in the RWV [14,23]. Additionally, in contrast to the 2-D Huh7 mon- olayers, TJ markers localized to apicalateral and/or baso- lateral planes in the 3-D Huh7 aggregates consistent with localization patterns observed in primary hepatocytes [6,43] and normal liver tissues [44]. Finally, CD26, a cell surface ectopeptidase that localizes to the apical plane of polarized cells [45], was non-detectable in 2-D Huh7 monolayers, while, apical staining of this marker was apparent in distinct areas of 3-D Huh7 aggregates (Fig. 3). Taken together, these data demonstrate that the expres- sion and distribution of cell adhesion and TJ proteins, including known HCV entry receptors, is enhanced and more polarized in 3-D Huh7 cultures than in 2-D monol- ayers. Increased d Huh7 RWV Figure 2 HCVcc Infection of Huh7 3-D Aggregates f f gg g Because it has been suggested that hepatocyte polarization is inversely related to the permissiveness of the cell for HCVcc infection [10,11], we sought to determine if Huh7 3-D cultures were permissive for HCVcc infection. As such, 3-D Huh7 cultures were inoculated with HCVcc JFH-1 1, 7, or 14 days post RWV-seeding and culture supernatant and cellular RNA were harvested at various time points p.i. for titration of extracellular viral titers and RTqPCR analysis of viral RNA, respectively. Fig. 4A illus- trates that HCV not only infected the Huh7 3-D aggre- gates, but that the kinetics of HCV RNA expansion and Page 4 of 8 (page number not for citation purposes) Page 4 of 8 (page number not for citation purposes) Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates Figure 3 Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregat Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodie specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sectio which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which th section was taken. The scale bar equals 20 μm. Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates Figure 3 Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggrega Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibod specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y secti which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which t section was taken. The scale bar equals 20 μm. Reorganiza Figure 3 g p g j p gg g g Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates. HCVcc Infection of Huh7 3-D Aggregates Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodies specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z section was taken. The scale bar equals 20 μm. HCVcc infection as cells infected 1 day post RWV seeding (Fig. 4B-C), suggesting that differentiation and polariza- tion does not negatively affect HCVcc infection in this 3- D cell culture model. infectious virus production increased exponentially to lev- els comparable to those reported using the robust 2-D Huh7 system [2,46]. To determine the percentage of cells expressing HCV proteins, indirect immunofluorescence analysis of infected 3-D Huh7 aggregates was performed. Fig. 4B shows that the majority of Huh7 cells were positive for the HCV E2 glycoprotein and that the entire aggregate was permissive for HCV infection rather than just the cells at the periphery, demonstrating that HCV can spread throughout the aggregates. Importantly, Fig. 4D and 4E illustrate that aggregates allowed to differentiate in the RWV for 7 or 14 days were as equally permissive for Page 5 of 8 (page number not for citation purposes) http://www.virologyj.com/content/6/1/103 Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 Robust HCVcc infection in 3-D Huh7 RWV cultures Figure 4 Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul- ture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohis- aggregates relative to Huh7 monolayers (Fig. 2). Addition- ally, we observed increased expression and organization of cellular HCV receptors, cell adhesion, tight junction and polarity-specific proteins, and the loss of cancer-asso- ciated nuclear localization of β-Catenin, in RWV 3-D Huh7 aggregates as compared to 2-D monolayers (Fig. 3). These data therefore suggest that the RWV environment promotes differentiation of Huh7 cells down a more hepatocyte-like route. Importantly, since these 3-D Huh7 cultures remain highly permissive for HCVcc infection, this system represents a new in vitro cell culture system for the study of HCV infection and antiviral drug studies in more polarized, xenobiotically-competent cells. Relevant to the study of HCV, expression of the HCV receptors CD81 and SR-B1 were both diffuse and poorly organized in 2-D cultured Huh7s cells, while their expres- sion was increased and localized to apical TJ regions and basolateral-sinusoidal surfaces in 3-D aggregates. Like- wise, TJ proteins, which typically localize to the apical sur- face in polarized hepatocytes [43], were more concentrated at the apical surface of 3-D Huh7 aggregates as compared to monolayer cultures. Notably however, the TJ protein CLDN1, a recently identified HCV receptor [7], not only localized to TJs, but was also present at both api- cal and basolateral surfaces in 3-D aggregates. This locali- zation pattern is in agreement with other studies [47] and the model proposed by Reynolds et al., describing tight- junctional (apical) and nonjunctional (basolateral) forms of CLDN1 in polarized hepatocytes [44]. As suggested by Mee et al, it may be that these non-junctional pools of CLDN1 have a direct role in HCV entry [11]. Interestingly, Battle et al., have demonstrated a correlation between HNF4α and cell adhesion and TJ molecules expression and organization [48]. Whether this is also the case in the 3-D Huh7 aggregates, which have increased HNF4α expression (Fig. 2A) remains to be determined. http://www.virologyj.com/content/6/1/103 Nonethe- less, the ability of 3-D cultured Huh7 cells to better organ- ize cell adhesion and TJ proteins is a phenotype consistent with other RWV-cultured cell types [14,21,23]. As such, RWV-cultured Huh7 cells provide an appropriate model for investigating HCV entry, particularly the interaction, organization, and stoichiometry of HCV receptors and TJ proteins. Additional analyses to determine the extent of differentiation and polarization of 3-D Huh7 aggregates is still warranted and a focus of ongoing studies. Robust HCVcc infection in 3-D Huh7 RWV cultures Figure 4 Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul- ture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohis- tochemical analysis of 10-fold serially diluted culture superna- tants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 sec- ondary antibody. Images were captured via confocal micros- copy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z sec- tion was taken. The scale bar equals 20 μm. Robust HCVcc infection in 3-D Huh7 RWV cultures Figure 4 Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul- ture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Discussion Here we demonstrate that Huh7 cells cultured in RWV bioreactors form multi-layered tissue-like aggregates that are phenotypically distinct from traditional Huh7 2-D monolayers (Fig. 1 and 2). Specifically, the RWV-environ- ment promoted increases in hepatocyte-specific, as well as Phase I and II metabolic gene transcripts in 3-D Huh7 Page 5 of 8 (page number not for citation purposes) Page 5 of 8 (page number not for citation purposes) Virology Journal 2009, 6:103 Robust HCVcc infection in 3-D Huh7 RWV cultures Figure 4 Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul- ture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohis- tochemical analysis of 10-fold serially diluted culture superna- tants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 sec- ondary antibody. Images were captured via confocal micros- copy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z sec- tion was taken. The scale bar equals 20 μm. p g The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors' contributions BS and VT participated in the design of the study, per- formed the experiments and drafted the manuscript. SLU designed the study and participated in drafting the manu- script. All authors read and approved the final manu- script. 14. Lamarca HL, Ott CM, Honer Zu Bentrup K, Leblanc CL, Pierson DL, Nelson AB, Scandurro AB, Whitley GS, Nickerson CA, Morris CA: Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion. Placenta 2005, 26:709-720. 15. Goodwin TJ, Prewett TL, Wolf DA, Spaulding GF: Reduced shear stress: a major component in the ability of mammalian tis- sues to form three-dimensional assemblies in simulated microgravity. J Cell Biochem 1993, 51:301-311. http://www.virologyj.com/content/6/1/103 Infectivity titers, expressed as FFU/ml (bars), were determined by immunohis- tochemical analysis of 10-fold serially diluted culture superna- tants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 sec- ondary antibody. Images were captured via confocal micros- copy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z sec- tion was taken. The scale bar equals 20 μm. To date, attempts to study HCV in polarized cells have been limited to colorectal adenocarcinoma Caco-2 cells [10] or HepG2 cells [11], neither of which support robust HCVcc infection. Although an inverse relationship between cell polarization and HCV entry into polarized Caco-2 [10] and HepG2 [11] cells has been observed no such phenotype was observed in 3-D Huh7 aggregates. Specifically, 3-D Huh7 aggregates, infected at various Page 6 of 8 (page number not for citation purposes) Page 6 of 8 (page number not for citation purposes) http://www.virologyj.com/content/6/1/103 Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 ice grant CA-133266 from the National Cancer Institute and the University of Illinois Chicago Council to support Gastrointestinal and Liver Disease (UIC GILD). VTC was supported by an Institutional Ruth L. Kirchstein National Research Service Award (DK-007788-07) from the National Insti- tute of Diabetes and Digestive and Kidney Diseases. ice grant CA-133266 from the National Cancer Institute and the University of Illinois Chicago Council to support Gastrointestinal and Liver Disease (UIC GILD). VTC was supported by an Institutional Ruth L. Kirchstein National Research Service Award (DK-007788-07) from the National Insti- tute of Diabetes and Digestive and Kidney Diseases. stages of differentiation (e.g. day 1, 7 or 14 post seeding), were equally permissive for HCVcc infection (Fig. 4B–E). Abbreviations 8. Liu S, Yang W, Shen L, Turner JR, Coyne CB, Wang T: Tight junc- tion proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J Virol 2009, 83:2011-2014. HCV: hepatitis C virus; JFH-1: Japanese Fulminant Hepa- titis; RWV: rotating wall vessel; 3-D: three dimensional; 2- D: two-dimensional; HCVcc: hepatitis C virus cell-cul- tured produced; MOI: multiplicity of infection; FFU: focus forming units; RTqPCR: real-time quantitative PCR; SR- B1: scavenger receptor class B member 1; CLDN1: clau- din-1; ZO-1: zona occludens 1; H&E: hematoxylin and eosin; p.i.: post infection; HNF: hepatocyte nuclear fac- tors; α1AT: alpha-1-antitrypisn; TTR: transthyretin; CYP: cytochrome P450s; UGT: UDP-glucuronosyltransferase; TJ: tight junction. p J 9. Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong YP, Rice CM: Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 2009, 457:882-886. 10. Mee CJ, Grove J, Harris HJ, Hu K, Balfe P, McKeating JA: Effect of cell polarization on hepatitis C virus entry. J Virol 2008, 82:461-470. 11. Mee CJ, Harris HJ, Farquhar MJ, Wilson G, Reynolds G, Davis C, van ISC, Balfe P, McKeating JA: Polarization restricts hepatitis C virus entry into HepG2 hepatoma cells. J Virol 2009, 83:6211-6221. 12. Schwarz RP, Goodwin TJ, Wolf DA: Cell culture for three-dimen- sional modeling in rotating-wall vessels: an application of simulated microgravity. J Tissue Cult Methods 1992, 14:51-57. References 1. Chisari FV: Unscrambling hepatitis C virus-host interactions. Nature 2005, 436:930-932. 2. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, Wie- land SF, Uprichard SL, Wakita T, Chisari FV: Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2005, 102:9294-9299. Conclusion Growing evidence suggests interplay between TJ protein expression, localization and function and HCV infection. Although, the current HCV infectious 2-D Huh7 cell cul- ture system does not amend itself well to elucidating these dynamic relationships, the highly HCV-permissive 3-D Huh7 cell culture system described herein more closely mimics the differentiated and polarized state of hepato- cytes. As such the RWV 3-D Huh7 cell culture system should prove useful for understanding the dynamic rela- tionship between HCV and TJ protein expression as well as elucidating how HCV interacts with and disrupts key aspects of hepatocyte physiology. 3. Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA, Rice CM: Com- plete replication of hepatitis C virus in cell culture. Science 2005, 309:623-626. 4. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich HG, Mizokami M, Bartenschlager R, Liang TJ: Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005, 11:791-796. g 5. Moradpour D, Penin F, Rice CM: Replication of hepatitis C virus. Nat Rev Microbiol 2007, 5:453-463. 6. Decaens C, Durand M, Grosse B, Cassio D: Which in vitro models could be best used to study hepatocyte polarity? Biol Cell 2008, 100:387-398. 7. Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B, Hatziioannou T, McKeating JA, Bieniasz PD, Rice CM: Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 2007, 446:801-805. http://www.virologyj.com/content/6/1/103 Furthermore, 3-D aggregates treated with PMA, a known disruptor of TJ formation [49], were no more permissive for HCV infection as compared to untreated parallel aggregates (data not shown), suggesting that the TJ barri- ers formed in 3-D Huh7 aggregates are not inhibitory for HCVcc infection. Competing interests 13. Nickerson CA, Goodwin TJ, Terlonge J, Ott CM, Buchanan KL, Uicker WC, Emami K, LeBlanc CL, Ramamurthy R, Clarke MS, Van- derburg CR, Hammond T, Pierson DL: Three-dimensional tissue assemblies: novel models for the study of Salmonella enter- ica serovar Typhimurium pathogenesis. Infect Immun 2001, 69:7106-7120. Acknowledgements Stevenson BR, Keon BH: The tight junction: morphology to molecules. Annu Rev Cell Dev Biol 1998, 14:89-109. 23. Carterson AJ, Honer zu Bentrup K, Ott CM, Clarke MS, Pierson DL, Vanderburg CR, Buchanan KL, Nickerson CA, Schurr MJ: A549 lung epithelial cells grown as three-dimensional aggregates: alter- native tissue culture model for Pseudomonas aeruginosa pathogenesis. Infect Immun 2005, 73:1129-1140. 44. Reynolds GM, Harris HJ, Jennings A, Hu K, Grove J, Lalor PF, Adams DH, Balfe P, Hubscher SG, McKeating JA: Hepatitis C virus recep- tor expression in normal and diseased liver tissue. Hepatology 2008, 47:418-427. 45. Darmoul D, Lacasa M, Baricault L, Marguet D, Sapin C, Trotot P, Bar- bat A, Trugnan G: Dipeptidyl peptidase IV (CD 26) gene expression in enterocyte-like colon cancer cell lines HT-29 and Caco-2. Cloning of the complete human coding sequence and changes of dipeptidyl peptidase IV mRNA lev- els during cell differentiation. J Biol Chem 1992, 267:4824-4833. 46. Sainz B Jr, Chisari FV: Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma- derived cells. J Virol 2006, 80:10253-10257. 4 C A i i i p g f 24. Walther I: Space bioreactors and their applications. Adv Space Biol Med 2002, 8:197-213. 25. Gastaminza P, Kapadia SB, Chisari FV: Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles. J Virol 2006, 80:11074-11081. els during cell differentiation. J Biol Chem 1992, 267:4824-4833. 46. Sainz B Jr, Chisari FV: Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma- derived cells. J Virol 2006, 80:10253-10257. p J 26. Zhong J, Gastaminza P, Chung J, Stamataki Z, Isogawa M, Cheng G, McKeating JA, Chisari FV: Persistent hepatitis C virus infection in vitro: coevolution of virus and host. J Virol 2006, 80:11082-11093. 47. J 47. Rahner C, Mitic LL, Anderson JM: Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut. Gastroenterology 2001, 120:411-422. 27. Sainz B Jr, Barretto N, Uprichard SL: Hepatitis C Virus infection in phenotypically distinct Huh7 cell lines. PLoS ONE 2009 in press. 48. Battle MA, Konopka G, Parviz F, Gaggl AL, Yang C, Sladek FM, Dun- can SA: Hepatocyte nuclear factor 4alpha orchestrates expression of cell adhesion proteins during the epithelial transformation of the developing liver. Proc Natl Acad Sci USA 2006, 103:8419-8424. p 28. Acknowledgements g y J 16. Gomez-Lechon MJ, Donato MT, Castell JV, Jover R: Human hepa- tocytes in primary culture: the choice to investigate drug metabolism in man. Curr Drug Metab 2004, 5:443-462. We thank Drs. Heather L. LaMarca and Kerstin Hönzer zu Bentrup for helpful discussions, Dr. Francis Chisari for Huh7 cells, Dr. Takaji Wakita for the JFH-1 containing plasmid (pJFH-1), Dr. Dennis Burton for the mono- clonal anti-HCV E2 human antibody (C1), Dr. Mei Ling Chen for assistance with confocal microscopy and Patricia A. Mavrogianis for paraffin embed- ding and sectioning of 3-D aggregates. g 17. Reid LM, Fiorino AS, Sigal SH, Brill S, Holst PA: Extracellular matrix gradients in the space of Disse: relevance to liver biol- ogy. Hepatology 1992, 15:1198-1203. 18. gy p gy 18. Zeilinger K, Sauer IM, Pless G, Strobel C, Rudzitis J, Wang A, Nussler AK, Grebe A, Mao L, Auth SH, Unger J, Neuhaus P, Gerlach JC: Three-dimensional co-culture of primary human liver cells in bioreactors for in vitro drug studies: effects of the initial cell quality on the long-term maintenance of hepatocyte-specific functions. Altern Lab Anim 2002, 30:525-538. This work was supported by Public Health Service grant AI-070827 from the National Institute of Allergy and Infectious Diseases, Public Health Serv- Page 7 of 8 (page number not for citation purposes) Page 7 of 8 (page number not for citation purposes) Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 http://www.virologyj.com/content/6/1/103 19. Freshney RI: Culture of animal cells: a manual of basic technique 4th edi- tion. New York, N.Y.: Wiley-Liss; 2000. 40. Coyne CB, Bergelson JM: Virus-induced Abl and Fyn kinase sig- nals permit coxsackievirus entry through epithelial tight junctions. 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Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extrac- tion. Anal Biochem 1987, 162:156-159. 49. 49. Schmitt M, Horbach A, Kubitz R, Frilling A, Haussinger D: Disrup- tion of hepatocellular tight junctions by vascular endothelial growth factor (VEGF): a novel mechanism for tumor inva- sion. J Hepatol 2004, 41:274-283. 30. Choi S, Sainz B Jr, Corcoran P, Uprichard SL, Jeong H: Characteri- zation of increased drug metabolism activity in dimethyl sul- foxide (DMSO)-treated Huh7 hepatoma cells. Xenobiotica 2009, 39:205-217. 50. 50. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25:402-408. 31. Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki Z, Gas- taminza P, Chisari FV, Jones IM, Fox RI, Ball JK, McKeating JA, Knete- man NM, Burton DR: Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 2008, 14:25-27. 32. Jochheim A, Hillemann T, Kania G, Scharf J, Attaran M, Manns MP, Wobus AM, Ott M: Quantitative gene expression profiling reveals a fetal hepatic phenotype of murine ES-derived hepa- tocytes. Int J Dev Biol 2004, 48:23-29. 33. Nacer-Cherif H, Bois-Joyeux B, Rousseau GG, Lemaigre FP, Danan JL: Hepatocyte nuclear factor-6 stimulates transcription of the alpha-fetoprotein gene and synergizes with the retinoic- acid-receptor-related orphan receptor alpha-4. Biochem J 2003, 369:583-591. 34. Kamiya A, Kinoshita T, Ito Y, Matsui T, Morikawa Y, Senba E, Nakashima K, Taga T, Yoshida K, Kishimoto T, Miyajima A: Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. Embo J 1999, 18:2127-2136. 35. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW, Pemberton PA, Remold-O'Donnell E, Salvesen GS, Travis J, Whisstock JC: The ser- pins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhi- bition, novel functions, and a revised nomenclature. J Biol Chem 2001, 276:33293-33296. Acknowledgements Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 8 of 8 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Publish with BioMed Central and every scientist can read your work free of charge 36. Dickson PW, Aldred AR, Menting JG, Marley PD, Sawyer WH, Sch- reiber G: Thyroxine transport in choroid plexus. J Biol Chem 1987, 262:13907-13915. 37. Nakata K, Tanaka Y, Nakano T, Adachi T, Tanaka H, Kaminuma T, Ishikawa T: Nuclear receptor-mediated transcriptional regu- lation in Phase I, II, and III xenobiotic metabolizing systems. Drug Metab Pharmacokinet 2006, 21:437-457. g 38. Martinez-Jimenez CP, Jover R, Donato MT, Castell JV, Gomez-Lechon MJ: Transcriptional regulation and expression of CYP3A4 in hepatocytes. Curr Drug Metab 2007, 8:185-194. p y g 39. Connolly-Andersen AM, Magnusson KE, Mirazimi A: Basolateral entry and release of Crimean-Congo hemorrhagic fever virus in polarized MDCK-1 cells. J Virol 2007, 81:2158-2164.
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Consequential LCA and LCC using linear programming: an illustrative example of biorefineries
˜The œinternational journal of life cycle assessment
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ERROR: type should be string, got "https://doi.org/10.1007/s11367-019-01650-6\nThe International Journal of Life Cycle Assessment (2019) 24:2191–2205 https://doi.org/10.1007/s11367-019-01650-6\nThe International Journal of Life Cycle Assessment (2019) 24:2191–2205 LCI METHODOLOGY AND DATABASES Abstract Purpose This paper aims to demonstrate how LCA can be improved by the use of linear programming (LP) (i) to determine the\noptimal choice between new technologies, (ii) to identify the optimal region for supplying the feedstock, and (iii) to deal with\nmultifunctional processes without specifying a certain main product. Furthermore, the contribution of LP in the context of\nconsequential LCA and LCC is illustrated. Methods We create a mixed integer linear program (MILP) for the environmental and economic assessment of new technologies. The model is applied in order to analyze two residual beech wood-based biorefinery concepts in Germany. In terms of the optimal\nconsequences for the system under study, the principle of the program is to find a scaling vector that minimizes the life cycle\nimpact indicator results of the system. We further transform the original linear program to extend the assessment by life cycle\ncosting (LCC). Thereby, two multi-objective programming methods are used, weighted goal programming and epsilon constraint\nmethod. Results and discussion The consequential case studies demonstrate the possibility to determine optimal locations of newly\ndeveloped technologies. A high number of potential system modifications can be studied simultaneously without matrix inver-\nsion. The criteria for optimal choices are represented by the objective functions and the additional constraints such as the available\nfeedstock in a region. By combining LCA and LCC targets within a multi-objective programming approach, it is possible to\naddress environmental and economic trade-offs in consequential decision-making. Conclusions This article shows that linear programming can be used to extend standard LCA in the field of technological choices. Additional consequential research questions can be addressed such as the determination of the optimal number of new production\nplants and the optimal regions for supplying the resources. The modifications of the program by additional profit requirements\n(LCC) into a goal program and Pareto optimization problem have been identified as promising steps toward a comprehensive\nmulti-objective LCSA. Keywords Eco-efficiency . Epsilon constraint . Goal programming . Mixed integer . Multi-objective optimization . Spatial LCA Keywords Eco-efficiency . Epsilon constraint . Goal programming . Mixed integer . Multi-objective opt Consequential LCA and LCC using linear programming: an illustrative\nexample of biorefineries Maik Budzinski1\n& Mattia Sisca1,2 & Daniela Thrän1,3 Received: 20 November 2017 /Accepted: 11 June 2019\n# The Author(s) 2019\n/Published online: 24 June 2019 * Maik Budzinski\nmaik.budzinski@ufz.de 1 Introduction In contrast,\nattributional LCA focuses on describing the life cycle of a\nproduct by attributing the environmental flows to the product\nunder study. The choice for either determining consequences or attribu-\ntional impacts also affects the way of modeling in terms of the\nmultifunctional problem. This problem arises when a process\nprovides more than one product that is not used within the\nsystem under study (Heijungs and Frischknecht 1998;\nHeijungs and Guinée 2007). In this case, the environmental\nburdens associated with the multifunctional process need to be\nallocated to different products. Several procedures have been\ndeveloped to solve the multifunctional problem (Heijungs and\nSuh 2002). Consequential LCA approaches prefer to use sub-\nstitution method to deal with the problem, whereas attribution-\nal LCA is a synonym for using the partitioning method\n(Ekvall and Weidema 2004; Thomassen et al. 2008; Schmidt\n2010). The ISO standard suggests the expansion of the system\nas one method to deal with multifunctional unit processes\n(ISO 2006). Also, partitioning method is recommended,\nwhere dividing unit processes and expanding the product sys-\ntem cannot be avoided. Several authors have identified that\nsystem expansion is conceptually equivalent to substitution\n(Heijungs and Guinée 2007; Tillman et al. 1994). Thereby,\nthe expansion of additional functions related to the co-\nproducts can either be carried out by subtracting the functions\nfrom the according process (substitution aka avoided burden\nmethod) or including the functions into the final demand vec-\ntor (system expansion by expanding the functional unit). In\nany way, dealing with multifunctional processes by using\npartitioning and/or substitution method depends on a model\nchoice (Majeau-Bettez et al. 2015). The choice of a certain\nmodel to deal with multifunctional processes is usually argued\nby the invertibility of the technology matrix (avoid rectangular\nmatrix) and the argumentation that the model represents real\nworld more closely than others would do (Suh et al. 2010). However, to solve systems with more products than processes\n(rectangular technology matrix), linear programming is a suit-\nable way (Heijungs and Suh 2002). The aim of this paper is, hence, to demonstrate how LCA\ncan be extended by the use of linear programming (i) to de-\ntermine the optimal choice between new technologies, (ii) to\nidentify the optimal region for supplying the feedstock, and\n(iii) to deal with multifunctional processes without specifying\na certain main product. 1 Introduction Responsible editor: Sangwon Suh Electronic supplementary material The online version of this article\n(https://doi.org/10.1007/s11367-019-01650-6) contains supplementary\nmaterial, which is available to authorized users. Electronic supplementary material The online version of this article\n(https://doi.org/10.1007/s11367-019-01650-6) contains supplementary\nmaterial, which is available to authorized users. Life cycle assessment (LCA), and in particular consequential\nLCA, is regarded as an appropriate tool for the assessment of\nenvironmental impacts of new bio-based technologies (e.g.,\nPawelzik et al. 2013). Despite the publication of various con-\nsequential modeling approaches, consequential LCA is still\nfar from a proper systematization (Zamagni et al. 2012). Although the use of consequential and attributional LCA for\ndecision-making is still being discussed (Ekvall et al. 2016;\nHertwich 2014; Plevin et al. 2014), all approaches are based\non the definition of consequential LCA that “is designed to\ngenerate information on the consequences of decisions”\n(Ekvall and Weidema 2004). Consequential LCA attempts to * Maik Budzinski\nmaik.budzinski@ufz.de 1\nHelmholtz Centre for Environmental Research – UFZ,\nPermoserstraße 15, 04318 Leipzig, Germany 2\nPolitecnico di Milano, Piazza Leonardo da Vinci 32,\n20133 Milan, Italy 3\nDeutsches Biomasseforschungszentrum (DBFZ) gemeinnützige\nGmbH, Torgauer Straße 116, 04347 Leipzig, Germany 2192 Int J Life Cycle Assess (2019) 24:2191–2205 of LCA with linear programming (LP) has a longer tradition. Azapagic and Clift (1995, 1998) introduced LP in the field of\nprocess engineering. Thereby, LP is used to allocate environ-\nmental burdens to multiple co-products by means of the mar-\nginal values at the solution of the LP model. Recent studies\nexist in the field of biomass utilization. Vadenbo et al. (2017)\nused a multi-objective optimization model to determine the\noptimal activity levels of a set of biomass process options. Kostin et al. (2012) carried out an assessment with a mixed-\ninteger linear programming approach for ethanol production\nchains. Based on a rectangular choice-of-technology model\n(Duchin and Levine 2011), Kätelhön et al. (2016) built a tech-\nnology choice model (TCM) for consequential LCA of rice\nproduction. Despite the attempts to incorporate LCA into a\nconsistent multi-objective framework combining linear pro-\ngramming and life cycle sustainability assessment (LCSA)\n(e.g., Gong and You 2017; Steubing et al. 2011; Liu et al. 2010), a conceptual discussion on how consequential LCA\nand LCC can be extended by LP is still scarce. determine how environmental flows will change due to the\nconsequences of a decision (Curran et al. 2005). 1 Introduction In doing so, we use the matrix-based\nnotation of Heijungs and Suh (2002) and create a rectangular\nmixed integer linear program and carry out a case study to\nillustrate its applicability. Thereby, two wood-based\nbiorefinery concepts are analyzed in terms of the optimal con-\nsequences for the total system under study. Furthermore, we\nmodify the original linear program into a weighted goal pro-\ngramming problem and a Pareto optimization model those\nextend the environmental focus on sustainability by adding\nthe economic perspective through a life cycle costing (LCC)\napproach. 3.1 Goal and scope Goal and scope\nstudy concerns two exemplar\nepts and data are based on\n6). The first biorefinery co\nnosolv lignin, and biomethan\nuces the same products as the\ns replaced by ethanol. The an\nmultifunctional biorefinery p\ne 1. The goal of the exempl\nly, we want to determine th\nopriate to reduce environmen\nnt production system. Second\nh site the biorefineries shoul\nly, to identify districts for\ntocks. 1\nAnnual input and output of b\nBi\nethane (m3)\n5.0\nene (kg)\n4. olysis lignin (MJ)\n1.9\nol (MJ)\n0 The study concerns two exemplary biorefinery concepts. The\nconcepts and data are based on Budzinski and Nitzsche\n(2016). The first biorefinery concept produces ethylene,\norganosolv lignin, and biomethane. The second configuration\nproduces the same products as the first one except for ethylene\nthat is replaced by ethanol. The annually produced amounts of\nthe multifunctional biorefinery processes are illustrated in\nTable 1. The goal of the exemplary case study is threefold. Firstly, we want to determine the biorefinery that is more\nappropriate to reduce environmental impacts in regard to the\ncurrent production system. Secondly, we want to determine at\nwhich site the biorefineries should be located (Fig. 1). And\nfinally, to identify districts for supplying woody biomass\nfeedstocks. An interesting but not often recognized characteristic of\nLCA is the existence of a price model beside the quantity\nmodel (As = f), which is similar to input-output models. This\nbecomes obvious when a price vector α is introduced (cf. Heijungs et al. 2013). Using this price vector, it is possible\nto compute the value added v per process. Table 1\nAnnual input and output of biorefinery processes\nBiorefinery 1\nBiorefinery 2\nBiomethane (m3)\n5.05·107\n5.05·107\nEthylene (kg)\n4.14·107\n0\nHydrolysis lignin (MJ)\n1.96·109\n1.96·109\nEthanol (MJ)\n0\n1.87·109\nOrganosolv lignin (kg)\n6.22·107\n6.22·107\nSodium hydroxide (kg)\n−5.04·105\n−5.04·105\nMagnesium sulfate (kg)\n−3.57·105\n−3.57·105\nPhosphorus (kg)\n−3.57·105\n−3.57·105\nSulfur (kg)\n−3.57·105\n−3.57·105\nSulfuric acid (kg)\n−3.76·106\n−3.76·106\nFodder yeast (kg)\n−2.32·106\n−2.32·106\nLime, packed (kg)\n−3.84·104\n−3.84·104\nRefrigerant (kg)\n−4.94·104\n0\nHeat, natural gas (MJ)\n−5.86·107\n0\nElectricity, natural gas (kWh)\n−1.08·109\n−1.35·109\nChemical factory (unit)\n−1.05·108\n−9.52·107\nTreated wastewater (m3)\n−3.33·10−2\n−3.33·10−2\nWater (kg)\n−2.84·106\n−3.16·106\nBeech wood (kg dm)\n−4.00·108\n−4.00·108 Table 1\nAnnual input and output of biorefinery processes v ¼ A0α\nð4Þ ð4Þ h ¼ Qg The so-called multifunctional problem arises if a process\ngenerates more than one product and if and only if the deliv-\nered functions are not used in the same proportion in the sys-\ntem (Heijungs and Frischknecht 1998). The most common\napproaches to solve the multifunction problem can be classi-\nfied into partitioning method and substitution method\n(Heijungs and Guinée 2007). By applying these procedures,\nthe technology matrix A becomes square, since an equal\namount of rows and columns exists. The partitioning method\ndivides the multifunctional process into processes that are\nmono-functional by the use of allocation factors. Several ways\nto gain these factors are possible, e.g., due to physical or\nmonetary characteristics of the products. The substitution\nmethod requires the definition of a mono-functional process,\nwhich provides the avoided product to the multi-functional\nprocess. Adding this process to the technology matrix A\nmakes its inversion possible. 2 Computational structure of LCA and LCC The computational structure of LCA is comprehensively de-\nscribed by Heijungs and Suh (2002). Mathematically, the first\nstep of life cycle inventory (LCI) analysis is the determination\nof the scaling vector s. The components of this vector scale\nprocesses of a system up or down such that the output of unit\nprocesses exactly matches the final demand. The final demand\nvector f includes the reference flows r, which are defined\nwithin the goal and scope of a LCA study. The scaling vector\ncan be determined by the multiplication of the inverse of tech-\nnology matrix A with the final demand vector. A major issue for standard life cycle assessment, which\naddresses one state of a particular product system, might be\nthe fact that no optimal production mixes can be determined. Usually, in LCA, this point is addressed by creating a small\nnumber of scenarios analyzing different product system alter-\nnatives iteratively (e.g., Budzinski and Nitzsche 2016; Renouf\net al. 2018). However, such procedure can be very time-con-\nsuming. Particularly in consequential LCA, this could be an\nissue. In many cases, not only the consequences of a potential\ndecision need to be described, but rather the optimal decision\nitself shall be identified. To overcome limitations in LCA,\namong other methods such as fuzzy programming (Tan et al. 2008) or graph methods (Vance et al. 2015), the combined use s ¼ A−1 f; with f i ¼\n> 0if i∈r\n0otherwise\n\u0001\nð1Þ s ¼ A−1 f; with f i ¼\n> 0if i∈r\n0otherwise\n\u0001\nð1Þ ð1Þ 2193 Int J Life Cycle Assess (2019) 24:2191–2205 g ¼ Bs ð3Þ 3 Exemplary case study The next step of the LCI analysis is to specify the inventory\nvector g according to the reference flows. This is done by\nmultiplying the intervention matrix B with the scaling vector. In the following sections, we demonstrate how the previously\nmentioned equations can be modified to create a rectangular\nchoice-of-technology model in regard to the goal and scope of\nthe study (Sect. 3.1). Therefore, we define a mixed integer\nlinear program (Sect. 3.2) and determine the results of an\nexemplary case study (Sect. 3.3). To include LCC aspects,\nwe transform the original problem into a weighted goal pro-\ngramming problem and a Pareto optimization model in Sects. 3.4 and 3.5, respectively. ð2Þ 1 http://www.cechemnet.de/Netzwerk/cechemnet_en v ¼ A0α To determine the value added in terms of a reference flow,\nthe elements of the scaling vector s can be multiplied by the\nvalue added of the corresponding processes, where 1 is the\nsummation operator (vector of ones). v0\nscaled ¼ 1 diag v\n0\n\u0003 \u0004\ns\n\u0003\n\u0004\nð5Þ ð5Þ ð5Þ This value added vector can then be used to determine life\ncycle costs (LCC). Heijungs et al. (2013) define life cycle cost\nof a composite final demand as l ¼ ∑j−vscaled\nj\n, where j runs\nover all case such that technical coefficient aij > 0, where ref-\nerence flows i is such that fi ≠0. In other words, the life cycle\ncosts of reference flows, which are specified in the final de-\nmand vector, are the negative sum of the corresponding entries\nin the scaled value added vector. 2194 Int J Life Cycle Assess (2019) 24:2191–2205 For simplicity reasons, we focus on four chemical parks of\nSchwarzheide. We assume that all chemical parks can provide\nFig. 1 Potential location for the two biorefinery types and annual availability of residual beech wood from forests within German districts (scenario\n100%) the two biorefinery types and annual availability of residual beech wood from forests within German districts (scenario Fig. 1 Potential location for the two biorefinery types and annual availability of residual beech wood from forests within German districts (scenario\n100%) Schwarzheide. We assume that all chemical parks can provide\nthe required infrastructure and utilities in the same manner. Furthermore, we suppose that either one or none of the\nbiorefinery concepts can be built at a location. Data on resid-\nual beech wood are estimated at the level of 37 administrative\ndistricts in Germany according to Polley and Kroiher (2006). For simplicity reasons, we focus on four chemical parks of\nthe Central European Chemical Network1 as potential plant\nlocations, which would in principal allow the building of the\ntwo biorefinery concepts: Leuna, Böhlen, Zeitz, and 2195 Int J Life Cycle Assess (2019) 24:2191–2205 This residual wood is currently not used and remains in forests\nafter chopping, which might be possibly used as feedstock for\nbiorefineries without inducing feedstock rivalry (Michels\n2009). Since the 100% use of available beech wood residuals\nis quite optimistic, we introduce a second scenario in which\nthe availability in each region is reduced by 50% (cf. Michels\n2009). v ¼ A0α The transport distances from the districts to the four\npotential plant locations can be covered by train and/or truck. In this study, we assume that up to 200 km, the wood is\ntransported only by truck. For distances greater than\n200 km, the wood is additionally transported by train. Thereby, we further assume that a minimal distance (between\n10 and 30 km depending on the availability of train network)\nis covered by truck. Varying transport distances for other pre-\nproducts of the biorefineries are neglected. multi-output processes are considered in the upstream system\nand, hence, is more general. The overall program can be summarized by the formulas 8\nto 11 considering that the scaling factors of biorefinery pro-\ncesses s1, s2, s3, s4, s5, s6, s7, and s8 are integers. Thereby, Eq. 8\nrepresents the objective function of minimizing environmental\nimpacts. Equation 9 illustrates the system under study includ-\ning all potential biorefineries, feedstock regions, and transpor-\ntation alternatives. Equation 10 includes the capacity con-\nstraints for feedstock availability in the regions. Equation 11\nconsiders the fact that only one of the two biorefinery options\ncan be built at a potential location. By using the linear pro-\ngramming model, we are interested in the reduction of the\ntotal environmental impacts of the current system. Hence,\nthe objective function results from Eqs. 2 and 3, and the target\nis to find a vector s that minimizes the total impacts h of the\nsystem, which is expressed by matrix A. Avisualization of the\nmodeling principle including the sub-matrices of A is given in\nFig. 2. The fundamental principle of the model is to check\nwhether environmental impacts can be reduced by biorefinery\n1 or 2 considering 4 potential locations (ABRS) and 37 regions\nof wood supply (AS). If not, the model would only choose\ncurrent processes (ARef) to provide the components of the final\ndemand. We assume that the new biorefinery products compete with\nexisting products of the current system. Hence, we define\nsubstitutable reference products for each biorefinery product:\nbiomethane vs. natural gas, bio-based ethylene vs. fossil eth-\nylene, hydrolysis lignin vs. lignite briquettes, ethanol vs. pet-\nrol, and organosolv lignin vs. polyol. The definition of these\navoided products is a long-discussed issue in LCA. Since the\ngeneral societal aim is to move towards a bio-based economy,\nwe use fossil-based references that provide the same\nfunctions. v ¼ A0α The amounts of reference flows are specified in regard to\nthe German demand of these products within the current sys-\ntem. Thereby, the reference flows need to be specified in the\nmanner that the products of current processes must provide the\nsame functions as of the biorefinery products. In the example,\nwe assume that this is guaranteed, e.g., by referring to MJ in\nthe case of ethanol and gasoline. Minimize h ¼ QBs\nð8Þ\nsubject to Minimize h ¼ QBs\nð8Þ ð8Þ e h\nQ s\nð8Þ\nsubject to subject to subject to subject to As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n:\nif i∈r\nif i∈T; S\notherwise\nð9Þ\n0≤s≤c\nð10Þ\ns1 þ s2 ≤1\ns3 þ s4 ≤1\ns5 þ s6 ≤1\ns7 þ s8 ≤1\nð11Þ As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n:\nif i∈r\nif i∈T; S\notherwise\nð9Þ As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n: ð9Þ For LCIA, we chose the category climate change taking\ninto account the three substances CO2, CH4, and N2O. ð10Þ ð11Þ As ¼ f A ¼\nABRS\n0\n0\n0\nARef\n−A*\nup\nAup\n−A**\nup\n−A***\nup\n0\n0\n0\nAS\n−A*\nS\n0\n−A*\nT\n0\n0\nAT\n0\n2\n664\n3\n775\nð12Þ by the inequation ð12Þ ð7Þ 3.2 The linear programming model According to Heijungs and Suh (2002), the principle of ex-\ntending LCA by LP is to relax the balance equations into\ninequations. Hence, we can replace the equation with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℕ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℕ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℕ The rectangular technology matrix Awith the dimension of\nproducts-by-processes consists of several sub-matrices. As ¼ f\nð6Þ\nby the inequation\nAs≥f:\nð7Þ ð6Þ As≥f: By using this formulation, the solution of such program\nwould allow flows that are greater than those specified in the\nfinal demand vector. Regarding the special case in which pro-\ncesses are defined to be strictly mono-functional, the formu-\nlation as As = f would also work for solving the LP. The for-\nmulation in Eq. (7) also provides solutions for cases in which These sub-matrices have the following dimensions: These sub-matrices have the following dimensions: ABRS\nir × jBRS (5 × 8)\nARef\nir × jRef (5 × 5)\nAup\niup × jup (17 ×\n17) ABRS\nir × jBRS (5 × 8)\nARef\nir × jRef (5 × 5)\nAup\niup × jup (17 ×\n17) −A*\nup\niup × jBRS (17 ×\n8)\n−A**\nup\niup × jS (17 × 37)\n−A***\nup\niup × jT (17 ×\n296)\n−A*\nT\niT × jBRS (8 × 8)\nAT\niT × jT. (8 × 296)\n−A*\nS\niS × jT. (37 × 296)\nAS\niS × jS.(37 × 37). with,\nir\nbiorefinery/reference product\niup\nproduct of upstream process\niT\ntransported wood\niS\nsupplied wood in a district\njBRS\nbiorefinery process\njRef\nsubstitutable reference process\njup\nupstream process\njS\nregion for supplying wood\njT\ntransport option\nThe columns of matrix ABRS represent the biorefinery op-\ntions jBRS of each type at each potential location. The rows of\nthis matrix represent the products ir which can also be pro-\nduced by current reference processes jRef. Matrix ARef\nincludes current available processes jRef that produce the same\nfunctions ir as the biorefinery processes. To address the choice for a certain district, which deliver\nthe residual beech wood, the model is extended by the matri-\nces AT, −A*\nT, AS and −A*\nS. Matrix AS is an identity matrix to\nconsider the different alternatives of beech wood supply from\nthe 37 districts. Matrices −A*\nS and ATare needed to include the\nalternatives of beech wood transport from each district to each\none of the 8 biorefinery alternatives. The linkage of the these\nalternatives to the biorefinery options is represented by matrix\n−A*\nT. Upstream processes in matrix Aup, which are needed to\nprovide the pre-products of the biorefineries, the cultivation,\nand transportation of wood and the reference products, com-\nplete the rectangular A matrix. As≥f: The program is designed as an LCA for a final demand fr\non reference flows r, which are characterized by the products\nof the biorefinery system and the corresponding reference\nproducts (biomethane/natural gas, bio-based ethylene/fossil\nethylene, hydrolysis lignin/lignite briquettes, ethanol/petrol,\norganosolv lignin/polyol). The amounts for the multiple com-\nponents of the functional unit are taken from statistical sources\n(e.g., VCI 2018; SDK 2017) and represent those of a current\nGerman demand. Other components of the final demand\nFig. 2 Simplified flowchart\nInt J Life Cycle Assess (2019) 24:2191–2205\n2196 Int J Life Cycle Assess (2019) 24:2191–2205 2196 Fig. 2 Simplified flowchart Fig. 2 Simplified flowchart includes current available processes jRef that produce the same\nfunctions ir as the biorefinery processes. To address the choice for a certain district, which deliver\nthe residual beech wood, the model is extended by the matri-\nces AT, −A*\nT, AS and −A*\nS. Matrix AS is an identity matrix to\nconsider the different alternatives of beech wood supply from\nthe 37 districts. Matrices −A*\nS and ATare needed to include the\nalternatives of beech wood transport from each district to each\none of the 8 biorefinery alternatives. The linkage of the these\nalternatives to the biorefinery options is represented by matrix\n−A*\nT. ir\nbiorefinery/reference product\niup\nproduct of upstream process\niT\ntransported wood\niS\nsupplied wood in a district\njBRS\nbiorefinery process\njRef\nsubstitutable reference process\njup\nupstream process\njS\nregion for supplying wood\njT\ntransport option Upstream processes in matrix Aup, which are needed to\nprovide the pre-products of the biorefineries, the cultivation,\nand transportation of wood and the reference products, com-\nplete the rectangular A matrix. The program is designed as an LCA for a final demand fr\non reference flows r, which are characterized by the products\nof the biorefinery system and the corresponding reference\nproducts (biomethane/natural gas, bio-based ethylene/fossil\nethylene, hydrolysis lignin/lignite briquettes, ethanol/petrol,\norganosolv lignin/polyol). The amounts for the multiple com-\nponents of the functional unit are taken from statistical sources\n(e.g., VCI 2018; SDK 2017) and represent those of a current\nGerman demand. Other components of the final demand The columns of matrix ABRS represent the biorefinery op-\ntions jBRS of each type at each potential location. The rows of\nthis matrix represent the products ir which can also be pro-\nduced by current reference processes jRef. f S;T ¼ 0 is introduced. We further define the first eight scaling factors for the\nbiorefinery processes to be integers. This choice is neces-\nsary, since the data collection for the biorefineries is based\non process simulation for a specific capacity. The inter-\nmediate flows and environmental interventions associated\nwith this capacity are not up- or downscalable in a linear\nmanner. The program is completed by introducing lower\nbounds (set to 0) and a vector of upper bounds c. In\nregard to the availability of residual beech wood in each\ndistrict, the data of Fig. 1 is considered as upper bounds\nfor sjS. The upper bounds c for the scaling factors of\nbiorefinery processes (sjBRS ) were set to 1, since we as-\nsume that only one biorefinery can be built at a chemical\npark. To study the origin of wood supply it is possible to analyze\nthe corresponding scaling factors of the transport processes jT. These factors represent the contribution to the overall wood\ndemand of the biorefineries. Figure 3a illustrates the solutions\nfor the 100% scenario. Thereby, the biorefinery in Zeitz is\ndelivered from regions in the middle west of Germany and\nfrom regions in the middle south. In contrast, the biorefinery\nin Schwarzheide is delivered by regions in the northeastern\npart of Germany. The LCI data for upstream biorefienry processes and\nfor the substitutable reference system is taken from the\necoinvent 3.2 database (cut-off model). The whole prob-\nlem and the corresponding data can be studied in more\ndetail in the Electronic Supplementary Material\n(MILP_LCA.xlsx). Due to the reduced wood availability in the 50% scenario,\nregions supplying the feedstock to biorefinery 1 in Leuna are\ndistributed throughout Germany (Fig. 3b). Hence, a large\ncatchment area is required to fulfill the demand of the\nbiorefinery in the scenario with reduced residual beech wood\navailability. 3.3 Results To solve the mixed integer linear program, the intlinprog\nsolver in Matlab was used. An additional scenario is an-\nalyzed in which the available amount of beech wood is\nreduced by 50%. The results for 100% scenario (1.14 Mt\ndm beech wood in Germany) indicate that two\nbiorefineries with a capacity of 0.4 Mt dm are built at\nthe considered chemical parks. Since the scaling factors\nfor biorefineries are defined as integers, not the total\navailable amount of beech wood is exhausted. The opti-\nmal locations to build the biorefineries are Zeitz and\nSchwarzheide. It is visible that for both scenarios, the\nethylene-producing biorefinery 1 is more preferable to\nreduce greenhouse gas emissions than the ethanol-\nproducing biorefinery 2 (Table 2). When reducing the\navailable residual wood for all districts by 50%, another\nlocation becomes more preferable than those of the 100%\nscenario. This is an interesting result, since one might\nassume that one of the locations identified in the 50% As≥f: Matrix ARef Int J Life Cycle Assess (2019) 24:2191–2205 2197 vector f are set to zero. The design of an LCA for a demand on\nthe functional unit does not take into account the linkage of\ndownstream processing with the modeled upstream process-\ning so that economy-wide impacts are cut off (Suh 2004). However, the modeling in regard to the functional unit is a\ngeneral principle of LCA (Peters and Hertwich 2006). To en-\nsure the balance between supply and demand of transported\nbeech wood, the constraint\n0\n0\nAS\n−A*\nS\n0\n−A*\nT\n0\n0\nAT\n0\n\u0005\n\u0006\ns ¼\nf S;T ¼ 0 is introduced. would be chosen. The total reduction of the impacts on\nclimate change compared to the impacts of the current\nsystem (hRef = QRef ∙BRef ∙ARef\n−1 ∙fr) is 4.76 Mt/a CO2\neq. in the 100% scenario and 2.38 Mt/a CO2 eq. in the\n50% scenario. The contribution analysis for the 100% scenario shows\nthat current substitutable processes still provide the ma-\njor part of the overall GHG emissions (lignite production\n88.48%, natural gas production 8.24%, petrol production\n1.95%, ethylene production 1.05%, polyol production\n0.25%) whereas the biorefinery system plays a minor\npart (biorefineries 0.12%, wood production −0.20%, oth-\ner upstream processes including transport 0.12%). Due to\nthe feedstock availability of German beech wood resid-\nuals, only a small ratio of current GHG emissions could\nbe reduced. Detailed information on contribution analy-\nses is provided in the Electronic Supplementary Material\n(MILP_LCA.xlsx). f S;T ¼ 0 is introduced. 3.4 Inclusion of LCC by multi-objective optimization For example, it might also reasonable to explicitly take\ninto account the profit for reference processes competing with\nbiorefineries, or to use discounted cash flow analysis taking\ninto account the time value of money (e.g., net present value). However, additional data must be collected. For simplicity\nreasons, we chose the standard approach of LCC in this study. Minimize whdþ\nh þ wzd−\nz\nð15Þ\nsubject to Minimize whdþ\nh þ wzd−\nz\nð15Þ ð15Þ subject to subject to subject to As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n:\nif i∈r\nif i∈t\notherwise\nð16Þ As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n: ð16Þ QBs þ d−−dþ ¼ h\nð17Þ\nps þ d−−dþ ¼ z\nð18Þ\n0≤s≤c\nð19Þ\ns1 þ s2 ≤1\ns3 þ s4 ≤1\ns5 þ s6 ≤1\ns7 þ s8 ≤1\nð20Þ QBs þ d−−dþ ¼ h\nð17Þ\nps þ d−−dþ ¼ z\nð18Þ\n0≤s≤c\nð19Þ\ns1 þ s2 ≤1\ns3 þ s4 ≤1\ns5 þ s6 ≤1\ns7 þ s8 ≤1\nð20Þ QBs þ d−−dþ ¼ h\nð17Þ\nps þ d−−dþ ¼ z\nð18Þ ð17Þ ð19Þ 0≤s≤c To deal with multiple objectives in linear programming,\nbasically, three groups can be distinguished: a priori, interac-\ntive, and a posteriori methods (Hwang and Masud 1979). The\nfirst class requires the definition of preferences between goals\nat the beginning of the solution process. The latter class re-\nquires the prioritization at the end after the presentation of all\nPareto optimal solutions. To illustrate the pros and cons of the\nbroad range of applications, we modify the linear program in\nthe next sections choosing weighted goal programming for an\na priori approach and epsilon-constraint method for an a\nposteriori approach. ð20Þ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℤ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℤ Table 3 shows the result of the GP model for both scenar-\nios. Contrary to the LP model, the optimal solution is the\nethanol concept (biorefinery 2) for both scenarios. The opti-\nmal chemical parks of the chosen biorefineries are identical to\nthose of the LP model. The regions that deliver the wood\nfeedstock to the plants are also identical. Therefore we omit\nto illustrate them again in the map. 3.4 Inclusion of LCC by multi-objective optimization For h,\nwe chose 0, since the aim of a sustainable economy can be\nregarded to be carbon neutral. For the profit target z, we\nchoose an arbitrarily large value (1.00E + 15 €) which is\nequivalent to maximize the objective function. The weighting\nfactors w result in a normalization and prioritization of the\ntarget deviations. For greenhouse gas emissions, we use the\nweighting factor wh = 0.025 which is the value of expected\ndamage costs in terms of €/kg CO2-eq (De Bruyn et al. 2010). The choice of this factor is concerned by uncertainty,\ncomparable to those related to the monetization of environ-\nmental impacts. Due to the illustrative character of this study,\nwe do not assess this in detail. However, robust optimization\nmay take uncertainty of parameters explicitly into consider-\nation (Wang and Work 2014). The weighting factor wz is set to\n1, since the unit of the profit target is €. Equation 13 consists of two major terms. The first term\nconsiders the cash-flows related directly to the biorefinery\nprocesses jBRS including revenues for biorefinery products processes jBRS including revenues for biorefinery products\n( ∑\niBRS¼1\nIBRS\nABRS\niBRS;jBRS \u0002 αiBRS ), payments for pre-products\n( ∑\niup¼1\nIup\nA*\niup;jBRS \u0002 αiup ), payments for wood ( ∑\niT¼1\nIT\nA*\niT;jBRS \u0002 αiT ),\nand other payments such as for personal and insurance\n( ∑\nK\nk¼1\nFk jBRS ). The second term represents the payments related\nto the transportation options jT of wood. This expression can\nbe simplified in matrix notation as Maximize z ¼ pËCs;\nð14Þ ð14Þ where the components of vector p specify the profit of pro-\ncesses j. This LCC approach is congruent to the proposed one by\nHeijungs et al. (2013), but differs in the way that only those\ncosts are taken into account, which are relevant for the invest-\nment decision for the biorefinery options. This choice is con-\ncerned with taking the view of potential investors maximizing\nthe profit of a biorefinery. Thereby, only the costs are consid-\nered that are relevant for the decision on the profitability of the\nbiorefinery (including costs for transportation and additional\ninputs). Besides to this simple LCC approach, however, there\nare further possibilities to consider cost information by the\nmodel. 3.4 Inclusion of LCC by multi-objective optimization Since we have not considered transportation and other costs so\nfar in this case study, we adopt the model by introducing an\nadditional objective for life cycle costing. In doing so, we\nintroduce for the specific biorefineries an additional input fac-\ntor matrix F with subcategories k (expressed in monetary\nunits): personal costs, taxes, insurance, and administration. As Heijungs et al. (2013) showed, the consideration of costs\nfor the inputs and outputs of the processes of the technology\nmatrix A can be carried out by using a price vector α. Since\nwe focus on the investor’s perspective, price information is\nonly required for products that are relevant for the profitability\nof the biorefineries. The additional objective function, which\nincludes costs for processed and produced commodities of the\nbiorefinery processes as well as the costs for transported beech\nwood, can be derived as Int J Life Cycle Assess (2019) 24:2191–2205 2198 Table 2\nResults of the mixed integer linear program, per annum Table 2\nResults of the mixed integer linear program, per annum\n100% scenario\n50% scenario\nBiorefinery 1, Leuna (pc.)\n0\n1\nBiorefinery 2, Leuna (pc.)\n0\n0\nBiorefinery 1, Zeitz (pc.)\n1\n0\nBiorefinery 2, Zeitz (pc.)\n0\n0\nBiorefinery 1, Böhlen (pc.)\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n0\nBiorefinery 1, Schwarzheide (pc.)\n1\n0\nBiorefinery 2, Schwarzheide (pc.)\n0\n0\nNatural gas (m3)\n9.580053·1010\n9.585027·1010\nFossil ethylene (kg)\n5.017197·109\n5.058598e·109\nLignite (MJ)\n3.214008·1013\n3.214204·1013\nPetrol (MJ)\n7.340000·1011\n7.340000·1011\nPolyol (kg)\n4.342704·108\n4.964352·108\nh (kg CO2-eq.)\n7.152422·1011\n7.156019·1011\nPotential savings h −hRef (kg CO2-eq.)\n−7.193181·108\n−3.596181·108 Maximize z ¼\n∑\njBRS¼1\nJ BRS\n∑\niBRS¼1\nIBRS\nABRS\niBRS;jBRS \u0002 αiBRS\n \n! −\n∑\niup¼1\nIup\nA*\niup;jBRS \u0002 αiup\n \n! −\n∑\niT ¼1\nIT\nA*\niT ;jBRS \u0002 αiT\n \n! −∑\nK\nk¼1\nFk jBRS\n \n! \u0002 sjBRS−∑\njT¼1\nJ T\n∑\niup¼1\nIup\nA***\niup;jT \u0002 αiup\n \n! \u0002 sjT\nð13Þ ð13Þ Fig. 3 Optimal regions to supply beech wood to the biorefineries Fig. 3 Optimal regions to supply beech wood to the biorefineries Fig. 3 Optimal regions to supply beech wood to the biorefineries Fig. 3 Optimal regions to supply beech wood to the biorefineries 2199 Int J Life Cycle Assess (2019) 24:2191–2205 respectively. The target function of the goal program mini-\nmizes the deviations in terms of the desired values. 3.4 Inclusion of LCC by multi-objective optimization The reason for choosing\nbiorefinery 2 is the higher priority to minimize the negative\ndistance to the economic target value (d−\nz ) compared to the\npriority of minimizing the positive distance to the environ-\nmental target value (dþ\nh ). Thus, the total annual amount of\nenvironmental savings is reduced from 0.72 Mt/a and 0.36 Mt/\na CO2-eq to 0.37 Mt/a and 0.18 Mt/a CO2-eq in the 100% 3.4.1 Goal programming In weighted goal programming, the objectives are taken ex-\nplicitly into account as constraints (Miller and Blair 2009). The principle of goal programming (GP) is to use slack vari-\nables dh and dz that measure the deviation from the desired\nenvironmental and economic target values h and z, Int J Life Cycle Assess (2019) 24:2191–2205 2200 Table 3\nResults of the goal program, per annum\n100% scenario\n50% scenario\nBiorefinery 1, Leuna (pc.)\n0\n0\nBiorefinery 2, Leuna (pc.)\n0\n1\nBiorefinery 1, Zeitz (pc.)\n0\n0\nBiorefinery 2, Zeitz (pc.)\n1\n0\nBiorefinery 1, Böhlen (pc.)\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n0\nBiorefinery 1, Schwarzheide (pc.)\n0\n0\nBiorefinery 2, Schwarzheide (pc.)\n1\n0\ndþ\nh (kg CO2-eq.)\n7.155941·1011\n7.157785·1011\nd−\nz (€)\n9.999991·1013\n9.999995·1013\nProfit z−d−\nz (€)\n9.174736·107\n4.509840·107\nPotential savings hRefS−dþ\nh (kg CO2-eq.)\n−3.674181·108\n−1.830181·108 Table 3\nResults of the goal program, per annum process-based LCC. However, a possible way is to implement\nan additional constraint in the LP model that ensures the ex-\nceeding of a minimal positive profit value. scenario and the 50% scenario, respectively. The annual\nprofits are 92 Mio € and 45 Mio € in the 100% scenario and\nthe 50% scenario, respectively. When introducing economic\naspects, this result is similar to the one of Budzinski and\nNitzsche (2016) who also concluded that the ethanol-\nproducing concept has a better economic performance than\nthe ethylene-producing concept. However, in Budzinski and\nNitzsche (2016), only the ethanol biorefinery is profitable,\nwhich is contrary to the results of this study. The reason for\nthat is the authors’ use of a dynamic cost calculation approach\nthat takes the time value of money into account. Thereby, the\ninternal rate of return on the investment must be at least equal\nthe minimum rate of return a decision-maker is willing to\naccept. The time value of money usually is not considered in 3.4.2 Epsilon-constraint method Comparing this difference with the\noverall GHG emissions of the system, one might assume that\nthe results are not reliable in terms of data inaccuracies. To\naddress this point, we have a look at the reference flows of the\nfinal demand vector. The chosen amounts represent an esti-\nmated total demand for Germany and, hence, are quite high. However, lower values would not lead to different results and\nwould not reduce uncertainty. In fact, the choice of values for\nthe considered reference flows is arbitrary. The determination\nof optimal locations for biorefineries is, hence, not affected by\nthis choice. Since we here assume identical conditions at the\npotential chemical parks, the identification of optimal loca-\ntions is only determined by the transport distances and the\ncorresponding impacts and costs. On the contrary, the choice\nfor the type of biorefinery in terms of environmental impacts\nis mainly dominated by its emissions and those of the refer-\nence processes. In terms of profit, the optimal solution is\nmainly dominated by the corresponding costs for biorefinery\n(pre)-products. In conclusion, the results for the optimal\nchoices of locations for biorefineries may be considered being\nrobust in the face of data uncertainties in other parts of the\nmodel. A problem that might result is numerical issues when\nsolving a problem with large discrepancies in order of Pareto optimal solutions are solutions that cannot be im-\nproved in any of the two objectives without degrading the\nother objective. The Pareto optimal solutions of the 100%\nscenario are illustrated in Fig. 4. In this example, three\nPareto optimal solutions are identified. The dominated solu-\ntions, which are not Pareto optimal, are below the blue line. However, more comprehensive problems can result in various\nnon-dominated solutions. Solution P1 suggests biorefinery 1\n(ethylene) in Zeitz and in Schwarzheide. In contrast to this\nenvironmentally most preferable solution, P3 contains the\nmost preferable solution in terms of profit (Table 4). Thereby, biorefinery 2 (ethanol) is located in Zeitz and\nSchwarzheide. The solution P2 can be interpreted as a com-\npromise between these extreme solutions of maximal profit\nand minimal impacts on climate change. Furthermore, the\nsolution may become worthwhile due to the fact that both\nbiorefinery concepts (ethylene and ethanol) would be built\nin Zeitz and Schwarzheide. This mixture of technologies\nmight be also interesting for decision-making. 3.4.2 Epsilon-constraint method A disadvantage of goal programming might be the require-\nment of defining of preferences at the beginning of the solu-\ntion process. Alternatively, a posteriori methods are available\nto prioritize between goals after the generation of all Pareto\nefficient solution. In doing so, epsilon-constraint method has\nbeen already used in the field of LCA (e.g., Azapagic and Clift\n1999). Here, in this study, we apply the epsilon-constraint\nmethod in GAMS (Mavrotas 2009a, b). Thereby, the target Fig. 4 Pareto front (100%\nscenario). h: environmental\nimpacts; z: profit Fig. 4 Pareto front (100%\nscenario). h: environmental\nimpacts; z: profit Fig. 4 Pareto front (100%\nscenario). h: environmental\nimpacts; z: profit 2201 Int J Life Cycle Assess (2019) 24:2191–2205 Table 4\nResults of the epsilon-constraint method (100% scenario), per annum\nP1\nP2\nP3\nh (kg CO2-eq.)\n7.154561·1011\n7.155251·1011\n7.155941·1011\nz (€)\n5.183191·107\n7.178963·107\n9.174736·107\na)\nb)\nBiorefinery 1, Leuna (pc.)\n0\n0\n0\n0\nBiorefinery 2, Leuna (pc.)\n0\n0\n0\n0\nBiorefinery 1, Zeitz (pc.)\n1\n0\n1\n0\nBiorefinery 2, Zeitz (pc.)\n0\n1\n0\n1\nBiorefinery 1, Böhlen (pc.)\n0\n0\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n0\n0\n0\nBiorefinery 1, Schwarzheide (pc.)\n1\n1\n0\n0\nBiorefinery 2, Schwarzheide (pc.)\n0\n0\n1\n1 Table 4\nResults of the epsilon-constraint method (100% scenario), per annum function of profit (Eq. 14) is additionally involved in the op-\ntimization problem as solution is nearly located to another such as P2 in the 50%\nscenario, clearly is an advantage of a posteriori approaches\nover a priori methods. Minimize h sð Þ; −z sð Þ\nð\nÞ:\nð21Þ ð21Þ Minimize h sð Þ; −z sð Þ\nð\nÞ: A special look requires the influence of transportation dis-\ntances on the overall result (profit and environmental im-\npacts). In this study the transportation of feedstock only has\na limited influence on the overall profit of the biorefinery. For\ninstance, the difference between biorefinery type 2 in Leuna\ncompared to the location in Böhlen is 177,280 € per year\n(Table 5). In regard to the potential reduction of GHG emis-\nsions, the difference is even less significant. Here, the\nbiorefinery 2 in Leuna has a reduction potential at 2000 kg\nCO2 eq. per year higher than in Böhlen (Pareto point 2 and 3\nof 50% scenario, Table 5). 3.4.2 Epsilon-constraint method In contrast to\ngoal programming in which only one solution is determined in\naccordance with the a priori defined priority order, Pareto\noptimization allows the decision-maker to deal with trade-\noffs after investigating the non-dominated solutions. The non-dominated solutions of the 50% scenario are illus-\ntrated in Fig. 5. The most beneficial solution P1 in terms of\nimpacts on climate change is given by the biorefinery 1 locat-\ned in Leuna. Biorefinery 2 in Leuna is the most profitable\nsolution P3. Contrary to the 100% scenario, the compromise\nsolution P2 is near to P3 suggesting biorefinery 2 in Böhlen. An interesting fact is that P3 (in both scenarios) is the solution\nof the goal programming approach (Sect. 3.4.1). However, the\nidentification of all non-dominated solutions, even if the Int J Life Cycle Assess (2019) 24:2191–2205 2202 extension of LCA is to provide a broader and systematic as-\nsessment of consequences. The implicit environmental com-\nparison of new bio-based technologies with fossil reference\ntechnologies can be regarded as a feature that has not been\nprovided by other optimization models within the field of\nLCA. The LCC formulation used four our purpose is congru-\nent to the suggestion of (Heijungs et al. 2013), since we only\nfocus on the processes that provide the reference flows. However, it differs in the way that we only need to collect\ndata for costs which are relevant for the decision-maker as a\nbiorefinery investor and neglecting the costs for current tech-\nnologies. This and the consideration of environmental targets\ncan be interpreted as an eco-efficiency approach. However,\ninstead of simply creating the fraction between an economic\nvalue and an environmental value (ISO 2012), the approach in\nthis study allow to assess a specific target achievement, i.e. being less pollutant than current available technologies. magnitude. Back substituting the solution vectors s into Eqs. 9\nand 16, however, identified the results´ reliability in terms of\naccuracy. magnitude. Back substituting the solution vectors s into Eqs. 9\nand 16, however, identified the results´ reliability in terms of\naccuracy. Fig. 5 Pareto front (50%\nscenario). h: environmental\nimpacts; z: profit 4 Discussion LCA has been developed for the assessment of environmental\nimpacts of a product. To broaden the scope of LCA, Udo de\nHaes et al. (2004) propose three general strategies: the use of\nLCA in a toolbox, hybrid analysis, and the extension of LCA. Using LCA in a toolbox, the limitations shall be overcome by\nadditional separate models that are used without a data link. The extension of LCA is considered with one consistent linear\nmodel. Thereby, LCA and the other tool are fully compatible. Hybrid modeling as a mixture of both approaches that com-\nbines LCAwith other models and linking these by data flows. The extension with linear programming (LP) leads to a con-\nsistent linear model that determines the optimal choice among\nothers for the total system under study. Interdependent choices\nin different regions can be studied simultaneously without\nmatrix inversion, since with LP even rectangular systems\ncan be solved. The criteria for choices are represented by the\nobjective function (minimizing impacts on climate change)\nand the additional constraints (e.g., available feedstock in a\nregion). It is shown that the modification of the program by\nadditional profit requirements (LCC) into a goal program and\na Pareto optimization approach also enables to incorporate\nmultiple objectives within the decision-making process. Thereby, regional biomass availability and transport logistic\noptions can be taken into consideration. The benefit of this Dealing with multiple objectives, GP needs an a priori\nweighting of the different goals. In contrast, epsilon constraint\nmethod uses the concept of Pareto efficiency in which the\nsolution is optimal in which an increase of a target would\nresult in a decrease of another target. The advantage of GP is\nthat one optimal solution can be determined whereas in the a\nposteriori method, the decision-maker is encouraged to decide\nfor a compromise. Besides these two methods for multiple\nobjective decision making, there exist further methods, which\ncannot be discussed here. A general overview including the\npros and cons is given by Hwang and Masud (1979). Rectangular LP models for choosing technologies have\nbeen used in input-output economics. Duchin and Levine\n(2011, 2012) introduced a rectangular model to study the Fig. 5 Pareto front (50%\nscenario). h: environmental\nimpacts; z: profit Fig. 5 Pareto front (50%\nscenario). 4 Discussion But instead of sectors as in input-output\nmodels, the columns of the technology matrix in LCA repre-\nsent processes, which are usually modeled in more detail. Furthermore, these processes can be multi-functional (e.g.,\nKätelhön et al. 2016), which is contrary to input-output\nmodeling in which the assumption of homogenous sector out-\nputs is implied by creating the technical coefficient matrix\n(Miller and Blair 2009). In our example, only the biorefinery\nprocesses are multi-functional. Solving the multifunctional\nproblem for these technologies can be interpreted as a system\nexpansion approach. Furthermore, this approach is equivalent\nto substitution method, since the implicit aim of the introduced\nmodels is the substitution of current processes by less pollut-\nant processes. To compare the biorefineries, the systems are\nexpanded by current substitutable processes which produce\nthe same type of products. Due to that a programming prob-\nlem results, in which we seek to find the optimal substitution\nof current processes by a set of biorefinery alternatives. Other\nprocesses in the example are mono-functional. However, if\nother multioutput processes would be considered within the\nupstream processes of the biorefinery or the reference system,\nthe multi-functionality can be solved by the program\ninterpreting it as a kind of surplus method (Heijungs and\nSuh 2002). When minimizing the total environmental impacts\nof the system under study, larger amounts of supplied products\nwould be allowed compared with those in the final demand. It\nis crucial to take this into consideration, since more products\nand hence more functions are possible in the final supply\nvector of the optimized system. On the other hand, if products\nof current technologies are not provided by the new technol-\nogies, the program would ensure that at least the amounts of\nthe current system are generated. In this case a study would\nonly be meaningful, if processes are modeled in a sufficient\ndetailed manner. In other words, it must be decided which Compared to common consequential LCA approaches\nwhich use substitution method to solve the multifunctional\nproblem, the definition of determining products wherein all\nother co-products are summarized into one avoided product\ngroup (e.g., Weidema 2001; Suh et al. 2010) is not necessary. Here, in contrast, all products of the new technologies are\nconsidered as determining products without distinguishing\nbetween the multiple products. For each biorefinery product,\nsubstitutable reference products are determined. 4 Discussion h: environmental\nimpacts; z: profit Int J Life Cycle Assess (2019) 24:2191–2205 2203 Table 5\nResults of the epsilon-constraint method (50% scenario), per annum\nP1\nP2\nP3\nh (kg CO2-eq.)\n7.15709438·1011\n7.15778471·1011\n7.15778469·1011\nz (€)\n2.514068·107\n4.492112·107\n4.509840·107\nBiorefinery 1, Leuna (pc.)\n1\n0\n0\nBiorefinery 2, Leuna (pc.)\n0\n0\n1\nBiorefinery 1, Zeitz (pc.)\n0\n0\n0\nBiorefinery 2, Zeitz (pc.)\n0\n0\n0\nBiorefinery 1, Böhlen (pc.)\n0\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n1\n0\nBiorefinery 1, Schwarzheide (pc.)\n0\n0\n0\nBiorefinery 2, Schwarzheide (pc.)\n0\n0\n0 Table 5\nResults of the epsilon-constraint method (50% scenario), per annum process, e.g., a certain ethylene plant, is substitutable by the\nnew process. Even in LCA, which uses more disaggregated\nprocesses than input-output analysis does, this is usually not\nthe case. For instance, in our example only one process for\nethylene generation represents a bundle of ethylene plants. Furthermore, the ethylene production is mono-functional,\nwhich does not represent real-life complexity, since ethylene\nusually is produced with other co-products (e.g., propylene). Including those co-products, however, can lead to binding\nconstraints that are not achievable by the new technologies. In our example this becomes obvious if the bundled process of\nfossil ethylene production would additionally provide propyl-\nene. Since it seems not realistic to achieve a model that repre-\nsents all production processes at a plant level, the only viable\nway is to make a model choice. The question is thus, what\nimplicit assumption is appropriate in terms of the goal of the\nstudy (Zamagni et al. 2012). A general discussion is beyond\nthe scope of this article. However, in terms of the predominant\ngoal of the exemplary case studies, to identity the best alter-\nnative from a set of new biorefinery options, we argue that\nusing aggregated mono-functional processes with average da-\nta seems to be sufficient. To increase the reliability of future\ncase studies, however, the assessments should be enhanced by\nsensitivity analyses using different approaches for allocating\nthe environmental impacts to the substitutable reference prod-\nucts (e.g., partitioning by physical and monetary factors). optimal choice of technologies. Before that, Carter (1970)\napplied a square choice-of-technology model using linear pro-\ngramming in a similar manner. Our model works in principle\nthe same way. References Azapagic A, Clift R (1995) Life cycle assessment and linear program-\nming – optimization of product system. Comput Chem Eng 19:229–\n234 Azapagic A, Clift R (1998) Linear programming as a tool in life cycle\nassessment. 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Delft, CE\nDelft, March 2010 4 Discussion In our opin-\nion, these choices for all products of the biorefineries seem\ncloser to reality than whether clustering several co-products 2204 Int J Life Cycle Assess (2019) 24:2191–2205 Acknowledgements This work has been carried out within the project\nSpitzencluster BioEconomy (031A078A). Acknowledgements This work has been carried out within the project\nSpitzencluster BioEconomy (031A078A). into one avoided product group or declaring the multi-\nfunctional processes to be mono-functional by using\npartitioning method. Funding information The German Federal Ministry of Education and\nResearch financially supported this project. Towards a comprehensive LCSA framework, some\npotential directions for further research shall be\nbroached. The characterization matrix Q can be easily\nmodified by corresponding characterization factors to\ntake into account additional environmental impact cate-\ngories (e.g., midpoint or endpoint categories). Targets for\nvarious impact categories can be considered separately\nwithin a multi-objective framework. On the other hand,\nnormalization and weighting could be introduced within\na single environmental target function. Thus the optimi-\nzation could be carried out in terms of a single environ-\nmental score which takes into account various normal-\nized and weighted impact indicator results. In the same\nmanner various social categories could be dealt with. By\nimposing a single score for each of the three pillars of\nsustainability (LCA, LCC, social LCA), the assessment\nof technological alternatives would be extended toward a\ncomprehensive LCSA within a multi-objective frame-\nwork. By identifying environmental, social, and econom-\nic benefits of new technologies (especially the compari-\nson with existing technologies those produce equivalent\nproducts), this framework would be also suitable to sup-\nport methods for estimating the maturity of technologies\nsuch as the technology readiness level (Hicks et al. 2009). Open Access This article is distributed under the terms of the Creative\nCommons Attribution 4.0 International License (http://\ncreativecommons.org/licenses/by/4.0/), which permits unrestricted use,\ndistribution, and reproduction in any medium, provided you give\nappropriate credit to the original author(s) and the source, provide a link\nto the Creative Commons license, and indicate if changes were made. 5 Conclusions Duchin F, Levine SH (2011) Sectors may use multiple technologies si-\nmultaneously: the rectangular choice-of-technology model with\nbinding factor constraints (revised). Rensselaer working papers in\neconomics number 1101 This article showed how (mixed integer) linear program-\nming can be used to extend standard LCA towards com-\nprehensive decision-making. Additional consequential re-\nsearch questions can be addressed such as the determi-\nnation of the optimal number of new production plants\nand the optimal region for supplying feedstocks while\nalso taking into consideration transport logistic options. The implicit environmental comparison of new bio-based\ntechnologies with fossil reference technologies can be\nregarded as a feature that has not been provided by other\noptimization models within the field of LCA. The exten-\nsion of LCA by linear programming remains a consistent\nlinear model, which is able to broaden the scope for\nconsequential assessments. The benefit of this extension\nof LCA is to provide a broader and systematic assess-\nment of consequences. 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DECHEMA Gesellschaft für Chemische Technik und\nBiotechnologie. http://www.fnr-server.de/ftp/pdf/berichte/\n22001307.pdf. Accessed Nov 2017 Wang R, Work D (2014) Application of robust optimization in matrix-\nbased LCI for decision making under uncertainty. Int J Life Cycle\nAssess 19(5):1110–1118 Weidema B (2001) Avoiding co-product allocation in life-cycle assess-\nment. J Ind Ecol 4(3):11–33 Miller RE, Blair PE (2009) Input-output analysis. Foundations and ex-\ntensions. Cambridge University Press Zamagni A, Guinée J, Heijungs R, Masoni P, Raggi A (2012) Lights and\nshadows in consequential LCA. 5 Conclusions Kluwer Academic Publishers Int J Life Cycle Assess (2019) 24:2191–2205 2205 Polley H, Kroiher F (2006) Struktur und regionale Verteilung des\nHolzvorrates und des potenziellen Rohholzaufkommens in\nDeutschland im Rahmen der Clusterstudie Forst- und\nHolzwirtschaft. 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POTENSI JASA LINGKUNGAN PADA KAWASAN TAMAN WISATA ALAM KLAMONO, KABUPATEN SORONG
Jurnal Kehutanan Papuasia/Jurnal Kehutanan Papuasia (Journal of Papuasia Forestry)
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Kurey, dkk Kurey, dkk Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Abstrak Penelitian ini bertujuan untuk mengetahui jenis - jenis komoditi dan bentuk aktifitas pemanfaatan jasa lingkungan pada kawasan Taman Wisata Alam Klamono. Metode yang digunakan adalah metode deskriptif dengan teknik observasi dan wawancara. Observasi dilakukan terhadap komoditi jasa lingkungan yang ada pada kawasan Taman Wisata Alam Klamono dan wawancara dilakukan pada masyarakat di Kampung Klawana. Hasil penelitian menunjukkan bahwa komoditi jasa lingkungan terdiri atas a). potensi sumber air yang meliputi sungai Klagene dan sungai Klawana. b). Keanekaragaman hayati meliputi potensi flora dan potensi fauna c). Penyerapan karbon meliputi tumbuhan berkayu dan tumbuhan non kayu d). Panorama bentang alam. Aktivitas pemanfaatan jasa lingkungan meliputi sumber pemanfaatan tata air, flora, fauna, tumbuhan berkayu, tumbuhan non kayu dan panorama bentang alam. Potensi pengembangan jasa lingkungan dapat dinilai dengan mengunakan kriteria penilaian daya tarik, aksesibilitas, kondisi sosial ekonomi masyarakat, keamanan pengelolaan, dan sarana prasarana. Hasil skoring mengindikasikan bahwa Taman Wisata Alam Klamono dapat dikembangkan tetapi perlu pertimbangan beberapa aspek strategis oleh pemerintah setempat. Kata kunci: jasa lingkungan, wisata alam, keanekaragaman hayati, flora fauna, penyerapan karbon (The Potency of Ecosystem Services in Klamono Nature Park, District of Sorong) Akwila Johanes Kurey1 Yohanes Y. Rahawarin1 dan Dominggas M.H. Renwarin1 Jurusan Kehutanan, Fakultas Kehutanan Universitas Papua Manokwari, Papua Barat, 98314. Tlp/Fax: +62986211065. Penulis Korespondensi: Email: jo.rahawarin@gmail.com Diterima: 15 Mei 2019| Disetujui: 01 Juli 2019 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Abstract This study works toward varieties of commodity and types of ecosystem service usage activity in Klamono nature park. The study has applied descriptive method through observation and interview techniques. The observation is focusing on the ecosystem service commodities in surrounding area and interview is carried out towards the local inhabitants of Klawana village. The result pointed out that ecosystem services consist of a). water resource potency that covered Klagene and Klawana rivers, b). natural resource, in particular the potency of flora and fauna, c). carbon absorption from woody and non woody plant species in the nature park, d). landscape panorama. The usage activities of ecosystem services covered water use, flora, fauna, woody and non woody plants, as well as ecosystem landscape. The prospect of ecosystem service development can be assessed by applying criteria such as attractiveness, accessibility, socio-economy status of the inhabitant community, management security, and facilities and infrastructure. Scoring 124 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Kurey, dkk Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) results indicated that Klamono nature park can be developed, however it required a number of strategic considerations by the local government. Keywords: ecosystem service, natural tourism, natural resource, flora fauna, carbon absorption PENDAHULUAN endemis di Sumatera dan 10 marga endemis di pulau Jawa. Sejauh ini telah diketahui 154 jenis mamalia, dimana 93 jenis merupakan endemis yang terdiri atas 41 jenis dari kalangan monotremata dan marsupialia, serta 52 jenis dari kalangan kelelawar dan binatang pengerat (Petocz 1987). Potensi yang begitu tinggi membutuhkan upaya perlindungan, pengawetan dan pemanfaatan yang lestari untuk menjaga keanekaragamannya. Hutan merupakan salah satu sumberdaya alam yang multifungsi dalam menunjang kehidupan umat manusia yang tidak hanya terbatas sebagai tempat konservasi keanekaragaman hayati dan pemeliharaan fungsi ekosistem, tetapi juga menghasilkan barang dan jasa bagi masyarakat (Rahawarin 2010). Hutan sangat penting bagi kehidupan di muka bumi, terutama bagi kehidupan generasi mendatang. Kesalahan dalam pengelolaan hutan berarti menyiksa kehidupan generasi mendatang. Untuk mencegah kesalahan dalam pengelolaan hutan, maka fungsi hutan harus dipelajari dan dimengerti secara holistik (utuh). Pengelolaan hutan bukan hanya sekedar menetapkan hutan sebagai perlindungan tanah, iklim, sumber daya air dan pemenuhan kebutuhan akan kayu dan produk lainya. Namun, pengelolaan hutan harus ditujukan untuk mendayagunakan semua lahan demi kepentingan negara, bahkan dunia (Arief 2005). Kawasan konservasi selain berfungsi sebagai kawasan yang dilindungi, juga mempunyai fungsi lain sebagai sistem penyangga kehidupan dan sebagai sarana kegiatan pengembangan penelitian dan ilmu pengetahuan, pendidikan, wisata alam dan penunjang budidaya. Upaya melestarikan sumberdaya alam hayati dan ekosistemnya bukan hanya menjadi tanggung jawab pemerintah tetapi merupakan tanggung jawab semua lapisan masyarakat. Hal ini sesuai dengan amanat Undang-Undang Nomor 41 tahun 1999 pasal 68 ayat 1 dan 2 serta pasal 29 ayat 1 yang menyatakan bahwa masyarakat selain mempunyai hak menikmati kualitas lingkungan hidup yang dihasilkan hutan, memanfaatkan hutan dan hasil hutan sesuai dengan peraturan yang berlaku, tetapi juga berkewajiban ikut serta dalam menjaga kawasan hutan dari gangguan dan kerusakan (Pangesti 2005). Tujuan dari penelitian ini antara lain untuk mengetahui jenis-jenis komoditi jasa lingkungan, mengetahui aktivitas pemanfaatan komoditi jasa lingkungan Salah satu wilayah di Indonesia yang memiliki sumber daya hutan yang paling potensial adalah Papua. Formasi hutan papua kaya akan jenis, general dan famili khas yang tidak dijumpai pada formasi lain dan dapat dijadikan tanaman berguna bagi manusia. Jumlah flora papua diperkirakan 20.000-25.000 jenis dengan keendemikan diperkirakan antara 60-90% (Johns 1997; Lekitoo et al. 2017). Terdapat 124 marga tanaman berbunga endemis, bila dibandingan dengan 59 marga endemis di Kalimantan; 17 marga 125 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk sebanyak 5 orang. Variabel Penelitian Variabel yang diamati dalam pelaksanaan penelitian ini meliputi: jenis komoditi jasa lingkungan, aktivitas pemanfaatan jasa lingkungan, dan kemungkinan pengembangan pemanfaatan jasa lingkungan. Teknik Pengumpulan Data Data yang dikumpulkan terdiri atas data primer dan data sekunder. Data primer dikumpulkan dengan teknik observasi, dokumentasi dan wawancara. Data primer meliputi: jenis komoditi jasa lingkungan, keberadaan jasa lingkungan, lokasi timbulnya jasa lingkungan yang dihasilkan, dan kegiatan pemanfaatan jasa lingkungan. Data sekunder dikumpulkan dengan cara pencatatan dan dokumentasi dari instansi terkait serta studi pustaka berupa laporan penelitian yang berkaitan dengan topik penelitian. Metode dan Teknik Penelitian Penelitian ini dilakukan dengan menggunakan metode deskriptif dengan teknik observasi lapang dan wawancara. Observasi dilakukan terhadap komoditi jasa lingkungan yang ada pada kawasan TWAK, sedangkan wawancara dilakukan terhadap masyarakat yang memanfaatkan jasa lingkungan. PENDAHULUAN Responden umum adalah masyarakat yang tinggal di kampung Klawana. Jumlah responden yang diwawancarai sebanyak 30% dari total jumlah masyarakat Kampung Klawana sebanyak 110 KK yang berada di sekitar kawasan TWAK. dan mengkaji kemungkinan pemanfaatan jasa lingkungan di kawasan Taman Wisata Alam Klamono (TWAK). Pelaksanaan Penelitian Kriteria yang dinilai adalah daya tarik, aksesbilitas, kondisi lingkungan dan sosial masyarakat, keamanan dan pengelolaan, sarana dan prasarana. Pada setiap kriteria memiliki nilai dan bobot. Nilai setiap kriteria (S) yang diperoleh dari hasil akan dikalikan dengan bobot (B) kriteria dengan jumlah nilai Pengelolaan dan Analisis Data Data yang dikumpulkan dianalisis mengunakan kriteria penelitian potensi obyek dan daya tarik alam yang telah dimodifikasi dari pedoman kriteria penilaiaan pengembangan (Direktorat Jendral Perlindungan Hutan dan Konservasi Alam, Departemen Kehutanan, 2003/dan Direktorat Produk Wisata Direktorat Jendral Destinasi Pariwisat Departemen Kebudayaan dan Pariwisata 2007). Kemudian data yang dianalisis diuraikan dan ditampilkan dalam bentuk tabel dan gambar. Tempat dan Waktu Penelitian ini dilaksanakan di areal Taman Wisata Alam Klamono Kabupaten Sorong dengan lamanya waktu penelitian sekitar 3 minggu, yaitu dari tanggal 26 Januari - 16 Februari tahun 2019. Yang menjadi obyek dalam penelitian ini adalah potensi jasa lingkungan pada kawasan TWAK, sementara subyek penelitian adalah masyarakat yang berada pada kampung disekitar kawasan TWAK. @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Penentuan Responden Penentuan responden dibagi menjadi dua yaitu responden kunci (key respondent) dan responden umum (common respondent). Responden kunci terdiri dari aparat kampung sebanyak 2 orang dan tokoh masyarakat sebanyak 3 orang sehingga total responden kunci 126 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk N = Jumlah nilai unsur unsur kriteria @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA N = Jumlah nilai unsur unsur kriteria unsurunsur kriteria (N). jumlah nilai unsur-unsur kriteria diperoleh dari nilai yang sudah ada pada tabel penilaiaan yang disesuaikan dengan kondisi dan karakter lokasi obyek penelitian dan dapat dihitung mengunakan rumus seperti berikut : Penilaian potensi jasa lingkungan berdasarkan modifikasi dari Pedoman Kriteria Penilaiaan Pengembangan Direktorat Jendral Perlindungan Hutan dan Konservasi Alam, Departemen Kehutanan (2003) dan Direktorat Produk Wisata Direktorat Jendral Destinasi Pariwisat Departemen Kebudayaan dan Pariwisata (2007), dengan kriteria penilaian seperti pada tabel 1. S = B × N S = B × N Keterangan : S = Nilai kriteria S = Nilai kriteria B = Bobot nilai kriteria Tabel 1. Daftar kriteria penilaian jasa lingkungan yang telah dimodifikasi No. Unsur yang dinilai Bobot 1. Daya tarik 6 2. Aksesibilitas 5 3. Kondisi lingkungan sosial ekonomi masyarakat 5 4. Keamanan dan pengelolaan 3 5. Sarana dan prasarana 3 Nilai maximum akan diperoleh dari jumlah total skor tertinggi, sedangkan nilai minimum diperoleh dari jumlah total skor terendah. Hasil perhitungannya berupa kategori dan selang penilaian potensi jasa lingkungan pada kawasan TWAK. Nilai maximum akan diperoleh dari jumlah total skor tertinggi, sedangkan nilai minimum diperoleh dari jumlah total skor terendah. Hasil perhitungannya berupa kategori dan selang penilaian potensi jasa lingkungan pada kawasan TWAK. Selanjutnya hasil penelitian akan dikategorikan berdasarkan nilai selang, dimana selang yang ditetapkan dikategorikan dalam 3 kategori, yaitu: baik, sedang dan buruk. Nilai selang akan dihitung dari rumus : Selang (S) = S max – S min/ Banyaknya kategori Selang (S) = S max – S min/ Selang (S) = S max – S min/ Banyaknya kategori Tabel 2. Kategori selang penilaiaan potensi jasa lingkungan kawasan TWAK No. Kategori Interval/Selang 1. Buruk 1090 – 1600 2. Sedang 1601 – 2110 3. Baik 2111 - 2620 obyek wisata alam. Kriteria penilaiaan masuk dikategorikan sebagai berikut : Kategori dan interval/selang diambil sebagai acuan standarisasi untuk menentukan layak dan tidaknya suatu obyek yang akan dikembangkan sebagai g g a. Kategori Buruk berarti bahwa potensi jasa lingkungan pada TWAK tidak dapat dikembangkan. 127 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk raya Sorong - Klamono tepatnya di Kilometer 37. b. Kategori Sedang berarti bahwa potensi jasa lingkungan dapat dikembangkan dan masih diperlukan pertimbangan kembali dari pemerintah. Sama seperti sungai Klagene, sungai Klawana juga merupakan sungai yg mengalir melewati kawasan TWAK. Kondisi sungai yang masih alami dengan banyaknya vegetasi berada di sepanjang kiri dan kanan sungai. N = Jumlah nilai unsur unsur kriteria Di sebut sungai Klawana karena sungai ini adalah sungai yang mengalir keluar dari TWAK ke kampung Klawana. c. Kategori Baik berarti bahwa potensi jasa lingkungan pada kawasan TWAK harus dikembangkan sebagai nilai keunggulan bagi kabupaten sorong. Data yang dikumpulkan diolah secara tabulasi dan dianalisis secara deskriptif dan selanjutnya ditampilkan dalam bentuk tabel dan gambar/foto. Jenis Komoditi Jasa Lingkungan Berdasarkanhasil observasi lapangan pada kawasan TWAK jenis komoditi jasa lingkungan meliputi yang ditemukan berupa: 1). tata air, 2). keanekaragaman hayati, 3). penyerapan karbon, dan 4). bentuk-bentuk pemanfaatan sumber daya alam dan jasa lingkungan. Berikut ini merupakan karakteristik dari masing masing jenis komoditi jasa lingkungan: Tata air Berdasarkan hasil pengamatan terdapat sumber air yang berada di dalam kawasan TWAK, diperoleh informasi berupa keberadaan sungai Klagene dan sungai Klawana yang merupakan sungai-sungai yang mengalir di dalam kawasan TWAK. Sungai Klagene dan sungai Klawana adalah merupakan anak sungai dari sungai induk pada Distrik Klamono yaitu sungai Klasofot. HASIL DAN PEMBAHASAN Kekayaan keanekaragaman hayati merupakan salah satu potensi pada aspek jasa lingkungan yang berpontensi dikembangkan sebagai parawisata dan ekowisata. Selain itu, terdapat juga kawasan konservasi yang memiliki potensi cukup tinggi yang tersebar di berbagai tipe ekosisitem yang terdapat di dalam kawasan TWAK. Potensi lingkungan yang berada di dalam kawasan TWAK berupa panorama alam, berbagai jenis flora dan fauna serta beberapa sungai yang mengalir di dalam kawasan. Potensi tersebut merupakan potensi untuk menunjang kegiatan wisata alam. Jenis Komoditi Jasa Lingkungan Jenis Komoditi Jasa Lingkungan @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Fauna Terdapat potensi berbagai jenis satwa khususnya jenis-jenis yang telah dilindungi undang- undang seperti Kakatua Jambul Kuning (Cacatua galerita triton), Kakatua Raja (Proboscinger aterrimus), Nuri Merah Kepala Hitam (Lorius lory), Cenderawasih (Paradiseae spp.), Kasuari (Casuarius casuarius), Rusa (Rusa timorensis) dan Kangguru Pohon (Dendrolagus sp.). Tumbuhan Berkayu (woody plant) kemudian diikuti oleh jenis Dillenia indica dan Vileburna rubescent masing- masing dengan nilai INP 10,841% dan 10,202%. Untuk tingkat pancang, jenis yang memiliki INP tertinggi adalah Cleytanthus papuana dengan 15,580%, kemudian disusul oleh jenis Sphatiostemon javaensis dengan nilai INP 12,845% dan jenis Villebrunea rubescent dengan nilai INP 12,674%. Sementara untuk tingkat pohon, jenis yang memiliki nilai INP tertinggi adalah Artocarpus altilis dengan nilai 25,695%, disusul oleh jenis Pometia pinnata dengan nilai INP 24,199% dan jenis Teijsmaniodendron bogoriense dengan nilai INP 14,031%. y ( y p ) Hasil pengamatan langsung yang dapat di amati dalam Kawasan TWAK, terdapat berbagai jenis tumbuhan berkayu, yang sebagai penyerap karbon dan penyedia oksigen. Terdiri dari 49 jenis tumbuhan berkayu dari 26 family yang terdapat di Kawasan TWAK Untuk menunjang jasa linkungan dan perlindungan kawasan Konsevasi dalam peningkatan tempat wisata TWAK terdapat tiga jenis famili tumbuhan berkayu yaitu, Fabaceae, Sapindaceae, dan Moraceae, yang dominan dalam kawasan TWAK. Jenis – jenis tumbuhan berkayu yang ditemukan memiliki potensi daya tarik yang sangat menarik baik dari karakter morfologi daun, batang, bunga dan buah. Pesona ini menjadi suatu hal yang unik dan jarang ditemukan ditempat lainnya. Hal ini di tunjukan dengan kelangkaan dan endemiknya jenis-jenis yang ditemukan pada kawasan tersebut. Jenis – jenis tumbuhan berkayu yang ditemukan beserta daya tarik jasa linkungan antara lain: Artocarpus altilis, Pometia pinnata, Teijsmaniodendron bogoriense, Ficus pungens, Melanolepis multiglandulosa, Ficus trachypison, Cleytanthus papuana, Artocarpus odoratasimus, Trichospermum javanicum, Macaranga mapa Spatiostemon javensis Penyerapan Karbon Potensi jasa lingkungan berupa penyerapan karbon merupakan salah satu jasa penting dalam kawasan konservasi yang juga berperan menyediakan oksigen da udara segar dari dalam kawasan TWAK. Dengan hadirnya berbagai jenis vegetasi hutan di Kawasan TWAK, memberikan potensi ketersediaan oksigen dan udara segar yang melimpah. Hasil identifikasi di lapangan, terdapat dua kelompok tubuhan yang petensial sebagai penyerap karbondioksida dan selanjutnya sebagai agen penyedia oksigen. Trichospermum javanicum, Macaranga mapa, Spatiostemon javensis, Tumbuhan Bukan Kayu (non woody plant) Flora Berbagai jenis tumbuhan di dalam kawasan ini merupakan jenis-jenis yang umumnya tumbuh di hutan dataran rendah. Kegiatan inventarisasi telah dilakukan di dalam kawasan TWAK (Maturbongs 2011) dengan perolehan data berdasarkan tingkatannya yaitu tingkat semai ditemukan sebanyak 102 jenis, pancang 102 jenis, dan pohon 102 jenis. Dari jenis data tersebut diperoleh nilai INP pada masing-masing tingkat vegetasi yang berbeda. Untuk tingkat semai, Cleytanthus papuana memiliki nilai INP tertinggi yakni 16,649%, Sungai Klagene berasal dari mata air yang berada di luar kawansan TWAK yang tepatnya di bawah kaki gunung Mariat dan mengalir di dalam kawasan sampai bermuara pada sungai induk sungai Klasifot atau yang biasa di sebut sungai induk Klamono. Keberadaan sungai Klagene dapat di lihat dari jalan 128 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk Bird Watching g Mengamati burung (bird watching) secara langsung di dalam hutan merupakan kegiatan wisata alam yang dapat memberikan pengetahuan. Selain itu pengunjung juga dapat melakukan pengambilan gambar/foto dari obyek- obyek satwa tersebut. Kegiatan bird watching di dalam TWAK dapat dilakukan dengan mengunjungi spot-spot pengamatan burung. Terdapat berbagai macam jenis burung yang dapat dijumpai di dalam dan sekitar kawasan termasuk jenis burung endemik seperti jenis Kakatua Jambul Kuning (Cacatua galerita triton), Kakatua Raja (Proboscinger aterrimus) Nuri Merah Kepala Hitam (Lorius lory). Pemanfaatan Tata Air Pemanafaatan tata air pada sungai Klagene telah dilakukan oleh masyarakat Kampung Klawana dan Wonosari. Sungai Klagene hanya dimanfaatkan sebagai patokan atau batas alam, yang memisahkan hak ulayat pada kawasan TWAK dari 2 marga suku Moy yaitu marga Mambringgofok dan Osok. Pemanfaatan tata air pada sungai Klawana juga dapat dilakukan oleh masyarakat Kampung Klawana dan Wonosari, namun sungai Klawana hanya dimanfaatkan sebagai tapal batas kedua kampung tersebut. Selain itu belum ada pemanfaatan lain yang dilakukan oleh Tumbuhan Bukan Kayu (non woody plant) Komposisi tumbuhan non kayu pada kawasan TWAK Kabupaten Sorong antara lain: rotan, palem, pandan, bambu, anggrek, paku-pakuan, herba dan liana yang merupakan tumbuhan non potensial sebagai peyerap karbon dan menyediakan oksigen untuk menujang potensi jasa 129 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk lingkungan dan perlindungan kawasan konservasi pada kawasan TWAK. Secara detail, jenis-jenis tumbuhan bukan kayu yang ditemukan antara lain dari jenis rotan (Calamus aruensis Bacc., Calamus kayensis Bacc., Korthalsia), dari jenis palm (Arenga macrocalyx Ex., Aracea sp., caryato rumpicna Mart., Licuala telifera., Licuala sp., Metroxilon sagoo Rotbb., Pigafeta filaris, Pinanga punicea, Rhopaloblaste sp.), jenis pandanus (Pandanus tectorius Sol., Pandanus furcatus Roxb., Pandanus conoides Lam., Pandanus odoratissimus, Freycinetia sp.), jenis bambu (Bambusa vurgaris, Schozostacchyum brachyclaudum Kurz., Schozostachyum lima, Schozostachyum zollingeri, neolaleba atra) dan jenis anggrek (Bullbophyllum sp., Calanthe sp., Colagyne asperata, Corimborkis veratrifolia, Dendrabium bofalce, Dendrobium bracteosum, Gramatophyllum scriptum BL., Gramatophyllum speciosum LINDL., Phalaenopsis amabilis, Pholidota imbricatai, Pomotocalpa sp., dan Spothoglottis plicata BL.). lingkungan dan perlindungan kawasan konservasi pada kawasan TWAK. Secara detail, jenis-jenis tumbuhan bukan kayu yang ditemukan antara lain dari jenis rotan (Calamus aruensis Bacc., Calamus kayensis Bacc., Korthalsia), dari jenis palm (Arenga macrocalyx Ex., Aracea sp., caryato rumpicna Mart., Licuala telifera., Licuala sp., Metroxilon sagoo Rotbb., Pigafeta filaris, Pinanga punicea, Rhopaloblaste sp.), jenis pandanus (Pandanus tectorius Sol., Pandanus furcatus Roxb., Pandanus conoides Lam., Pandanus odoratissimus, Freycinetia sp.), jenis bambu (Bambusa vurgaris, Schozostacchyum brachyclaudum Kurz., Schozostachyum lima, Schozostachyum zollingeri, neolaleba atra) dan jenis anggrek (Bullbophyllum sp., Calanthe sp., Colagyne asperata, Corimborkis veratrifolia, Dendrabium bofalce, Dendrobium bracteosum, Gramatophyllum scriptum BL., Gramatophyllum speciosum LINDL., Phalaenopsis amabilis, Pholidota imbricatai, Pomotocalpa sp., dan Spothoglottis plicata BL.). masyarakat maupun Balai Konservasi Sumberdaya Hutan (BKSDH). Sejauh ini hanya dilakukan kegiatan pengawasan dan perlindungan oleh pemerintah dan pemegang hak ulayat. Jungle Tracking Kawasan TWAK menyediakan jalur tracking alam yang memikat. Di sepanjang jalur track, pengunjung dapat menikmati suasana alam dan udara yang segar, selain itu disepanjang jalur track bisa mengenal berbagai macam jenis tumbuhan endemik TWAK serta dapat berjumpa dengan satwa langka yang menarik dari beberapa jenis burung paruh bengkok yang ada di Papua. Fishing Terdapat beragam jenis ikan air tawar yang berada di sungai Klagewe di Kawasan TWA Klamono. Kegiatan memancing (fishing) merupakan salah satu kegiatan wisata alam yang dapat di lakukan kepada pengunjung. Hal menarik yang dapat dilakukan yaitu sambil 130 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk pusat kota kabupaten menuju kawasan TWAK dengan waktu tempuh 2-4 jam tergantung kendaraan darat apa yg digunakan. memancing pengunjung dapat menikmati keindahan alam, Tetapi pada pelaksanaan sendiri belum adanya pengelolaan yg baik dari pemanku kepentingan (stakeholder) dalam menentuan arah kebijakan pemanfaatan dan pengembangan Taman Wisata Alam Klamono. Dari kondisi lingkungan sosial ekonomi masyarakat sekitar juga sangat berperan penting dalam penyeimbangan potensi jasa lingkungan . unsur – unsur yang dinilai dari kondisi sosial ekonomi masyarakat antara lain: tata ruang wilayah objek, mata pencaharian, tingkat Pendidikan, dan tanggapan masyarakat terhadap pengembangan TWAK. Kondisi sosial ekonomi masyarakat sekitar berdasarkan hasil wawancara bahawa masyarakat pada kampung klawana cukup mendukung dengan (nilai 375) karena adanya sikap positif yang di tujukan oleh masyarakat sekitar. @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Pengembangan Pemanfaatan Jasa Lingkungan Kelayakan pengembangan dilakukan melalui beberapa kriteria untuk mengambarkan karakteristik potensi wisata alam yang layak dikembangkan atau sebeliknya belum dapat di kembangkan. Potensi wisata alam pada kawasan Taman Wisata Alam Klamono meliputi 5 (lima) kriteria, yaitu daya tarik, aksesibilitas, kondisi lingkungan sosial ekonomi masyarakat, keamanan dan pengelolaan dan sarana dan prasarana. j y Keamanan dan pengelolaan kawasan adalah salah satu hal penting dalam melestarikan potensi jasa lingkungan yang berada pada kawasan TWAK. Penilaian keamanan dan pengelolaan dalam kawasan dapat dinilai dgn kriteria penilaian sebagai berikut: keamanan Kawasan yang meliputi kebakaran, penebangan liar, perambahan, keamanan pengunjung dan pengolahan kawasan. Dari aspek keamanan dan pengelolaan di beri (nilai 240) dari segi keamanan masih kurang baik, karena masih adanya aktifitas manusia pada kawasan yang menyebapkan kebakaran akibat pembukaan lahan, penebangan liar yang di lakukan besar besaran dari dalam maupun di luar kawasan TWAK, oleh para pemilik hak ulaiyat maupun dari luar dengan. Daya tarik merupakan nilai dari obyek yang menjadi salah satu unsur utama dalam menarik wisatawan datang ke tempat tertentu. Penilaian terhadap unsur- unsur daya tarik di lakukan untuk mengetahui potensi daya tarik TWAK yang di nilai meliputi unsur keindahan alam, keunikan sumber daya alam, banyaknya sumber daya alam yang menojil, serta jenis kegiatan wisata alam. Dari skoring yang dilakukan, diperoleh nilai 450 yang masuk kategori cukup tinggi. Aksesibilitas merupakan salah satu faktor penting yang menentukan mudah atau tidaknya wisatawan datang ke daerah tujuan wisata misalnya sarana trasportasi dan jalan. Dari aksebilitas, TWAK di tunjang dengan aksesibilitas yang memadai (725) k arena mudan di akses mengunakan kendaraan mobil atau motor dengan waktu tempuh yang relatif cepat. Kondisi jalan darat yang cukup baik dari Sarana penunjang yang sangat diperlukan dalam menjalankan kegiatan wisata alam dengan tujuan memberikan rasa aman dan nyaman bagi pengunjung. Unsur-unsur kriteria sarana dan prasarana 131 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk umum yang berdomisili di Kampung Klawana, sebagian besar menyetujui apabila TWAK dikembangkan dan dilestarikan secara baik. Hal ini menjadi modal dasar yang tentunya untuk dikembangkan dimasa depan karena adanya persetujuan dan dukungan masyarakat setempat. Namun dimana saat ini dalam hal keamanan dan pengelolaan masih ada banyak terjadi pelanggaran misalnya penebangan liar yang di sengaja (ilegal logging), dan perambahan hutan (pengunaan lahan). Pengelolaan kawasan sendiripun belum ada misalnya pintu gerbang masuk kawasan, rambu rambu kawasan, pos penjagaan untuk keamanan kawasan. Pengembangan Pemanfaatan Jasa Lingkungan Selama ini dari pihak pemerintah dan balai besar konservasi sumberdaya hutan masih belum ada kesepakaan tentang status kawasan Taman Wisata Alam Klamono dengan pemegang hak ulayat (Mambringgofok). Sejauh ini justru baru diadakanya pendekatan seperti kegiatan dari BBKSDH melalui pembentukan kelompok tani hutan, mitra polhut di Kampung Klawana, Kampung Klamono dan Wonosari. Selain itu ada juga bantuan berupa ternak sapi pada tahun 2018, penangkaran buaya pada tahun 2018, pembibitan pada tahun 2018, dan pelatihan RHL, dalam bentuk uang tunai ataupun secara lansung diberikan. Oleh karena itu di harapkan kepada pemerintah setempat dan Balai Besar Konservasi Sumberdaya Hutan Provinsi Papua Barat untuk dapat mengembangkan dan meningkatkan aspek pengelolaan Kawasan terutama pengamanannya di sekitar kawasan TWAK. yang dinilai yaitu unsur sarana berupa rumah makan, sarana angkutan umum dan prasarana berupa jalan, jembatan, jaringan listrik, jaringan air minum dan jaringan telepon. Hasil penilaian sarana dan prasarana yg di nilai di sini adalah sarana dan prasarana yang berada di sekitar TWAK atau yang terdapat pada kampung klamono sedangkan pada kawasan TWAK sendiri belum adanya perhatian daan aktifitas aktifitas pada kawasanTWAK. Sarana dan prasarana (nilai 135) sarana rumah makan dan sarana angkot umum dapat diakses dengan mudah sebelum kita mengunjungi TWAK. Sedangkan prasarana seperti jalan, jembatan, jaringan listrik, jaringan air minum, dan jaringan telepon dapat membantu kita untuk sampai pada kawasan TWAK. umum yang berdomisili di Kampung Klawana, sebagian besar menyetujui apabila TWAK dikembangkan dan dilestarikan secara baik. Hal ini menjadi modal dasar yang tentunya untuk dikembangkan dimasa depan karena adanya persetujuan dan dukungan masyarakat setempat. Namun dimana saat ini dalam hal keamanan dan pengelolaan masih ada banyak terjadi pelanggaran misalnya penebangan liar yang di sengaja (ilegal logging), dan perambahan hutan (pengunaan lahan). Pengelolaan kawasan sendiripun belum ada misalnya pintu gerbang masuk kawasan, rambu rambu kawasan, pos penjagaan untuk keamanan kawasan. Selama ini dari pihak pemerintah dan balai besar konservasi sumberdaya hutan masih belum ada kesepakaan tentang status kawasan Taman Wisata Alam Klamono dengan pemegang hak ulayat (Mambringgofok). Sejauh ini justru baru diadakanya pendekatan seperti kegiatan dari BBKSDH melalui pembentukan kelompok tani hutan, mitra polhut di Kampung Klawana, Kampung Klamono dan Wonosari. Selain itu ada juga bantuan berupa ternak sapi pada tahun 2018, penangkaran buaya pada tahun 2018, pembibitan pada tahun 2018, dan pelatihan RHL, dalam bentuk uang tunai ataupun secara lansung diberikan. Pengembangan Pemanfaatan Jasa Lingkungan Oleh karena itu di harapkan kepada pemerintah setempat dan Balai Besar Konservasi Sumberdaya Hutan Provinsi Papua Barat untuk dapat mengembangkan dan meningkatkan aspek pengelolaan Kawasan terutama pengamanannya di sekitar kawasan TWAK. Hasil rekapitulasi nilai terhadap 5 unsur penilaian yang terlah di modifikasi modifikasi dari Pedoman Kriteria Penilaiaan Pengembangan Direktorat Jendral Perlindungan Hutan dan Konservasi Alam, Departemen Kehutanan (2003) dan Direktorat Produk Wisata Direktorat Jendral Destinasi Pariwisat Departemen Kebudayaan dan Pariwisata adalah sebesar 1925, yang terdiri dari unsur daya tarik bernilai 450, aksebilitas bernilai 725, soaial ekonomi masyarakat sekitar bernilai 375, keamana dan pengelolaan bernilai 240 dan sarana prasarana bernilai 135. Berdasaran kategori dan selang penilaian, potensi jasa lingkungan kawasan TWAK tergolong pada kategori penilaian sedang (1601– 2110). Hal ini berarti bahwa potensi jasa lingkungan pada kawasan TWAK dapat di kembangkan tetapi perlu pertimbangan kembali dari pemerintah. Berdasarkan hasil obserfasi lapangan dan wawancara dengan responden kunci dan responden DAFTAR PUSTAKA Arief A. 2005. Hutan dan kehutanan. Penerbit Kansius, Yogyakarta. DAFTAR PUSTAKA Arief A. 2005. Hutan dan kehutanan. Penerbit Kansius, Yogyakarta. 132 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk Lekitoo K, Peday HFZ, Panambe N and Cabuy RL. 2017. Ecological and ethnobotanical facet of ‘Kelapa Hutan’ (Pandanus spp.) and perspectives towards its existence and benefit. International Journal of Botany, 13: 103-114. BBKSDA Papua Barat. 2007. Buku Informasi Kawasan Bidang KSDA Wilayah I Sorong. Balai Besar Konservasi Sumberdaya Alam Papua Barat. Sorong. Departemen Kehutanan. 1999. Undang- Undang Nomor 41 Tahun 1999. Pemerintah Republik Indonesia, Jakarta. Maturbongs RG. 2011. Analisis kualitas obyek dan daya tarik wisata di Taman Wisata Alam Klamono Kabupaten Sorong. [Tesis]: Program Pascasarjana Universitas Gadjah Mada. Yogyakarta. Departemen Kehutanan. 2003. Pedoman analisis daerah operasi objek dan daya tarik wisata alam (ADO-ODTWA). Direktorat Jendral Perlindingan Hutan dan Konservasi alam. Bogor. Pangesti T. 2005. Wisata alam. Materi pelatihan konservasi sumber daya hayati. Balai Diklat Kehutanan, Bogor. Departemen Kebudayaan dan Pariwisata. 2007. Pedoman penilaian daya tarik wisata. Direktorat Jenderal Pengembangan Destinasi Pariwisata. Petocz RG. 1987. Konservasi alam dan pembangunan di Irian Jaya. Strategi Pemanfaatan Sumber Daya Alam Secara Rasional. PT. Temprint. Jakarta. Johns RJ. 1997. Biodiversity in Irian Jaya: Proposals for the listing of selected areas for priority conservation. (Disampaikan dalam Lokakarya Keanakragaman Hayati di Biak). Royal Botanical Gardens, Kew, Ricmond Surrey TW9, CAB England. Rahawarin YY. 2010. @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA 133 Pengembangan Pemanfaatan Jasa Lingkungan Forest land use by the community in Sorong Natural Tourism Park at Sorong City, West Papua Province. Biodiversitas, Vol 11 (4): 222-227. 133
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Prediction of Under-Detection of Paediatric Tuberculosis in the Democratic Republic of Congo: Experience of Six Years in the South-Kivu Province
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RESEARCH ARTICLE Editor: Nerges Mistry, Foundation for Medical Research, INDIA Received: June 17, 2016 Accepted: December 9, 2016 Published: January 6, 2017 Methods Copyright: © 2017 Andre´ 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. We collected TB notification data from 2010 to 2015 and analysed the space-time variations in notification for the different forms of TB among the 113 health facilities (HF) the South- Kivu Province, a region with a low HIV incidence. The different forms of TB notified were: smear positive pulmonary TB (SS+PTB), smear negative pulmonary TB (SS-PTB) and extra-pulmonary TB (EPTB). We further analysed the distribution of these different forms of the disease per age group and explored the possibility to predict the detection of PedTB. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by TB REACH grant (Stop TB Partnership) (http://www.stoptb. org/global/awards/tbreach/wave2.asp). Emmanuel Andre´1,2*, Yvette Lufungulo Bahati3, Eric Mulume Musafiri4, Olivier Bahati Rusumba5, Dimitri Van der Linden2, Francis Zech1 1 Institut de Recherche Expe´rimentale et Clinique, Universite´ Catholique de Louvain, Brussels, Belgium, 2 Cliniques Universitaires Saint-Luc, Brussels, Belgium, 3 Hoˆpital Provincial Ge´ne´ral de Re´fe´rence de Bukavu, Bukavu, Democratic Republic of Congo, 4 Coordination Provinciale Lèpre et Tuberculose, Bukavu, Democratic Republic of Congo, 5 Ambassadeurs de Lutte contre la Tuberculose, Bukavu, Democratic Republic of Congo a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * emmanuel.andre@uclouvain.be * emmanuel.andre@uclouvain.be Introduction Citation: Andre´ E, Lufungulo Bahati Y, Mulume Musafiri E, Bahati Rusumba O, Van der Linden D, Zech F (2017) Prediction of Under-Detection of Paediatric Tuberculosis in the Democratic Republic of Congo: Experience of Six Years in the South- Kivu Province. PLoS ONE 12(1): e0169014. doi:10.1371/journal.pone.0169014 In the field of tuberculosis (TB), and particularly in regard to paediatric TB (PedTB), clinical skills of health professionals play an important role in determining quality of care. In an era where novel diagnostic technologies and efficient treatment regimens are being made avail- able for the poorest, we must not divert our attention from the importance of clinical skills, as this deliverable remains the cornerstone of individualized patient care and ultimately the best assurance for optimal use of resources. The aim of our work was to study the epidemi- ology of PedTB and the determinants of PedTB under-detection in the South-Kivu Province of the Democratic Republic of Congo (DRC), a setting with nearly no technical resources allowing to support the clinical diagnosis of PedTB, i.e. chest X-rays, rapid molecular tests or culture laboratories. Editor: Nerges Mistry, Foundation for Medical Research, INDIA Editor: Nerges Mistry, Foundation for Medical Research, INDIA Conclusion Our observations strongly suggest that under-detection of PedTB is associated with insuffi- cient clinical skills and technical resources at the HF level which similarly affects other forms of the disease, in particular SS-TB. We demonstrated that, in the specific context of South- Kivu, under-detection of PedTB can be predicted by a low SS-TB/SS+PTB ratio in the adult population. In the context of severely under-resourced settings, this ratio could be used to rapidly identify HF that should benefit in priority from deeper evaluation, and eventually tar- geted interventions. Under-Detection of Tuberculosis among Children in DRC bacteriological confirmation (SS-TB) among adults, the number of TB cases notified by the HF and the fact that the HF was supported or not by Me´decins Sans Frontières (MSF). The proportion of SS-TB among adults was found to be a valid indicator for predicting the level of detection of PedTB at the same HF. Abbreviations: DRC, Democratic Republic of Congo; EPTB, Extra pulmonary tuberculosis; HZ, Health Zone; HF, Health facility; INH, Isoniazid; MSF, Me´decins Sans Frontières; MTB, Mycobacterium tuberculosis complex; NGO, Nongovernmental organization; PedTB, Paediatric tuberculosis; RIF, Rifampicin; SS+PTB, Smear- positive pulmonary tuberculosis; SS-PTB, Smear- negative pulmonary tuberculosis; SS-TB, Smear negative tuberculosis (SS-PTB and EPTB); TB, Tuberculosis; WHO, World Health Organization. Results Significant differences were observed between HF in regard to the proportion of paediatric TB and the proportion of SS-TB among adults. We found a strong correlation between the proportion of PedTB and three major factors: the proportion of TB cases with no Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 1 / 9 Introduction The World Health Organisation (WHO) estimates that there were 9.6 million new patients infected with tuberculosis (TB) in 2014. Globally, an estimated 10% of TB patients are children less than 15 years, but two third of these are currently un-diagnosed or un-notified. The chal- lenge of under-detection of TB is thus much more important among children than adults for which approximately one third of TB patients are currently missed [1]. Paediatric TB (PedTB) can rapidly evolve towards life-threatening manifestations such as miliary TB or meningitis [2]. Improving the capacity of health care providers to detect TB among children will not only save lives, but also have major repercussions in regard to the control of the epidemic. The Democratic Republic of Congo (DRC) lies among the few high TB burden countries which did not meet any of the 2015 targets in regard to TB control: incidence is not falling and neither prevalence nor mortality was reduced by 50% in comparison with 1990 figures. In 2015, DRC notified 111,683 TB cases (146.8/100,000), roughly half of the estimated numbers of cases estimated for this country by WHO. In this low-income country, children aged less than 15 years represent 46% of the population compared to 26% globally [3]. Despite the over- representation of this age group, only 11.4% of TB patients were less than 15 years in 2015. The aim of our work was to develop a method to rapidly identify health facilities (HF) which have a higher probability to be facing PedTB under-detection as a result of the inability of health-care providers to recognise this clinical entity. Such method would allow the design and implementation of targeted interventions, but also to evaluate their impact on PedTB detection. Materials and Methods Study setting The DRC presents a very large network of HF constituting the public health system. TB-related services are well integrated within primary health care services, which offer free access to TB diagnostics—including rapid molecular tests in a minority of HF—and treatment [4]. Clinical guidelines developed by the National Tuberculosis Programme of DRC (17) include recommendations for the clinical diagnosis of PedTB. These recommendations are rel- atively scanty, as they take into account the current situation of the vast majority of HF which 2 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Under-Detection of Tuberculosis among Children in DRC lack technical resources such as X-rays, molecular tests or tuberculin skin test (TST). There- fore, the diagnosis of PedTB is principally based on aspecific clinical symptoms and the notion of TB contact in the household. In practice, the use of rapid molecular diagnostic tests, which are being progressively scaled-up in the country since 2012, is currently limited to patients with presumptive drug resistant TB or HIV-TB coinfection. Due to this particular context, and considering that no drug resistance survey has been performed in DRC to date, the incidence of drug-resistant TB (and particularly among children) cannot be precisely estimated. Regular supervision activities are performed by the national and provincial teams, although some areas are inaccessible due to insecurity or the high costs associated with travelling in the most remote areas. The South-Kivu province lies along the eastern border of DRC and presents a population of over 6,6 million inhabitants, mainly living in rural areas. Health delivery is provided to the population through over 500 HF, among which 78 (2010–2013) to 113 (2014–2015) provide TB services: Ziehl-Nielsen microscopy and provision of first-line anti-TB drugs. Since 2012, ten laboratories located in urban HF have been progressively equipped with GeneXpert MTB/RIF technology. The province does not have a laboratory able to perform mycobacterial cultures—the most sensitive diagnostic method—and diagnostic samples are usually not sent to the National Reference Centre in Kinshasa unless drug resistance is suspected. g The province is divided in 34 HZ, and each HZ has 1 to 6 HF. A HF is responsible for a median of 51,500 habitants (percentiles 10 and 90: 17,800 and 97,300). The number of TB cases notified by each HF is largely variable: 10% of these report less than 7 cases per year, while 10% report more than 140 cases, the median being 32. Materials and Methods Study setting Among the 34 health zones (HZ), five are mainly urban (Bagira, Kadutu, Ibanda, Kamituga, and Uvira), while the remaining 29 HZ are located in rural areas. Some of the latter are relatively well served in terms of infrastructure and communications, while 11 are difficult to reach due to the absence of roads or on-going armed conflicts. Most of these isolated HZ are located in the centre and west of the province. As a result of the long-lasting humanitarian crisis in this region, the vast majority of the 34 HZ receive external support from non-governmental organisations (NGOs) for the delivery of pri- mary health care services. This support is generally integrated within the existing public system and fully consistent with the national guidelines. Four HF located in the HZ Shabunda, Fizi and Kimbi-Lulenge (since 2012), are nevertheless considered as exceptions: these are supported by Me´decins Sans Frontières (MSF) which applies a more “independent” policy in regard to the organisation of health services, including supplementation of the health structures with qualified medical staff, clinical guidelines and technical resources including laboratory equip- ment [5]. In regard to the diagnosis of PedTB, MSF recommends a documented growth assess- ment of the children suspected or exposed to TB. Furthermore, MSF supports sample transportation and provides technical resources such as X-rays, HIV testing, TST or investiga- tions for EPTB. During the study period, there was no significant difference in the population covered by each HF between rural, isolated (enclaved), or MSF-associated HF (p = 0.14). Diagnosis of smear negative and extra-pulmonary TB among adults Adults with SS-TB accounted for 21.8% of all cases notified and 28.5% of adult cases. With regard to the yearly notified cases of SS-TB for each HZ during the 6 years, the per- centage of SS-TB among adult cases was highly variable from one HF to the other. Moreover, we observed significant differences of the average percentages between rural HZ (15.7%), rural enclaved (19.4%), urban (37.5%), or MSF-supported HZ (34,7%). Notified incidence of TB Between 2010 and 2015, the South-Kivu province reported 28,878 cases of TB. Among the 26,337 patients for which clinical outcome was available, 3.73% deceased during the year fol- lowing diagnosis. This proportion was higher among SS-PTB (6.22%) and EPTB (4.89%) com- pared to SS+PTB (2.79%). During this period, the mean incidence of TB per 100,000 people per year was 84.5 new cases, among which 63.6% were adults with a pulmonary disease con- firmed by microscopy. A transient increase in notification was observed due to the intensifica- tion of active case-finding strategies supported by two TB REACH grants [7]. g g pp y g The notified incidence was largely dependent on the notification of adults with SS+PTB, accounting for up to 100% of all TB notified in some HZ. Data collection and statistical analysis We collected standard notification reports provided by the National TB Program. These reports include the notification of the different forms of TB, including smear positive pulmo- nary TB (SS+PTB), smear negative pulmonary TB (SS-PTB) and extra-pulmonary TB (EPTB). In this analysis, SS-PTB and EPTB among adults are considered jointly as smear-negative TB (SS-TB), while PedTB includes all forms of the disease among children under 15 years. We analysed the changes in the incidence and proportion of different clinical TB presenta- We analysed the changes in the incidence and proportion of different clinical TB presenta- tions for each HF over a period of 6 years from 2010 to 2015. Each HF and each year was 3 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Under-Detection of Tuberculosis among Children in DRC studied as a separate unit, with an internal correlation for the successive results of the same HF. The relations between the percentages of PedTB and diverse factors were examined using a generalised linear regression of a beta-binomial variable. Actually, the percentages of paediat- ric case followed a beta-binomial distribution, due to hyperdispersion of results between health areas. We used quasi-least squares, which is suitable for calculating internal correlations to adjust the regression, and chose the hypothesis of constant between-year correlation matrix. To calculate statistical significance, we used the “sandwich variance matrix” augmented by the bias correction proposed by Morel, Bokossa and Neerchal [6], which may be evaluated by the normal distribution. To study the relation between the percentages of PedTB cases with other factors, we first explored the curves of non-linear regressions and further selected the adequate expression to include these factors in linear univariate and multivariate regressions. We used the logarithm for the total number of TB cases reported for the populations surveyed by the HFs and the percentages of SS-TB adults. We report here only the results of linear regressions. The data being anonymised, ethical approval was not required for this retrospective study. PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Diagnosis of paediatric TB The proportion of children less than 15 years accounted for 14.6% of all TB cases notified in the South-Kivu province, compared to 11.4% at the national level (p<0.00001). In total, 4,219 PedTB cases were notified, among which 18.1% had a bacteriologically confirmed diagnosis and only two children were diagnosed with MDR-TB. In total, 56 MDR-TB cases were reported during the same period. The proportion of PedTB cases notified varied significantly between HZ, with a ratio vary- ing between 0% and 40%. This important variation cannot be explained only by demographic particularities, and rather suggests an important heterogeneity in the ability to diagnose PedTB between HF. Looking at the yearly notified cases of PedTB for each HF during the 6 years, we could not find any significant difference between urban, rural and isolated (enclaved) HZ (p = 0.074). The strongest association was found between the ratio of PedTB and the ratio of SS-PTB 4 / 9 Under-Detection of Tuberculosis among Children in DRC Fig 1. Correlation between the percentage of PedTB and the percentage of SS-TB among adults. Circles represent the number of TB cases notified for each Health Facility (HF) during one year (6 years are represented). A strong correlation (p<0.000001) was found between the proportion of SS-TB among adults and the proportion of PedTB. doi:10.1371/journal.pone.0169014.g001 Fig 1. Correlation between the percentage of PedTB and the percentage of SS-TB among adults. Circles represent the number of TB cases notified for each Health Facility (HF) during one year (6 years are represented). A strong correlation (p<0.000001) was found between the proportion of SS-TB among adults and the proportion of PedTB. doi:10.1371/journal.pone.0169014.g001 among adults (Fig 1) (p<0.000001). Furthermore, strong correlations were observed between PedTB detection and the total number of TB cases diagnosed (p = 0.000016) and with the pres- ence of MSF in the HF (p<0.00001). The size of the population covered by each HF has no sig- nificant correlation with the ratio of PedTB. Development and validation of a model predicting under-detection of paediatric TB For the final multivariate linear regression, we used the results as a model to predict the num- ber of PedTB cases for each HF and each year (Fig 2). This model is based on 3 variables easily available through the standard notification reports: the percentage of SS-TB among adults, the total number of TB cases notified by the HF during the year and an external support received by MSF. In the multivariate regression, these three factors remain independently significant: being in a HF with a low or great percentage of adults with SS-TB (p<0.000001), being or not in a MSF-supported HF (p<0.000001) and being in a HF with a low or great number of TB cases notified (p = 0.0011). The validity of this model was further confirmed by the analysis of the normalized Anscombe’s residues (data not shown). Under-Detection of Tuberculosis among Children in DRC Fig 2. Application of the model predicting under-detection of PedTB at the HF level based on 3 informations: percentage of SS-TB among adults, external support received by MSF and the total number of TB cases notified by the HF during the year. doi:10.1371/journal.pone.0169014.g002 Fig 2. Application of the model predicting under-detection of PedTB at the HF level based on 3 informations: percentage of SS-TB among adults, external support received by MSF and the total number of TB cases notified by the HF during the year. Fig 2. Application of the model predicting under-detection of PedTB at the HF level based on 3 informations: percentage of SS-TB among adults, external support received by MSF and the total number of TB cases notified by the HF during the year. doi:10.1371/journal.pone.0169014.g002 the disease which are notified through quarterly reports. This observation suggests that there is a high inequality between HF in regard to the ability to diagnose complex clinical presenta- tions of TB, including PedTB and SS-TB among adults. We could demonstrate with a strong level of evidence that the main factors which impact the detection of PedTB in South-Kivu, a setting with scarce technical resources, are linked with the clinical skills and the clinical experi- ence of the health professionals. Indeed, the HF which receive from MSF an important input in terms of qualified health professionals tend to diagnose more PedTB and other complex forms of the disease. Furthermore, HF which diagnose many cases of TB (all forms of the dis- ease), tend to be better skilled to recognise children affected by the disease. Of note, this critical importance of clinical skills may be exacerbated by the very poor infrastructure available in most HF: overall, only a very small proportion of TB suspects have access to X-ray, rapid molecular diagnostic or TST. Particularities of the study setting and implication for TB surveillance The South-Kivu province reports an incidence of 80.7/100,000 for all forms of TB, much lower than the 325/100,000 national incidence estimated by WHO. We found that the reported inci- dence is highly variable between HF, and particularly when looking at the different forms of 5 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Conclusion There is a critical lack of medical doctors in rural areas of Africa. In 2013, the South-Kivu province counted 408 doctors (among which 14 had a specialization degree) and 2,862 nurses for a population of over 6,6 million. In such context, nurses, laboratory technicians or the so- called “Rapidly Trained Agents” form the front line of primary health-care delivery and are inevitably faced to medical conditions that they have not been trained to recognize. Further- more, these professionals and the patients have very limited access to complementary tests such as X-Ray and accurate laboratory tests, which could eventually palliate part of these deficiencies. The direct consequences of this lack of clinical skills are under-detection, increased mortal- ity and continued transmission of the disease in the community [10]. As expected, SS-TB among adults are similarly affected by these insufficient clinical skills. In the South-Kivu prov- ince, these patients show an increased mortality during the first year after diagnosis. In such context, performing a continuous assessment of all provider’s level of “knowl- edge, attitudes and practices (“KAP”) related to TB would be the best option, but is not something that can be implementable in many difficult contexts such as the rural health zones of the South-Kivu province. Our findings suggest that a first screening of the facilities which would beneficiate the most from a complete assessment can be done based on the— already collected—data. We propose to use the ratio between SS-TB and SS+TB notification among adults as an indicator for insufficient clinical skills and under-detection of TB among children. This indicator should enable TB control programs and their partners to precisely and promptly identify those settings requiring additional support to tackle under-diagnosis of PedTB and other complex clinical presentations of the disease. The interventions should focus in parallel on the awareness of PedTB, active case finding strategies, strengthening of clinical skills oriented towards PedTB and finally the implementation of adequate technical resources such as X-rays, TST and rapid molecular laboratory assays. Improving clinical skills of health professionals should not be considered as the only priority, but the importance of these human skills should not be under-estimated as they remain the cornerstone of rational use of diagnos- tic and treatment resources. In DRC, availability of X-rays, TST, rapid diagnostic tests are still anecdotal when considering the thousands of health facilities located in rural areas. Conclusion Further- more, a recent study performed by the National Tuberculosis Programme revealed that only 47% of HF had access to anti-TB treatments with adapted paediatric drug formulation [11]. Continued efforts focusing on the quality of clinical skills are a keystone for tackling the dramatic burden of infectious diseases among children in low-resource settings [12–14]. We propose an innovative approach to rapidly identify those HF facing alarming indicators and which would beneficiate the most from additional human and technical resources. Importance of clinical skills The very strong association between under-detection of SS-TB among adults and under-detec- tion of PedTB is therefore to be explained by the fact that recognizing these complex clinical entities require (particularly in the absence of performant radiological investigations and labo- ratory assays) higher clinical skills than the common smear positive pulmonary form of the disease. Interestingly, four rural HF supported by MSF had the highest proportion of TB cases diagnosed (mean 26.7%), and seemed to perform better than urban facilities in regard to PedTB detection. The explanation of these high figures are probably the result of highly quali- fied and adequately supported health staff and free access to X-rays or laboratory testing. Insufficient clinical skills among health professionals has been reported as the main reason behind the frequent missed opportunities to diagnose and treat patients with TB that present to health facilities [8]. A qualitative study performed in Tanzania [9] reports that “inadequate 6 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Under-Detection of Tuberculosis among Children in DRC awareness of the burden of childhood TB, limited knowledge of the wide spectrum of clinical presentation and lack of clinical decision support strategies is detrimental to the health staff’s central responsibility of suspecting and referring children with TB especially in the early dis- ease stages”. The study authors stressed the importance of implementing strategies focusing on awareness among health staff and clinical skills in order to improve diagnosis. S1 Fig. Validity of the model predicting under-detection of PedTB. Graphical representa- tion of normalized Anscombe-s residues. (TIF) Supporting Information S1 Fig. Validity of the model predicting under-detection of PedTB. Graphical representa- tion of normalized Anscombe-s residues. (TIF) 7 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Under-Detection of Tuberculosis among Children in DRC S1 Dataset. Dataset. (XLS) S1 Dataset. Dataset. (XLS) Acknowledgments The authors would like to acknowledge the highly valuable comments made by Matthew Saun- ders and Esto Bahizire during the redaction of this manuscript. Furthermore, the authors would like to thank the staff of the Coordination Provinciale Lèpre et Tuberculose of the South-Kivu province for the hard work of supervision of the health structures of the province, which allowed to collect the information analysed in this manuscript. Finally, the authors would like to thank the TB patients of the South-Kivu province, which are actively involved in the implementation of sustainable solutions and working on a daily basis for ending the TB epidemic. Writing – review & editing: EA EMM OBR YLB DVDL FZ. Writing – review & editing: EA EMM OBR YLB DVDL FZ. PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Author Contributions Conceptualization: EA FZ YLB DVDL. Resources: EA EMM OBR YLB DVDL FZ. Resources: EA EMM OBR YLB DVDL FZ. Supervision: FZ DVDL. Supervision: FZ DVDL. Validation: EA EMM OBR YLB DVDL FZ. Validation: EA EMM OBR YLB DVDL FZ. Visualization: EA FZ DVDL. PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 References References 1. WHO. Global Tuberculosis Report 2015. 2015. 2. Kumar MK, Kumar P, Singh A. Recent advances in the diagnosis and treatment of childhood tuberculo- sis. J Nat Sci Biol Med. 2015; 6(2):314–20. doi: 10.4103/0976-9668.159988 PMID: 26283820 3. Department of Economics and Social Affairs UN. 2015 Revision of World Population Prospects 2015 [22/12/2015]. http://esa.un.org/unpd/wpp/. 4. Chaulet P. Integrating tuberculosis control activities into the primary health care system at district level. Bull Int Union Tuberc Lung Dis. 1989; 64(2):33–5. PMID: 2790281 5. Tuberculosis. Practical guide for clinicians, nurses, laboratroy technicians and medical auxiliaries: Me´decins Sans Frontières and Partners in Health; 2014. http://refbooks.msf.org/msf_docs/en/ tuberculosis/tuberculosis_en.pdf. 6. Morel JG, Bokossa MC, Neerchal NK. Small Sample Correction for the Variance of GEE Estimators. Biometrical Journal. 2003; 45(4):395–409. 1. WHO. Global Tuberculosis Report 2015. 2015. 2. Kumar MK, Kumar P, Singh A. Recent advances in the diagnosis and treatment of childhood tuberculo- sis. J Nat Sci Biol Med. 2015; 6(2):314–20. doi: 10.4103/0976-9668.159988 PMID: 26283820 3. Department of Economics and Social Affairs UN. 2015 Revision of World Population Prospects 2015 [22/12/2015]. http://esa.un.org/unpd/wpp/. 4. Chaulet P. Integrating tuberculosis control activities into the primary health care system at district level. Bull Int Union Tuberc Lung Dis. 1989; 64(2):33–5. PMID: 2790281 5. Tuberculosis. Practical guide for clinicians, nurses, laboratroy technicians and medical auxiliaries: Me´decins Sans Frontières and Partners in Health; 2014. http://refbooks.msf.org/msf_docs/en/ tuberculosis/tuberculosis_en.pdf. 6. Morel JG, Bokossa MC, Neerchal NK. Small Sample Correction for the Variance of GEE Estimators. Biometrical Journal. 2003; 45(4):395–409. 8 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014 January 6, 2017 Under-Detection of Tuberculosis among Children in DRC 7. Blok L, Sahu S, Creswell J, Alba S, Stevens R, Bakker MI. Comparative meta-analysis of tuberculosis contact investigation interventions in eleven high burden countries. PLoS One. 2015; 10(3):e0119822. doi: 10.1371/journal.pone.0119822 PMID: 25812013 8. Claassens MM, Jacobs E, Cyster E, Jennings K, James A, Dunbar R, et al. Tuberculosis cases missed in primary health care facilities: should we redefine case finding? Int J Tuberc Lung Dis. 2013; 17 (5):608–14. doi: 10.5588/ijtld.12.0506 PMID: 23575325 9. Bjerrum S, Rose MV, Bygbjerg IC, Mfinanga SG, Tersboel BP, Ravn P. Primary health care staff’s per- ceptions of childhood tuberculosis: a qualitative study from Tanzania. BMC Health Serv Res. 2012; 12:6. doi: 10.1186/1472-6963-12-6 PMID: 22229965 10. Marais BJ. Strategies to improve tuberculosis case finding in children. Public Health Action. 2015; 5 (2):90–1. doi: 10.5588/pha.15.0028 PMID: 26393106 11. PNLT. Rapport e´pide´miologique de la Tuberculose2012. 12. References Farmer P, Robin S, Ramilus SL, Kim JY. Tuberculosis, poverty, and "compliance": lessons from rural Haiti. Semin Respir Infect. 1991; 6(4):254–60. PMID: 1810004 13. Grange J, Zumla A. Tuberculosis and the poverty-disease cycle. J R Soc Med. 1999; 92(3):105–7. PMID: 10396250 14. Hansen C, Paintsil E. Infectious Diseases of Poverty in Children: A Tale of Two Worlds. Pediatr Clin North Am. 2016; 63(1):37–66. doi: 10.1016/j.pcl.2015.08.002 PMID: 26613688 9 / 9
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Ttyh1 Protein is Expressed in Glia In Vitro and Shows Elevated Expression in Activated Astrocytes Following Status Epilepticus
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Ttyh1 Protein is Expressed in Glia In Vitro and Shows Elevated Expression in Activated Astrocytes Following Status Epilepticus Elzbieta Wiernasz • Aleksandra Kaliszewska • Wojciech Brutkowski • Joanna Bednarczyk • Malgorzata Gorniak • Beata Kaza • Katarzyna Lukasiuk Received: 27 March 2014 / Revised: 26 September 2014 / Accepted: 8 October 2014 / Published online: 15 October 2014  The Author(s) 2014. This article is published with open access at Springerlink.com Abstract In a previous study, we showed that Ttyh1 protein is expressed in neurons in vitro and in vivo in the form of punctuate structures, which are localized to neu- ropil and neuronal somata. Herein, we provide the first description of Ttyh1 protein expression in astrocytes, oli- godendrocytes and microglia in vitro. Moreover, using double immunofluorescence, we show Ttyh1 protein expression in activated astrocytes in the hippocampus fol- lowing amygdala stimulation-induced status epilepticus. We demonstrate that in migrating astrocytes in in vitro wound model Ttyh1 concentrates at the edges of extending processes. These data suggest that Ttyh1 not only partici- pates in shaping neuronal morphology, as previously described, but may also play a role in the function of activated glia in brain pathology. To localize Ttyh1 expression in the cellular compartments of neurons and astrocytes, we performed in vitro double immunofluores- cent staining using markers for the following subcellular structures: endoplasmic reticulum (GRP78), Golgi appa- ratus (GM130), clathrin-coated vehicles (clathrin), early endosomes (Rab5 and APPL2), recycling endosomes (Rab11), trans-Golgi network (TGN46), endoplasmic reticulum membrane (calnexin), late endosomes and lyso- somes (LAMP1) and synaptic vesicles (synaptoporin and synaptotagmin 1). We found that Ttyh1 is present in the endoplasmic reticulum, Golgi apparatus and clathrin- coated vesicles (clathrin) in both neurons and astrocytes and also in late endosomes or lysosomes in astrocytes. The presence of Ttyh1 was negligible in early endosomes, recycling endosomes, trans-Golgi network, endoplasmic reticulum membrane and synaptic vesicles. Keywords Astrocytes  Gliosis  Microglia  Oligodendrocytes  Status epilepticus  Tweety Neurochem Res (2014) 39:2516–2526 DOI 10.1007/s11064-014-1455-3 Neurochem Res (2014) 39:2516–2526 DOI 10.1007/s11064-014-1455-3 ORIGINAL PAPER ORIGINAL PAPER Culture of Primary Astrocytes and Microglia Primary glial cell cultures were prepared from 1-day-old Wistar rat pups, as previously described [13, 14]. Briefly, the forebrains of the pups were aseptically removed and homogenized through mechanical dissociation. The cells were isolated from the cerebral cortices through trypsin- ization (0.025 % trypsin at 37 C for 20 min) and mechanically dissociated into a single cell suspension. The cells were plated onto poly-L-lysine-coated (#P4707, Sigma) 75-cm2 culture flasks at a density of 3 9 105 cells/ cm2 in culture medium comprising DMEM with Gluta- MAX and high-glucose at 4.5 g/l (#31966-021, Gibco), supplemented with 10 % fetal bovine serum (FBS, #10500- 064, Gibco), penicillin (100 U/ml) and streptomycin (100 lg/ml) (#15140-148, Gibco). The cells were cultured at 37 C, 95 % O2 and 5 % CO2. The culture medium was changed after 3 days and then every 4–5 days. After 9–10 days in culture, the confluent glial cells were shaken and centrifuged to recover microglia from astrocytes and oligodendrocytes in culture. The loosely adhered microglia were recovered through mild shaking and plated at a den- sity of 2–3 9 105 cells/cm2 in 24-well plates or 60-mm dishes. After changing the media to remove the nonad- herent cells, the adherent microglia were incubated for 48 h. The expression of Ttyh1 mRNA is restricted to neural tissue and the testis [8]. In the brain, Ttyh1 mRNA has been detected in neurons [9, 10]. The expression of Ttyh1 protein is also neuronal and has been detected in neurons in vivo and in vitro [6, 8, 11]. The overexpression of Ttyh1 protein in cell lines and neurons in vitro leads to excessive filopodia formation [8, 11]. The involvement of Ttyh1 has been implicated in aberrant neuronal structural plasticity in vivo, as increased Ttyh1 protein expression was observed in the molecular layer of the dentate gyrus during epileptogenesis [11]. In a previous study, we have determined that Ttyh1 protein forms complexes with proteins localized in the endoplasmic reticulum and Golgi [11]. Thus, the aim of the present study is to verify these results using double immunocytochemistry with markers of different cellular compartments in vitro. In addition, we analyzed the expression of Ttyh1 in glial cells in vitro and in vivo in the normal and injured brain following status epilepticus. Culture of Primary Astrocytes and Microglia The results show the ubiquitous expression of Ttyh1 protein in astrocytes, oligodendrocytes and microglia in vitro and the increased expression of Ttyh1 in astrocytes in the hippo- campus following status epilepticus. We also demonstrate that in migrating cultured astrocytes Ttyh1 is concentrated at the margins of extending processes. To remove oligodendrocytes from astrocytes, primary glial cells in 75-cm2 culture flasks were placed on a rotary shaker and shaken at 37 C for 24 h (200 rpm). The culture medium containing oligodendrocytes was removed, and the astrocytes were plated onto 10-cm2 culture dishes. After 3 days, the astrocytes were trypsinized and plated onto poly- L-lysine-coated glass coverslips (#1001/18, Glaswarenfab- rik Karl Hecht GmbH & Co) in 12-well plates at a con- centration of 75 9103 cells/ml and maintained in culture for 72 h, followed by fixation and immunocytochemistry. Culture of Primary Hippocampal Neurons Culture of Primary Hippocampal Neurons Hippocampal neurons were cultured as previously descri- bed [11, 12]. Briefly, the hippocampi were dissected from E18 to E19 Wistar rat embryos and dissociated through incubation in 0.04 % trypsin (#27250-0180, Gibco). The cell suspension was plated onto poly-D-lysine-coated (#P7280, Sigma) glass coverslips (#1001/18, Glaswaren- fabrik Karl Hecht GmbH & Co) in 12-well plates. The cells were cultured in Neurobasal G3 medium (#21103, Gibco), supplemented with B27 (#17504, Gibco), 10 mg/l genta- micin (#15-750-037, Gibco), 0.5 mM L-glutamine (#G8540, Sigma) and 25 lM L-glutamate (#G1626, Introduction Campbell et al. [1] first described the tweety (twe, tty) gene in the fli (flightless) genomic locus of Drosophila mela- nogaster [1]. Mutations of tty in Drosophila result in the loss of flight ability [1]. Homologs of the tweety (tty) gene have also been identified in Caenorhabditis elegans and mammals [2]. Three mammalian homologs, Ttyh1, Ttyh2 and Ttyh3, have been described [2–4]. Elzbieta Wiernasz and Aleksandra Kaliszewska have contributed equally to this work. E. Wiernasz  A. Kaliszewska  J. Bednarczyk  M. Gorniak  K. Lukasiuk (&) Laboratory of Epileptogenesis, The Nencki Institute of Experimental Biology, 3 Pasteur St, 02-093 Warsaw, Poland e-mail: k.lukasiuk@nencki.gov.pl E. Wiernasz  A. Kaliszewska  J. Bednarczyk  M. Gorniak  K. Lukasiuk (&) Laboratory of Epileptogenesis, The Nencki Institute of Experimental Biology, 3 Pasteur St, 02-093 Warsaw, Poland e-mail: k.lukasiuk@nencki.gov.pl Human and mouse homologs of the D. melanogaster tweety gene encode proteins predicted to be integral membrane proteins that share a high degree of sequence identity and common structural features with five or six transmembrane regions positioned in a similar arrange- ment. This arrangement comprises a pair of transmembrane regions, followed by a hydrophobic region and an addi- tional pair of transmembrane regions, which are followed by more hydrophobic and transmembrane regions [2]. The glycosylated N-terminus is located extracellularly, whereas W. Brutkowski Laboratory of Imaging Tissue Structure and Function, The Nencki Institute of Experimental Biology, Warsaw, Poland B. Kaza Laboratory of Molecular Neurobiology, Neurobiology Center, The Nencki Institute of Experimental Biology, Warsaw, Poland 12 3 Neurochem Res (2014) 39:2516–2526 2517 the involved in Ca2? binding, an acid-residue-rich C-ter- minus is located cytoplasmically [5, 6]. The highest level of sequence variation among Tweety-related proteins is observed at the C-terminus, which likely specifies the function of these proteins [3]. Sigma). After 5 days, the G3 medium was exchanged for G2 medium (G3 medium without L-glutamate), and the cells were maintained without changing the media until the 14th day of culture, when the cells were fixed and used for immunocytochemistry. The human Tweety proteins are putative maxi-chloride ion channels with differentially induced Cl- channel activity [4, 7]. Tweety homolog 1 (Ttyh1) is a volume- regulated Cl- channel, whose activity is regulated by cell swelling, whereas the activity of Ttyh2 and Ttyh3 is reg- ulated by ionomycin and calcium ions [4, 7]. Culture of Primary Astrocytes and Microglia Culture of Oligodendrocytes Rat oligodendrocyte precursor cells (OPCs) were prepared according to a standard shaking method based on the dif- ferential adherent properties of glia [15–17]. Briefly, the forebrains from 1-day-old Wistar rat pups brains were aseptically removed and homogenized through mechanical dissociation. The cells were plated onto poly-L-lysine- coated (#P4707, Sigma) flasks, maintained for 10 days in DMEM/F12 (containing 10 % fetal calf serum, 2 mM glutamine and antibiotics) and fed every 2–3 days by a complete media change. At 10 days after plating, the cul- ture flasks were placed onto a rotary shaker and pre-shaken at 37 C for 6 h to remove the microglia cells. The medium was changed, and the flasks were shaken at 37 C for an additional 18–20 h. The cell suspension containing OPCs was collected through centrifugation. The cell pellet was suspended in OPC medium SATO medium containing DMEM with GlutaMAX (#31966-021, Gibco) supple- mented with 100 lg/ml BSA, 6.2 ng/ml progesterone, 16 lg/ml putrescine, 5 ng/ml sodium selenite, 400 ng/ml T4, 400 ng/ml T3, 50 lg/ml holo-transferrin, 5 lg/ml insulin, 10 ng/ml PDGF, 10 ng/ml FGF, penicillin (100 U/ml) and streptomycin (100 lg/ml)] and plated onto poly- L-lysine-coated glass coverslips (#1001/18, Glaswarenfab- rik Karl Hecht GmbH & Co) in 12-well plates at a con- centration of 2 9 105 cells/ml. The cells were fed every other day with 10 ng/ml PDGF and 10 ng/ml FGF until the 8th day of culture. To obtain differentiated oligodendro- cytes, the OPCs were cultured for 7 additional days in differentiation medium (DMEM (#31966-021, Gibco) containing 10 % fetal bovine serum (FBS, #10106-151, Gibco), penicillin (100 U/ml) and streptomycin (100 lg/ ml) (#15140-148, Gibco)]. The medium was changed every 2–3 days. The differentiated oligodendrocytes were fixed and used for immunocytochemical staining. Immunocytochemistry Primary hippocampal neurons, astrocytes, microglia and oligodendrocytes in culture were fixed for 10 min in 4 or 2 % paraformaldehyde (#P6148, Sigma) in PBS containing 4 % sucrose (#S0389, Sigma), permeabilized with 0.5 % Triton X-100 (#X100, Sigma) in PBS for 15 min, blocked (60 min at RT) in 5 % bovine serum albumin (#A2153, Sigma) (BSA) in 0.25 % Triton X-100 in PBS, and incu- bated overnight at 4 C with primary antibodies diluted in PBS containing 3 % BSA and 0.25 % Triton X-100. Sub- sequently, the cells were washed in PBS and incubated (2–2.5 h) with secondary antibodies diluted in PBS con- taining 3 % BSA and 0.25 % Triton X-100. The cell nuclei were counter-stained with 4,6-diamidino-2-phenylindole, DAPI (1:1000, #D9542, Sigma). Each staining was per- formed using 3–5 independent cultures. Amygdala Stimulation-Induced Status Epilepticus Amygdala Stimulation-Induced Status Epilepticus Scratch Assay Astrocytes (105 cells) were seeded onto 18 mmØ glass coverslips pretreated with a solution of 100 lg/ml poly poly-L-lysine (Sigma) and incubated in astrocyte culture medium until reaching confluence. Confluent monolayers were scraped with a 200 ll pipette tip. Cells were fixed in 4 % paraformaldehyde in PBS with 4 % sucrose 6, 24 or 48 h following wounding. Immunocytochemistry for Ttyh1 and GFAP was performed as described below. 12 3 2518 Neurochem Res (2014) 39:2516–2526 VA, USA, #E363-3-2WT-SPC) was implanted into the left lateral nucleus of the amygdala under isoflurane anesthesia. For surface EEG recording, a stainless steel screw was implanted contralaterally into the skull over the right frontal cortex. After a 2-week recovery period, the animals were electrically stimulated via the intra-amygdala elec- trode to evoke SE. Stimulation consisted of a 100-ms train of 1-ms biphasic square-wave pulses (400 lA peak to peak) delivered at 60 Hz, every 0.5 s for 20 min (Master-8 Stimulator connected with an ISO-Flex stimulus isolation unit, A.M.P.I., Israel). SE was terminated at 1.5–2 h after stimulation through the intraperitoneal injection of diaze- pam (20 mg/kg; Relanium, Polfa SA, Warsaw, Poland). The sham-operated control animals were implanted with electrodes but did not receive electrical stimulation. Con- trol (n = 7) and stimulated (n = 7) rats were sacrificed 4 days later. Animals were video-EEG monitored (Comet EEG, Grass Technologies, West Warwick, RI) to confirm development of SE lasting at least 90 min. VA, USA, #E363-3-2WT-SPC) was implanted into the left lateral nucleus of the amygdala under isoflurane anesthesia. For surface EEG recording, a stainless steel screw was implanted contralaterally into the skull over the right frontal cortex. After a 2-week recovery period, the animals were electrically stimulated via the intra-amygdala elec- trode to evoke SE. Stimulation consisted of a 100-ms train of 1-ms biphasic square-wave pulses (400 lA peak to peak) delivered at 60 Hz, every 0.5 s for 20 min (Master-8 Stimulator connected with an ISO-Flex stimulus isolation unit, A.M.P.I., Israel). SE was terminated at 1.5–2 h after stimulation through the intraperitoneal injection of diaze- pam (20 mg/kg; Relanium, Polfa SA, Warsaw, Poland). The sham-operated control animals were implanted with electrodes but did not receive electrical stimulation. Con- trol (n = 7) and stimulated (n = 7) rats were sacrificed 4 days later. Scratch Assay Animals were video-EEG monitored (Comet EEG, Grass Technologies, West Warwick, RI) to confirm development of SE lasting at least 90 min. Culture of Oligodendrocytes Image Acquisition and Analysis Image Acquisition and Analysis The Carl Zeiss LSM780 Spectral Confocal System inter- faced with Axio Observer Z.1 inverted microscope equip- ped with Plan Apochromat 409/1.4 Oil DIC, Plan Apochromat 639/1.4 Oil DIC objectives, Ar ion (488 nm), DPSS diode (561 nm), diode (405 nm CW/pulsed) laser and Zen 2011 software were used for fluorescence image acquisition. The images were processed using ImageJ software (Ras- band, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland). The displayed images were not manipulated beyond adjusting the histogram. Deconvolu- tion was performed using Huygens Software (SVI, Hil- versum, The Netherlands). Colocalization analysis was performed with ImarisColoc (Bitplane, Zurich, Switzer- land). Every time colocalization channel containing only colocalized voxels was generated. Results are presented as a merge of fluorescent channel and colocalization channel for easier perception of colocalization sites. Pearson’s correlation coefficient was calculated to quantify the degree of colocalization between fluorophores. Ttyh1 Immunohistochemistry for western blot analysis using a standard protocol [11]. The experiment was repeated three times. Briefly, the cells were lysed in lysis buffer containing 50 mM KCl, 50 mM PIPES, 10 mM EGTA, 2 mM MgCl2, 0.5 % Triton X-100, 100 lM PMSF and 1 mM DTT. The brain tissue was homogenized in the same buffer using a TissueRuptor homogenizer (Qiagen). The lysates were centrifuged at 20,800g for 20 min at 4 C. The supernatants were col- lected and stored at -20 C. The protein concentration was determined using Protein Assay Dye Reagent (#500-0006, BioRad). The samples were separated using SDS-PAGE and transferred to nitrocellulose (#RPN303D, HybondTM- ECL, Amersham). For immunodetection, the nonspecific binding was blocked through incubation in 5 % non-fat milk in TBST (0.5 M Tris, 0.9 % NaCl, 0.1 % Tween 20, pH 8). Subsequently, the membrane was incubated in a solution of mouse anti-Ttyh1 antibody (1:500, #WH0057348M4, Sigma) in TBST overnight at 4 C, followed by incubation in a solution of secondary anti- mouse horseradish peroxidase-linked antibody (1:5,000, #NA931, Amersham Biosciences) in TBST for 2 h at RT. Chemiluminescent detection was performed using ECL (#RPN2209, Amersham) according to the manufacturer’s instructions. The protein molecular weights were estimated using prestained protein markers. To verify that equal amounts of protein were loaded, the membrane was strip- ped and reprobed with mouse anti-GAPDH antibody (1:300, #MAB374, Chemicon Millipore) in TBST over- night at 4 C, followed by incubation with secondary anti- mouse horseradish peroxidase-linked antibody. Immunohistochemistry All animal procedures were approved by the Ethical Committee on Animal Research of the Nencki Institute, and the experiments were conducted in accordance with the guidelines of Directive 2010/63/EU of the European Par- liament and the Council of the European Union (EU). Adult male Sprague–Dawley rats (290–320 g) were obtained from the The Mossakowski Medical Research Centre (Polish Academy of Sciences, Warsaw, Poland) and housed in a controlled environment (24 C, lights on 07:00–19:00) with free access to food and water. Status epilepticus (SE) was triggered through electrical stimula- tion of the amygdala as previously described [18], with some modifications [19]. Briefly, a stimulating and recording bipolar electrode (Plastic One Inc., Roanoke, The rats were anesthetized with morbital (1.25 ml/kg, i.p.) and perfused with 0.9 % NaCl (2 min, 30 ml/min), fol- lowed by perfusion with 4 % paraformaldehyde in 0.1 % phosphate buffer (PB; 0.1 M Na2HPO4, and 0.1 M NaH2 PO4, pH 7.4) for 20 min (30 ml/min) as previously described [11]. Next, the brains were removed, postfixed for 4 h in the same fixative, cryoprotected in 30 % sucrose in 0.02 M PB buffer for 3–4 days, frozen on dry ice and stored at -70 C. Coronal sections (30 lm) were cut and stored in a cryoprotectant solution (30 % ethylene glycol, 25 % glycerol, 0.05 M PB) at -20 C. Immunohistochemistry was performed on free-floating coronal brain sections using a standard procedure [11]. 123 2519 Neurochem Res (2014) 39:2516–2526 Briefly, for double immunofluorescence staining, after blocking for unspecific binding for 2 h with PBS contain- ing 3 % bovine serum albumin (BSA, Sigma) and 0.1 % Triton X-100, a mixture of primary antibodies (diluted in PBS containing 0.3 % BSA and 0.01 % Triton X-100) was applied, and the cells were incubated overnight at 4 C. Subsequently, the sections were incubated for 2 h with the appropriate secondary antibodies diluted in PBS (contain- ing 0.3 % BSA and 0.01 % Triton X-100), and the nuclei were counter-stained with 4,6-diamidino-2-phenylindole (DAPI), mounted on gelatin-coated slides and coverslipped in Vectashield (Vector Laboratories). For detection of Ttyh1 with 3,30-diaminobenzidine (DAB) as a chromo- phore, sections were incubated with anti-Ttyh1 antibody, and then in a solution of appropriate biotinylated antibody. For signal detections, sections were incubated in avidin– biotin solution (ABC kit Vectastain, #PK6100, Vector Laboratories) and then in 0.05 % 3,30-diaminobenzidine (DAB) solution containing 0.04 % H2O2. Antibodies The following primary antibodies were used in the present study: mouse anti-Ttyh1 (1:200, #WH0057348M4, Sigma), rabbit anti-Synaptotagmin 1 (1:100, #105102, Synaptic Systems), rabbit anti-Synaptoporin (1:500, #102002, Syn- aptic Systems), rabbit anti-Neuronal Class III b-Tubulin (1:750, #PRB-435P, Covance), rabbit anti-LAMP1 (1:500, #ab24170, Abcam), rabbit anti-GRP78 (1:200, #ab21685, Abcam), rabbit anti-Calnexin (1:40, #ab22595, Abcam), rabbit anti-GM130 (1:200, #ab52649, Abcam), rabbit anti- TGN46 (1:500, #ab16059), rabbit anti-Clathrin (1:500, #ab21679, Abcam), rabbit anti-Rab5 (1:500, #ab18211, Abcam), rabbit anti-Rab11 (1:100, #71-5300, Invitrogen), rabbit anti-APPL2 (home made, [20]), rabbit anti-Olig2 (1:500, #AB9610, Chemicon Millipore), rabbit anti-Iba1 (1:1,000, #019-19741, Wako), mouse anti-GFAP-Cy3 (1:1,000, #C9205, Sigma). Anti-IgG mouse (1:200, #553447, BD Pharmingen) anti-IgG rabbit (1:200, #ab46540, Abcam) were used as controls to test specificity of immunostainings. The following secondary antibodies were used in the present study: goat anti-mouse conjugated with Alexa Fluor 488 (1:200, #A11001, Molecular Probes), goat anti-rabbit conjugated with Texas Red (1:200, #T2767, Molecular Probes), horse anti-mouse conjugated with Texas Red (1:200, #TI2000, Vector Laboratories), and anti-mouse biotinylated antibody (1:200, #BA2001, Vector Laboratories). Western Blot Ttyh1-positive structures were present throughout the entire cell, in the soma and in branches. The cell nuclei were counter- stained with DAPI (blue). Scale bars 10 lm. The images represent maximum intensity Z projections of confocal sections. DAPI, 4,6- diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1, Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte lineage transcription factor 2 (Color figure online) oligodendrocyte immunostained with anti-Ttyh1 (green) and anti- Olig2 (red). Ttyh1-positive structures were present throughout the entire cell, in the soma and in branches. The cell nuclei were counter- stained with DAPI (blue). Scale bars 10 lm. The images represent maximum intensity Z projections of confocal sections. DAPI, 4,6- diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1, Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte lineage transcription factor 2 (Color figure online) Fig. 2 Western blot analysis of Ttyh1 protein expression in protein extracts isolated from microglia, cerebral astrocyte cultures and hippocampal neuronal cultures. A single band represents Ttyh1 protein (predicted molecular weight of 37 kDa), which was detected in all lysates. GAPDH (predicted molecular weight of 38 kDa) was used as a protein loading control. GAPDH—Glyceraldehyde 3-phos- phate dehydrogenase vesicle volume analysis was performed using Spots algo- rithm based on absolute intensity included in Imaris soft- ware. All the analysis were performed using at least five images, which were obtained from 3 to 5 independent experiments. Microscope pictures of DAB stained slices were taken using a Nikon Eclipse 80i microscope equipped with a 109 and 209 objective and Wikom Evolution WF camera. Fig. 2 Western blot analysis of Ttyh1 protein expression in protein extracts isolated from microglia, cerebral astrocyte cultures and hippocampal neuronal cultures. A single band represents Ttyh1 protein (predicted molecular weight of 37 kDa), which was detected in all lysates. GAPDH (predicted molecular weight of 38 kDa) was used as a protein loading control. GAPDH—Glyceraldehyde 3-phos- phate dehydrogenase Results Expression of Ttyh1 Protein in Neurons, Astrocytes, Microglia and Oligodendrocytes In Vitro compared with oligodendrocytes. We have observed dif- ferences in the size of the Ttyh1-immunopositive dots in all cell types. Previous studies have shown the expression of Ttyh1 mRNA in rat cortical neurons and the widespread expres- sion of Ttyh1 protein in neurons in vitro and in vivo [10, 11]. In the present study we evaluated the expression of Ttyh1 protein in glial cells (astrocytes, oligodendrocytes and microglia) cultured in vitro. Immunocytochemistry with the anti-Ttyh1 antibody revealed the abundant expression of Ttyh1 in astrocytes, oligodendrocytes and microglia in vitro. Pattern of expression was consistent from culture to culture. Images of representative cells are presented on Fig. 1. The western blot analysis confirmed the presence of Ttyh1 protein in extracts of isolated hippocampal neurons, cerebral astrocytes and microglia in vitro (Fig. 2). 123 Western Blot Proteins isolated from rat primary cultured neurons, astrocytes, microglia and rat brain tissue were processed 12 3 123 Neurochem Res (2014) 39:2516–2526 2520 Fig. 1 Localization of Ttyh1 protein in rat astrocytes, microglia and oligodendrocytes in vitro. a Double immunolabeling of primary astrocytes for Ttyh1 and GFAP. Ttyh1-positive structures were present in the entire cell, in astrocyte somata and in the branches along the filament network stained with GFAP. b Ttyh1 immunore- activity in microglia double-immunostained with anti-Ttyh1 (green) and anti-Iba1 (red). Fine Ttyh1-positive, dot-like structures were present throughout the entire cell. c Ttyh1 immunoreactivity in Fig. 1 Localization of Ttyh1 protein in rat astrocytes, microglia and oligodendrocytes in vitro. a Double immunolabeling of primary astrocytes for Ttyh1 and GFAP. Ttyh1-positive structures were present in the entire cell, in astrocyte somata and in the branches along the filament network stained with GFAP. b Ttyh1 immunore- activity in microglia double-immunostained with anti-Ttyh1 (green) and anti-Iba1 (red). Fine Ttyh1-positive, dot-like structures were present throughout the entire cell. c Ttyh1 immunoreactivity in oligodendrocyte immunostained with anti-Ttyh1 (green) and anti- Olig2 (red). Ttyh1-positive structures were present throughout the entire cell, in the soma and in branches. The cell nuclei were counter- stained with DAPI (blue). Scale bars 10 lm. The images represent maximum intensity Z projections of confocal sections. DAPI, 4,6- diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1, Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte lineage transcription factor 2 (Color figure online) oligodendrocyte immunostained with anti-Ttyh1 (green) and anti- Olig2 (red). Ttyh1-positive structures were present throughout the entire cell, in the soma and in branches. The cell nuclei were counter- stained with DAPI (blue). Scale bars 10 lm. The images represent maximum intensity Z projections of confocal sections. DAPI, 4,6- diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1, Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte lineage transcription factor 2 (Color figure online) Fig. 1 Localization of Ttyh1 protein in rat astrocytes, microglia and oligodendrocytes in vitro. a Double immunolabeling of primary astrocytes for Ttyh1 and GFAP. Ttyh1-positive structures were present in the entire cell, in astrocyte somata and in the branches along the filament network stained with GFAP. b Ttyh1 immunore- activity in microglia double-immunostained with anti-Ttyh1 (green) and anti-Iba1 (red). Fine Ttyh1-positive, dot-like structures were present throughout the entire cell. c Ttyh1 immunoreactivity in oligodendrocyte immunostained with anti-Ttyh1 (green) and anti- Olig2 (red). Expression of Ttyh1 in the Hippocampus in Control Animals and After Amygdala Stimulation-Induced Status Epilepticus We detected Ttyh1 immunoreactivity in astrocytes, microglia and oligodendrocytes, and this result prompted use to determine whether Ttyh1 is expressed in activated glial cells in vivo. Following amygdala stimulation induced status epilepticus, reactive astrocytes in hippocampus can be observed as early as after 24 h and persist for at least In astrocytes and oligodendrocytes Ttyh1 was present in dot-like structures in cell somata and in branches. Simi- larly, in microglia Ttyh1-positive structures were evenly distributed throughout the cytoplasm. Ttyh1 immunoreac- tivity in astrocytes and microglia was weaker when 123 123 2521 Neurochem Res (2014) 39:2516–2526 Fig. 3 Ttyh1 expression in the normal rat brain and at 4 days after amygdala stimulation-induced status epilepticus. DAB immunostain- ing with anti-Ttyh1 antibody in the hippocampus of a sham control brain (a) and at 4 days after status epilepticus (b). Fragments of pyramidal layer and stratum oriens of CA3 region are framed and enlarged in c (control brain) and d (stimulated brain). In the control brain exclusively neuronal somata within pyramidal layer are stained (indicated with arrowheads). At 4 days after status epilepticus astrocyte-like profiles (arrows) and neuronal somata within pyramidal layer (arrowheads) are revealed with immunostaining with anti-Ttyh1 antibody. Scale bars 500 lm for a and b, 50 lm for c and d brain exclusively neuronal somata within pyramidal layer are stained (indicated with arrowheads). At 4 days after status epilepticus astrocyte-like profiles (arrows) and neuronal somata within pyramidal layer (arrowheads) are revealed with immunostaining with anti-Ttyh1 antibody. Scale bars 500 lm for a and b, 50 lm for c and d Fig. 3 Ttyh1 expression in the normal rat brain and at 4 days after amygdala stimulation-induced status epilepticus. DAB immunostain- ing with anti-Ttyh1 antibody in the hippocampus of a sham control brain (a) and at 4 days after status epilepticus (b). Fragments of pyramidal layer and stratum oriens of CA3 region are framed and enlarged in c (control brain) and d (stimulated brain). In the control brain exclusively neuronal somata within pyramidal layer are stained (indicated with arrowheads). At 4 days after status epilepticus astrocyte-like profiles (arrows) and neuronal somata within pyramidal layer (arrowheads) are revealed with immunostaining with anti-Ttyh1 antibody. Scale bars 500 lm for a and b, 50 lm for c and d Fig. 3 Ttyh1 expression in the normal rat brain and at 4 days after amygdala stimulation-induced status epilepticus. Expression of Ttyh1 in the Hippocampus in Control Animals and After Amygdala Stimulation-Induced Status Epilepticus DAB immunostain- ing with anti-Ttyh1 antibody in the hippocampus of a sham control brain (a) and at 4 days after status epilepticus (b). Fragments of pyramidal layer and stratum oriens of CA3 region are framed and enlarged in c (control brain) and d (stimulated brain). In the control cells in the CA3 of animals subjected to amygdala stimu- lation (Fig. 4). The increase in Ttyh1 immunoreactivity in reactive astrocytes in the CA3 reflected the increased intensity of immunofluorescence and the increase in the number of Ttyh1-positive dots (Fig. 4d). 14 days [21]. We performed preliminary DAB immuno- stainings to detect Ttyh1 protein in slices from rats 24 h, 4 and 14 days after amygdala stimulation-induced status epilepticus and time-matched control animals. 4 days fol- lowing status epilepticus majority of immunostained hip- pocampal cells displayed star-like processes typical of hypertrophic reactive astrocytes. They were more or less evenly distributed among different layers (Fig. 3b, d). In hippocampi of control animals only sparse neuron-shaped somata were strongly labeled (Fig. 3a, c). They were found mainly in the pyramidal layer. In other timepoints we did not observe Ttyh1 expression in cells morphologically resembling astrocytes (data not shown). To test possibility that status epilepticus induces Ttyh1 expression also in oligodendrocytes or microglia, we per- formed double immunostainings using anti-Ttyh1 antibody and anti-Iba1 (microglia marker) or anti-Olig2 (oligoden- drocyte marker) antibody (data not shown). Ttyh1 immu- noreactivity in microglia in vitro was negligible in the control brain. Similarly, in the hippocampus of the stimu- lated brain, Ttyh1 colocalization with Iba1 was only occasionally observed. Ttyh1 immunostaining in oligo- dendrocytes in vivo was difficult to interpret. Because the oligodendrocytes insulating neuronal axons are thin and the oligodendrocyte marker Olig2 is specifically localized inside the nucleus, not in the branches, it is difficult to distinguish immunoreactivity in oligodendrocytes. We To confirm Ttyh 1 astrocytic localization, we performed double immunohistochemistry, using the anti-Ttyh1 and anti-GFAP antibody. Few Ttyh1-positive dots were observed in GFAP-positive astrocytes in the CA3 of the sham control animals. In contrast, numerous Ttyh1-im- munopositive elements were detected in GFAP-positive 12 3 3 2522 Neurochem Res (2014) 39:2516–2526 Fig. 4 Ttyh1 expression in astrocytes in the normal rat brain and at 4 days after amygdala stimulation-induced status epilepticus. a– c Ttyh1 expression in the hippocampal CA3 of a sham control brain. Expression of Ttyh1 in the Hippocampus in Control Animals and After Amygdala Stimulation-Induced Status Epilepticus a Confocal image of immunofluorescence with anti-Ttyh1 (green) and b the astrocyte marker anti-GFAP (red), c merged image of double immunofluorescence staining demonstrating Ttyh1, GFAP and cell nuclei counter-stained with DAPI (blue). d–f Ttyh1 expression in the hippocampal CA3 pyramidal layer at 4 days after status epilepticus. d Confocal image of immunofluorescence with anti-Ttyh1 and e anti- GFAP, f merged image of double immunofluorescence staining observed only few Ttyh1-positive dots in the close vicinity of oligodendrocyte nuclei in the control brain and after status epilepticus. deposited only in the part of the process most protruding toward the scratch. In wider lamellipodia it was concen- trated along the whole leading edge. Such uneven distri- bution of Ttyh1 in astrocytes along the scratch at any timepoint was observed only in fraction of cells. Expression of Ttyh1 in the Hippocampus in Control Animals and After Amygdala Stimulation-Induced Status Epilepticus a Confocal image of immunofluorescence with anti-Ttyh1 (green) and b the astrocyte marker anti-GFAP (red), c merged image of double immunofluorescence staining demonstrating Ttyh1, GFAP and cell nuclei counter-stained with DAPI (blue). d–f Ttyh1 expression in the hippocampal CA3 pyramidal layer at 4 days after status epilepticus. d Confocal image of immunofluorescence with anti-Ttyh1 and e anti- GFAP, f merged image of double immunofluorescence staining demonstrating Ttyh1, GFAP and cell nuclei counter-stained with DAPI. The yellow arrows on green channel (a, d) are pointing single, representative Ttyh1-positive structures. The same Ttyh1-positive structure, marked by the yellow arrows on merge channel (c, f), show the colocalization of Ttyh1 and GFAP. Note that in the control brain, colocalization was negligible, while Ttyh1-positive punctuate staining was observed throughout the astrocytes from the brain after status epilepticus. Scale bar 5 lm. The images represent single confocal sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein (Color figure online) rat brain and at epilepticus. a– am control brain. Ttyh1 (green) and image of double GFAP and cell expression in the tatus epilepticus. Ttyh1 and e anti- escence staining demonstrating Ttyh1, GFAP and cell nuclei counter-stained with DAPI. The yellow arrows on green channel (a, d) are pointing single, representative Ttyh1-positive structures. The same Ttyh1-positive structure, marked by the yellow arrows on merge channel (c, f), show the colocalization of Ttyh1 and GFAP. Note that in the control brain, colocalization was negligible, while Ttyh1-positive punctuate staining was observed throughout the astrocytes from the brain after status epilepticus. Scale bar 5 lm. The images represent single confocal sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein (Color figure online) demonstrating Ttyh1, GFAP and cell nuclei counter-stained with DAPI. The yellow arrows on green channel (a, d) are pointing single, representative Ttyh1-positive structures. The same Ttyh1-positive structure, marked by the yellow arrows on merge channel (c, f), show the colocalization of Ttyh1 and GFAP. Note that in the control brain, colocalization was negligible, while Ttyh1-positive punctuate staining was observed throughout the astrocytes from the brain after status epilepticus. Scale bar 5 lm. The images represent single confocal sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein (Color figure online) Fig. 4 Ttyh1 expression in astrocytes in the normal rat brain and at 4 days after amygdala stimulation-induced status epilepticus. a– c Ttyh1 expression in the hippocampal CA3 of a sham control brain. Scratch-Induced Changes in Localization of Ttyh1 in Cultured Astrocytes Ttyh1 Immunopositive Puncta Volume and Identity in Cultured Neurons and Astrocytes Ttyh1 Immunopositive Puncta Volume and Identity in Cultured Neurons and Astrocytes Reactive astrogliosis, observed in many pathological brain processes including epileptogenesis, can be induced in vitro by a scratch injury [22]. Astrocytes rapidly polarize after lesion and extend their processes perpendicularly to the scratch. We performed Ttyh1 immunofluorescent staining on astrocytes fixed 6, 24 and 48 h after wounding. We observed gradual filling of the area of the scratch by migrating astrocytes. In some migrating astrocytes we observed concentration of Ttyh1 protein at the margin of lamellipodia (Fig. 5). In narrow lamellipodia Ttyh1 was In neurons and in glial cells Ttyh1 is present in form of dot- like structures of different sizes. Hypothetically this het- erogeneity may reflect presence of Ttyh1 in different sub- cellular structures. Neurons and astrocytes are particularly interesting in terms of Ttyh1 expression, as in those types of cells we were able to detect prominent Ttyh1 immuno- reactivity in vivo. We asked whether in those two cell types Ttyh1 is present in the same subcellular structures. Firstly, 123 123 2523 Neurochem Res (2014) 39:2516–2526 Fig. 5 Scratch-induced changes in localization of Ttyh1 in cultured astrocytes. Cultured astrocytes were fixed 48 h after making a scratch and labeled for Ttyh1 (green) and GFAP (red). Yellow dashed line indicates the scratch. In presented astrocytes migrating toward the scratch Ttyh1 was particularly concentrated at the margins of lamellipodia. Ttyh1 formed deposits only in astrocyte processes most protruding toward the scratch (a) or along the leading edge (b). The cell nuclei were counter-stained with DAPI (blue). Scale bar 25 lm. The images represent maximum intensity Z projections of confocal sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein (Color figure online) Fig. 5 Scratch-induced changes in localization of Ttyh1 in cultured astrocytes. Cultured astrocytes were fixed 48 h after making a scratch and labeled for Ttyh1 (green) and GFAP (red). Yellow dashed line indicates the scratch. In presented astrocytes migrating toward the scratch Ttyh1 was particularly concentrated at the margins of lamellipodia. Ttyh1 formed deposits only in astrocyte processes most protruding toward the scratch (a) or along the leading edge (b). The cell nuclei were counter-stained with DAPI (blue). Scale bar 25 lm. The images represent maximum intensity Z projections of confocal sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein (Color figure online) Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was determined using Imaris software. Ttyh1 Immunopositive Puncta Volume and Identity in Cultured Neurons and Astrocytes Immunopositive puncta are classified into groups depending on their volume. Percent distribution of Ttyh1 vesicle volume in both neurons and astrocytes is similar, with most puncta having volume 0.05–149 lm3 and only few percent of puncta bigger than 0.35 lm3. Note that proportions of immuno- positive puncta in different size groups vary between neurons and astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05; ***p \ 0.001 We classified all Ttyh1 immunopositive puncta into one of five groups depending on their volume (Fig. 6). Distri- bution of Ttyh1 immunopositive puncta volume was roughly the same for neurons and astrocytes, with rela- tively small proportion of \0.05 lm3 puncta, highest pro- portion of 0.05–0.149 lm3 puncta and gradually decreasing contribution of 0.15–0.249, 0.25–0.349 and [0.35 lm3 puncta. However, percentages of puncta in different size groups varied between neurons and astrocytes as the puncta 0.05–0.149 lm3 were more abundant in neurons than in astrocytes, while small puncta of \0.05 lm3 and the large ones (0.25–0.349 and[0.35 lm3) were more abundant in astrocytes. In a previous study, in which we performed a pull-down assay using hippocampal protein lysates, we revealed that Ttyh1 protein specifically binds to a number of proteins, including a large group of proteins localized in the endo- plasmic reticulum or Golgi apparatus [11]. To further characterize the subcellular localization of Ttyh1 protein, we performed double immunocytochemistry on primary hippocampal neurons and primary cortical astrocyte cell cultures using a number of antibodies against specific subcellular markers. Ttyh1 protein has been detected in the Golgi apparatus (Pearson’s correlation coefficient PCC, neurons r = 0.30, astrocytes r = 0.32; Fig. 7a, e), endo- plasmic reticulum (PCC, neurons r = 0.27, astrocytes r = 0.47; Fig. 7b, f) and clathrin-coated vesicles (PCC, neurons r = 0.27, astrocytes r = 0.35; Fig. 7c, g) in both neurons and astrocytes. In astrocytes, Ttyh1 colocalized with LAMP1, the marker of late endosomes and lysosomes (PCC, r = 0.48; Fig. 7d). No colocalization of Ttyh1 with Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was determined using Imaris software. Immunopositive puncta are classified into groups depending on their volume. Percent distribution of Ttyh1 vesicle volume in both neurons and astrocytes is similar, with most puncta having volume 0.05–149 lm3 and only few percent of puncta bigger than 0.35 lm3. Ttyh1 Immunopositive Puncta Volume and Identity in Cultured Neurons and Astrocytes Note that proportions of immuno- positive puncta in different size groups vary between neurons and astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05; ***p \ 0.001 Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was determined using Imaris software. Immunopositive puncta are classified into groups depending on their volume. Percent distribution of Ttyh1 vesicle volume in both neurons and astrocytes is similar, with most puncta having volume 0.05–149 lm3 and only few percent of puncta bigger than 0.35 lm3. Note that proportions of immuno- positive puncta in different size groups vary between neurons and astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05; ***p \ 0.001 Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was determined using Imaris software. Immunopositive puncta are classified into groups depending on their volume. Percent distribution of Ttyh1 vesicle volume in both neurons and astrocytes is similar, with most puncta having volume 0.05–149 lm3 and only few percent of puncta bigger than 0.35 lm3. Note that proportions of immuno- positive puncta in different size groups vary between neurons and astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05; ***p \ 0.001 we addressed the issue indirectly. We compared distribu- tion of Ttyh1 immunopositive puncta volume in cultured neurons and astrocytes. Secondly, we tried to colocalize Ttyh1 with different subcellular markers in neurons and astrocytes. 12 3 2524 Neurochem Res (2014) 39:2516–2526 Fig. 7 Ttyh1 colocalization with subcellular organelle markers in primary cultures of hippocampal neurons and cortical astrocytes. The panels represent confocal images of immunofluorescent staining with anti-Ttyh1 (green) and antibodies directed against different organelle markers (red) and cell nuclei counter-stained with DAPI (blue). Intensity correlation-based analysis was performed on Imaris soft- ware. The colocalized areas, representing the overlay of green and red pixels, are shown as white dots (see yellow arrows that are pointing representative examples of white dots). The colocalization of Ttyh1- positive dot-like structures with the Golgi apparatus marker GM130 (a), endoplasmic reticulum marker, GRP78 (b), coated pits and vesicles marker, clathrin (c) in neurons and astrocytes. Ttyh1 colocalizes with LAMP1 (late endosome-lysosome marker) in astrocytes (d) and does not colocalize in neurons (h). Scale bar 5 lm. The images represent single confocal sections. LAMP1 was detected in neurons (PCC, r = 0.021; Fig. 7h). microgliosis occur in several brain areas, including hippo- campus [21, 23]. In hippocampal slices from rats 4 days after status epilepticus we observed Ttyh1 immunoreac- tivity in cells possessing star-like processes characteristic for hypertrophic astrocytes, while in control brain we observed immunostaining only in neurons. We confirmed astrocytic localization of Ttyh1 protein by collocalizing it with GFAP. We conclude that in physiological conditions Ttyh1 is expressed mainly in neurons, but in brain pathol- ogy it may be also be expressed by astrocytes. Previously we described Ttyh1 immunoreactivity in the hippocampi of animals 14 days after status epilepticus [11]. At this time- point we observed increased neuropilar Ttyh1 immunore- activity in inner molecular layer of dentate gyrus, but we did not detect immunostained astrocytic profiles despite the fact that astrogliosis in epileptic hippocampus is persistent and can be detected at this timepoint [21]. Therefore we hypothesize that astrocytic expression of Ttyh1 is charac- teristic for early phase of astrogliosis. Despite the lack of Ttyh1 expression in astrocytes in control brains, we have observed Ttyh1 expression in astrocytes in vitro which may indicate that these cells do not maintain a complete resting phenotype in dissociated culture. We did not observe We observed no colocalization of Ttyh1 protein with a number of other cell compartment-specific markers, including markers of early endosomes (Rab5 and APPL2), recycling endosomes (Rab11), trans-Golgi network (TGN46), endoplasmic reticulum membrane (calnexin) and synaptic vesicles (synaptoporin and synaptotagmin 1). Ttyh1 Immunopositive Puncta Volume and Identity in Cultured Neurons and Astrocytes GM130, 130-kDa cis-Golgi matrix protein; GRP78, 78-kDa glucose-regulated protein; LAMP1, lysosomal-associated membrane protein 1 (Color figure online) positive dot-like structures with the Golgi apparatus marker GM130 (a), endoplasmic reticulum marker, GRP78 (b), coated pits and vesicles marker, clathrin (c) in neurons and astrocytes. Ttyh1 colocalizes with LAMP1 (late endosome-lysosome marker) in astrocytes (d) and does not colocalize in neurons (h). Scale bar 5 lm. The images represent single confocal sections. GM130, 130-kDa cis-Golgi matrix protein; GRP78, 78-kDa glucose-regulated protein; LAMP1, lysosomal-associated membrane protein 1 (Color figure online) Fig. 7 Ttyh1 colocalization with subcellular organelle markers in primary cultures of hippocampal neurons and cortical astrocytes. The panels represent confocal images of immunofluorescent staining with anti-Ttyh1 (green) and antibodies directed against different organelle markers (red) and cell nuclei counter-stained with DAPI (blue). Intensity correlation-based analysis was performed on Imaris soft- ware. The colocalized areas, representing the overlay of green and red pixels, are shown as white dots (see yellow arrows that are pointing representative examples of white dots). The colocalization of Ttyh1- positive dot-like structures with the Golgi apparatus marker GM130 (a), endoplasmic reticulum marker, GRP78 (b), coated pits and vesicles marker, clathrin (c) in neurons and astrocytes. Ttyh1 colocalizes with LAMP1 (late endosome-lysosome marker) in astrocytes (d) and does not colocalize in neurons (h). Scale bar 5 lm. The images represent single confocal sections. GM130, 130-kDa cis-Golgi matrix protein; GRP78, 78-kDa glucose-regulated protein; LAMP1, lysosomal-associated membrane protein 1 (Color figure online) LAMP1 was detected in neurons (PCC, r = 0.021; Fig. 7h). Discussion Previous in vivo studies have indicated that in the brain, Ttyh1 protein is abundantly expressed in punctuate, dot-like structures localized in neuropil and neuronal somata [11], and Ttyh1 mRNA in the normal brain is expressed in neu- rons [10]. Therefore, we concluded that Ttyh1 is predomi- nantly a neuronal protein. However, in the present study, we detected Ttyh1 expression in astrocytes, microglia and ol- igodendrocytes in vitro and this result prompted use to determine whether Ttyh1 is expressed in activated glial cells in vivo. In amygdala stimulation model of temporal epilepsy neurodegeneration and consequent astrogliosis and 123 123 Neurochem Res (2014) 39:2516–2526 2525 is consistent with that of a previous study characterizing proteins that form complexes with Ttyh1 in the brain tissue [11]. The localization of Ttyh1 in the ER has also been demonstrated in HEK293 and HEK293T cells and in the mouse brain [6, 8]. The presence of Ttyh1 protein in the ER and Golgi may reflect Ttyh1 protein synthesis and post- translational modifications. It has been shown that glyco- sylation occurs in the Golgi and is important for the proper function of Tweety proteins [5]. A small population of Ttyh1-positive structures has been detected in endocytic vesicles and lysosomes. This interesting observation sug- gests that Ttyh1 is present in transport vesicles and may be involved in endocytosis and/or exocytosis. prominent Ttyh1 immunoreactivity in microglia and oli- godendrocytes in vivo, either in slices from control rats or from stimulated animals. However we can not exclude possibility that in other pathological conditions microglia and oligodendrocytes express Ttyh1. We performed a scratch injury on cultured astrocytes monolayer to mimic reactive astrogliosis in vitro. In some cells migrating into the scratched area we observed that Ttyh1 is concentrated at the leading edge of lammelipodia. It has been shown in HEK 293 cell line, that Ttyh1 was similarly concentrated at the peripheral membrane of cells, where it induced formation of filopodia and co-localized with a5 integrin subunit [8]. Integrins are crucial for cell migration and its attachment to extracellular matrix. Dur- ing the process of attachment of astrocytes, some integrin subunits can be found at the margins of the cell on lamellipodia [24, 25], which resembles pattern of Ttyh1 immunoreactivity in migrating astrocytes. Discussion Seizures acti- vate integrin signaling and induce a turnover in adhesive contacts [26, 27], which hypothetically may be linked to increased Ttyh1 expression observed during epileptogene- sis and epilepsy [10, 28]. We observed increased Ttyh1 immunoreactivity only in small proportion of migrating astrocytes, so it is possible that Ttyh1 is involved only in specific phase of cell migration and adhesion. Because Ttyh1 expression is primarily restricted to the brain [8] and Ttyh1 is highly expressed in neurons, we verified whether Ttyh1 is present in synaptic vesicles involved in the storage and subsequent release of neuro- transmitters at synapses. We were unable to detect colo- calization with the synaptic vesicles markers synaptoporin or synaptotagmin 1 in vitro. This observation is consistent with a previous in vivo study showing the sparse colocal- ization of Ttyh1 protein with presynaptic markers, and it is reasonable to conclude that the majority of Ttyh1-positive elements are not presynaptically localized in the brain [11]. This conclusion is not in agreement with the results of Morciano et al. [9] who co-isolated Ttyh1 protein with the docked synaptic vesicles. However, it is possible that the immunofluorescence used in the present study was not sensitive enough to detect minute amounts of synaptic vesicle-bound Ttyh1 or that Ttyh1 is present at presynaptic sites only under specific conditions. As we have showed in our previous paper [11], primary hippocampal neurons after 8 days in culture show abundant expression of Ttyh1 protein with characteristic differences in size and distribution of Ttyh1-immunopositive dots. We could distinguish two populations of Ttyh1-immunoposi- tive dots: a population of large and a population of small dots. The large Ttyh1-positive dots were localized to sites adjacent to neuronal nuclei, and the small dots were local- ized along neurites. Here, we used older neuronal cultures (14DIV). In comparison to the younger culture (8DIV), older neurons showed less abundant expression and more homogenous distribution of Ttyh1 dots of different sizes. We conclude that the number and distribution of Ttyh1 immunopositive puncta in neurons in vitro depends on the culture age. Such unequal distribution has not been observed in glial cells in vitro. Percent distribution of Ttyh1 immunopositive puncta volume in neurons and astrocytes was comparable. There were differences between neurons and astrocytes in percentage of immunopositive puncta in specific size groups, but it is difficult to asses whether these differences have any functional meaning. References 13. Zawadzka M, Kaminska B (2005) A novel mechanism of FK506- mediated neuroprotection: downregulation of cytokine expression in glial cells. Glia 49:36–51 1. Campbell HD, Schimansky T, Claudianos C, Ozsarac N, Kaspr- zak AB, Cotsell JN, Young IG, de Couet HG, Miklos GL (1993) The Drosophila melanogaster flightless-I gene involved in gas- trulation and muscle degeneration encodes gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans and humans. Proc Natl Acad Sci USA 90:11386–11390 14. Przanowski P, Dabrowski M, Ellert-Miklaszewska A, Kloss M, Mieczkowski J, Kaza B, Ronowicz A, Hu F, Piotrowski A, Kettenmann H, Komorowski J, Kaminska B (2014) The signal transducers Stat1 and Stat3 and their novel target Jmjd3 drive the expression of inflammatory genes in microglia. J Mol Med (Berl) 92:239–254 2. Campbell HD, Kamei M, Claudianos C, Woollatt E, Sutherland GR, Suzuki Y, Hida M, Sugano S, Young IG (2000) Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: a novel gene family encoding predicted transmembrane proteins. Genomics 68:89–92 15. McCarthy KD, de Vellis J (1980) Preparation of separate as- troglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85:890–902 16. Chen Y, Balasubramaniyan V, Peng J, Hurlock EC, Tallquist M, Li J, Lu QR (2007) Isolation and culture of rat and mouse oli- godendrocyte precursor cells. Nat Protoc 2:1044–1051 3. Rae FK, Hooper JD, Eyre HJ, Sutherland GR, Nicol DL, Cle- ments JA (2001) TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma. Genomics 77:200–207 17. Cole R, de Vellis J (2001) Preparation of astrocyte, oligo- dendrocyte, and microglia cultures from primary rat cerebral cultures. In: Fedorof S, Richardson A (eds) Protocols for neural cell culture, 3rd edn. Humana Press Inc., New Yersey, pp 117–127 4. Suzuki M, Mizuno A (2004) A novel human Cl(-) channel family related to Drosophila flightless locus. J Biol Chem 279:22461–22468 18. Nissinen J, Halonen T, Koivisto E, Pitkanen A (2000) A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Res 38:177–205 5. He Y, Ramsay AJ, Hunt ML, Whitbread AK, Myers SA, Hooper JD (2008) N-glycosylation analysis of the human Tweety family of putative chloride ion channels supports a penta-spanning mem- brane arrangement: impact of N-glycosylation on cellular pro- cessing of Tweety homologue 2 (TTYH2). Biochem J 412:45–55 19. References Bot AM, De˛bski KJ, Lukasiuk K (2013) Alterations in miRNA levels in the dentate gyrus in epileptic rats. PLoS ONE 8:e76051 20. Miaczynska M, Christoforidis S, Giner A, Shevchenko A, Ut- tenweiler-Joseph S, Habermann B, Wilm M, Parton RG, Zerial M (2004) APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment. Cell 116:445–456 6. Kumada T, Yamanaka Y, Kitano A, Shibata M, Awaya T, Kato T, Okawa K, Abe T, Oshima N, Nakahata T, Heike T (2010) Ttyh1, a Ca(2 ?)- binding protein localized to the endoplasmic reticulum, is required for early embryonic development. Dev Dyn 239:2233–2245 21. Jurewicz E, Bednarczyk J, Bot A, Lukasiuk K, Filipek A (2013) Status epilepticus induces long lasting increase in S100A6 expression in astrocytes. Neurochem Res 38:1941–1948 7. Suzuki M (2006) The Drosophila tweety family: molecular can- didates for large-conductance Ca2?-activated Cl- channels. Exp Physiol 91:141–147 22. Yu AC, Lee YL, Eng LF (1993) Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis. J Neurosci Res 34:295–303 8. Matthews CA, Shaw JE, Hooper JA, Young IG, Crouch MF, Campbell HD (2007) Expression and evolution of the mammalian brain gene Ttyh1. J Neurochem 100:693–707 23. Guzik-Kornacka A, Sliwa A, Plucinska G, Lukasiuk K (2011) Status epilepticus evokes prolonged increase in the expression of CCL3 and CCL4 mRNA and protein in the rat brain. Acta Neurobiol Exp (Wars) 71:193–207 9. Morciano M, Beckhaus T, Karas M, Zimmermann H, Volknandt W (2009) The proteome of the presynaptic active zone: from docked synaptic vesicles to adhesion molecules and maxi-chan- nels. J Neurochem 108:662–675 24. Tawil N, Wilson P, Carbonetto S (1993) Integrins in point con- tacts mediate cell spreading: factors that regulate integrin accu- mulation in point contacts vs. focal contacts. J Cell Biol 120:261–271 10. Stefaniuk M, Lukasiuk K (2010) Cloning of expressed sequence tags (ESTs) representing putative epileptogenesis-related genes and the localization of their expression in the normal brain. Neurosci Lett 482:230–234 25. Peng H, Shah W, Holland P, Carbonetto S (2007) Integrins and dystroglycan regulate astrocyte wound healing: the integrin b1 subunit is necessary for process extension and orienting the microtubular network. Dev Neurobiol 68:559–574 11. Stefaniuk M, Swiech L, Dzwonek J, Lukasiuk K (2010) Expression of Ttyh1, a member of the Tweety family in neurons in vitro and in vivo and its potential role in brain pathology. J Neurochem 115:1183–1194 26. Discussion In conclusion, the data presented in this work indicate the Ttyh1 might be involved in neuronal and glial cell functions. Particularly, the elevated expression of Ttyh1 in astrocytes following damaging brain insult implies some unknown role for Ttyh1 protein in brain pathology. There is little information on the potential molecular function of Ttyh1, thus additional studies are required to determine the precise role of this protein. Acknowledgments This research was supported through funding from Polish National Science Centre Grant 2011/03/NZ4/00302, the Nencki Institute and the EU-funded grant BIO-IMAGINE: BIO- IMAGing in research INnovation and Education (EU FP7 Grant Agreement No. 264173). The authors would like to thank Prof. Marta Miaczynska (Laboratory of Cell Biology, International Institute of Molecular and Cell Biology) for anti-APPL2 antibody, Dr. Mal- gorzata Zawadzka and Dr. Bartosz Wylot (Laboratory of Molecular Neurobiology, Nencki Institute) for assistance with oligodendrocyte cultures. The analysis of Ttyh1 colocalization with the markers of cellular compartments in neurons and astrocytes in vitro revealed that Ttyh1 is present in several subcellular com- partments, including the lumen of the endoplasmic reticu- lum, the cis network of the Golgi apparatus, clathrin-coated pits and vesicles and late endosomes-lysosomes. This result Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. 12 3 2526 Neurochem Res (2014) 39:2516–2526 References References Gall CM, Lynch G (2004) Integrins, synaptic plasticity and epi- leptogenesis. Adv Exp Med Biol 548:12–33 12. Araujo IM, Carreira BP, Pereira T, Santos PF, Soulet D, Inacio A, Bahr BA, Carvalho AP, Ambrosio AF, Carvalho CM (2007) Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor- mediated neurodegeneration via calpain activation in hippocam- pal neurons. Cell Death Differ 14:1635–1646 p g p 27. Wu X, Reddy DS (2012) Integrins as receptor targets for neu- rological disorders. Pharmacol Ther 134:68–81 28. Lukasiuk K, Kontula L, Pitka¨nen A (2003) cDNA profiling of epileptogenesis in the rat brain. Eur J Neurosci 17:271–279 123 123
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pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
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pH Dependence of Acrylate-Derivative Polyelectrolyte Properties Thomas Swift Abstract There are many polymers formed of acrylate monomers in existence. Here we interrogate four commonly-used examples and study how their solution properties are pH dependent, or how their state of ionisation can affect their solution properties. Poly(acrylic acid) and poly(methacrylic acid) are both polyelectrolytes, with ioni- sable functional groups that make them stimuli responsive, changing their hydro- dynamic volume. Poly(acrylamide) is a mass-produced material used in a variety of industrial applications, often with an anionic and cationic co-monomer, which dictates both its efficacy and impact on the environment. Poly(N-isopropyl acryl- amide) is a thermally responsive material with applications in smart bioengineering. In solution, these materials can interact with each other due to competing hydrogen bonding interactions. However, this interpolymer complexation is dependent on both the ionisation, and the conformational state, of the polymers involved. This review focuses on the results from fluorescence tagging and turbidimetric techniques. Keywords: poly(acrylic acid), poly(methacrylic acid), poly(acrylamide), poly(N-isopropylacrylamide), stimuli responsive, interpolymer complexation, hydrodynamic volume, solution properties 1. Introduction The chemical structure of ionisation (or dissociation/neutralisation) is thus: RCOOH ⇋ RCOO − + H + (1) (1) and the dissociation constant (α) can be described by the Henderson- Hasselbalch equation and the dissociation constant (α) can be described by the Henderson- Hasselbalch equation α = ( [X] + [ H + ] − [ OH − ] ) / [RCOOH] (2) pH = p K a + log {α / (1 − α) } (3) α = ( [X] + [ H + ] − [ OH − ] ) / [RCOOH] (2) α = ( [X] + [ H + ] − [ OH − ] ) / [RCOOH] (2) pH = p K a + log {α / (1 − α) } (3) (3) where X is the ionising (titrating) species and pKa the dissociation constant; the pH at which 50% of the carboxylic groups have been ionised. However, for a poly- acid, this is a more contentious issue than studying small molecules due to each acid group is affected by the presence of neighbouring repeat units, which thus modify their titration behaviour. In general, the first COOH group on a polymer backbone shows a similar pKa to a small molecule analogue. However, as the polymer chain becomes increasingly ionised, the building negative charge constrains further deprotonation, and the pKa value alters with increasing pH. In this behaviour, particularly polymeric electrolytes show divergent behaviour from small molecules, and Katchalksy and Spitnik proposed a revision to the Henderson Hasselbalch Equation [4]. pH = p K a + n log {α / (1 − α) } (4) (4) pH = p K a + n log {α / (1 − α) } where n is a constant dependent on the ionic strength of the solution and the strength of the polyacid. In a stationary solution, this plot should produce a straight line (slope n, intercept pKa). However, this is rarely observed, particularly in aque- ous solutions, and this was the first indication researchers had that many polymeric macromolecules undergo a conformational rearrangement on the nanoscale in response to chemical ionization [4, 5]. Over the years, this has proven fertile ground for research, with poly(carboxylic acid)s receiving particular attention in the literature as they are excellent, chemically distinct, model systems [3, 4, 5–13]. 1. Introduction A common feature of the many polymer systems formed from acrylate mono- mers is their hydrophilicity; apparent either from their increased absorbency, wettability or increased solubility. Whilst the latter is often overlooked in materials science, it is of vital importance to a range of industries, as a multitude of polyacry- lates form vital components in commercial products too varied to list, but including dispersants, adhesives, emulsifiers, lubricants, flocculants, thickeners, surfactants, sensors, delivery agents, coatings, chromatographic phases, grouting, passivation and many more. As of 2018, the multi-million tonne polyacrylate global market is still rising with an annual growth greater than 6% [1]. Research over the last 20 years into controlled radical polymerisation, and copolymerisation, has provided increased insight into the distinct properties of these materials. However, even 50 years after the initial patenting of poly(acrylic acid) [2], new discoveries about its fundamental properties are still being made [3]. In solution many, but not all, acrylate copolymers act as polyelectrolytes, con- taining ionisable repeat units; and thus show some form of stimuli-response to pH. 1 Acrylate Polymers for Advanced Applications The solution forces that govern these properties are the same that give function to biological macromolecules (i.e., peptides, proteins, DNA) and so many polyelectro- lytes have been used as simple models for these more complex systems. However, due to their applications are so widespread and varied, it is essential to any chemist or engineer working with these sensitive materials to acquire some understanding of the need to control their pH. Depending on the nature of these ionisable repeat units, a polymer can be clas- sified as a ‘weak’ or ‘strong’ polyelectrolyte, governed by the pKa of the ionisable groups. As samples containing carboxylic acid repeat units dissociate relatively easily, they fall into the former category. 1. Introduction However, even non-responsive systems, such as poly(acrylamide), have been found to demonstrate responsible macromolecular behaviour in the presence of corre- sponding polymer systems via a process of interpolymer complex formation [14]. Many polyacrylates engage in hydrogen bond driven complex interactions. The field has proven to be extremely complex due to the multitude of competing factors that affect this often weak, almost always labile, interface. This chapter will discuss recent advances in the study of pH dependent poly- acrylate solution behaviour, examining our improvements in understanding of 2 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 weak polyelectrolyte systems. Critically this review limits itself to studies of linear polymer systems, as the properties of branched, or crosslinked, macromolecules are fundamentally different [15, 16] and warrant further, separate discussion. Figure 1. Polyacid chemical structures. g Polyacid chemical structures. 2. Poly(carboxylic acids) 3 Acrylate Polymers for Advanced Applications macromolecular swelling results in dramatically changed properties between the compact/swollen polymer. Compared to this, the equivalent deprotonation and subsequent anionic charge drive PAA to adopt an extended state with a relatively smooth transition, with only small changes to polymer physical properties save additional anionic potential. These conformational responses to external stimuli can be viewed as ‘smart behaviour’ and have led to the incorporation of acrylic acid and methacrylic acid monomers being incorporated into a range of copolymer systems to act as triggers and solvating groups in a range of applications. Due to the increased compaction, and hydrophobicity, of its globular state, PMAA can solubilise low molar mass organic compounds in solution [12, 17, 32], which is a property not shared by PAA [17, 32, 33]. This is particularly evidenced by the fluorescence emission vibrational fine structure of the aromatic label pyrene. The pyrene excited state emits multiple emission bands, and the relative intensity of bands 1 and 3 vary with different solvents, thus when dispersed in a solution it can give an indication of system polarity [34, 35]. For example, the I3/I1 ratio is known to vary between 0.55 (water) and 1.7 (n-pentane) [26]. This feature has been used in the study of many polymer systems, and commonly used by spectroscopists to study macromolecular aggregate structures such as colloids [36], microemulsions [37], micelles [38] and microgels [39, 40]. For example when a 10−6 M solution of pyrene was dispersed in an aqueous solution of PAA, the I3/I1 ratio did not shift from ≈0.55 between pH 3 and 10, identical to the ratio seen for a dispersion in water. This reflects the fact that any interaction between the fluorophore and the polymer does not alter the microenvironment of the label, and confirms the exis- tence of PAA in a water-swollen conformation across the entire pH range. In PMAA at low pH, however, a I3/I1 ratio of 1.1 is commonly observed [12], indicating the compact hypercoiled polymer provides hydrophobic shielding from the aqueous solvent. When the pH of pyrene/PMAA solution is increased, this ratio begins to decrease at pH 5, indicating the conformational rearrangement of the polymer, until at pH 6 the probe is released into the solution, returning the fluorescence emission ratio to the state seen in both pure water and PAA. 2. Poly(carboxylic acids) The two most comprehensively studied synthetic poly(carboxylic acid)s within the literature are poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) respectively. Both contain a carboxylic acid repeat unit that dissociated to form a negatively charged anion in low pH aqueous solutions. The additional methyl group on the methacrylic acid functional group gives PMAA a degree of amphiphilic behaviour [17] depending on the degree of ionisation (Figure 1). p g g g This additional hydrophobicity dominates the solution properties of PMAA, leading to the aforementioned ‘anomalous’ Henderson Hasselbalch titration behav- iour [4, 5, 7, 9], whilst PAA has long been considered a more ‘ideal’ system [18] as it does not undergo as dramatic a macroscopic switch. As the carboxylic acid group can only be classed as hydrophilic when the functional monomer is protonated, PMAA undergoes a rapid swelling as the pH is increased, becoming an entirely hydrophobic material with increasing anionic charge along the backbone. Extensive investigations have been carried out into its behaviour using diverse methods and techniques: pontentiometry [4, 5, 7, 10, 19], viscometry [8, 11], Raman spec- troscopy [20], scattering methods [21–23] and fluorescence probe interrogation techniques [3, 24–26]. The combined research has shown that PMAA undergoes a dramatic conformational change between pH 4 and 6, (corresponding to an α (degree of ionisation) between 0.1 and 0.3), whilst PAA adopts a relatively smooth swelling process in the same pH range (initiating at the same degree of ionisation). In acidic media, due to the increased hydrophobicity, PMAA adopts a globular, contracted structure designed to minimise unfavourable interactions between the hydrophobic backbone and side chain and the aqueous solution, whilst PAA has been described as a random, statistical coil [6, 7, 9]. The PMAA shows significantly increased compaction due to the hydrophobic methyl backbone [8, 13, 22, 24–29], that has been shown to induce hypercoiling [8]. This has two net effects—increased hydrophobic density gives it both greater solubilisation potential but at the cost of reduced solubility and mobility. As the degree of ionisation is increased from pH 4 to 6 the PMAA anionic units begin populating the macromolecule backbone, resulting in a transition between pH 5 and 6 where repulsive units between these charges initiate a macroscopic switch from the compact to the water swollen (described in multiple places as ‘rod like’ [30, 31]) state. Due to the increased initial compaction in PMAA, this Figure 1. g Polyacid chemical structures. 2. Poly(carboxylic acids) As such differences in behaviour between low and high molar mass PAA materials have been observed, such as stark changes in the polymer behaviour at oil-water interfaces [27, 48]. Figure 2. Conformation of polyacid with different degrees of ionisation. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 Figure 2. Conformation of polyacid with different degrees of ionisation. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 Figure 2. Conformation of polyacid with different degrees of ionisation. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 Figure 2. Conformation of polyacid with different degrees of ionisation. More recent data indicates that the length scale of the chain plays a role in this transition. For instance, whilst in 0.1 M NaCl the hydrodynamic radii of PAA scales with molar mass [46] the conformational rearrangement of the chai non ionisation in low ionic strength liquids only occurs above a known molar mass lower limit [3]. Current results suggest this is a salt dependent phenomenon [41, 47] and has not been observed in PMAA (although increasing polymer size does slow the kinetics of polymer reconfiguration [27]). As such differences in behaviour between low and high molar mass PAA materials have been observed, such as stark changes in the polymer behaviour at oil-water interfaces [27, 48]. 2. Poly(carboxylic acids) This experiment confirms both the increased solubilisation potential of PMAA over PAA and also the fact that the transition occurs over a broad pH range. p g However, the electrostatic potential of these polyelectrolytes cannot be so simply described as indicating that the swollen/collapsed state is neutral/charged as there is an evident near neighbour effect present in polymers that is not seen in comparative small molecule systems [41]. This has been evidenced by the different acid dissociation titration behaviours seen in PMAA when comparing different polymer tacticities [42]. In dilute solutions intrachain interactions across the macromolecule tend to dominate its properties—the molecule can be considered a single long chain surrounded by counter ions, and their solution properties are thus governed by their corresponding electrostatic interactions, which are well described by a range of mathematical theories [43, 44]. To summarise: due to electrostatic repulsion ionisation of acrylate polyelectrolytes occurs over a much wider pH range than observed in the equivalent small molecule, and at the ‘stated’ pKa only a fractional ionisation of repeat units will carry a negative charge. For example, potentiometric titrations of PAA found that, at pH 4.5 (pKa of acrylic acid and the point at which conformational change will occur) only 1/10th of the acrylate repeat units in the polymer will carry this fractional charge [3, 45]. The polymer will continue to ionise up to pH 11 with no further polymer swelling observed despite increasing electrostatic potential of the system. Therefore, it is inappropriate to suggest that the conformational change is driven purely by elec- trostatic potential, as if this was solely the case further rearrangements at greater degrees of ionisation would be observed (Figure 2). 4 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 More recent data indicates that the length scale of the chain plays a role in this transition. For instance, whilst in 0.1 M NaCl the hydrodynamic radii of PAA scales with molar mass [46] the conformational rearrangement of the chai non ionisation in low ionic strength liquids only occurs above a known molar mass lower limit [3]. Current results suggest this is a salt dependent phenomenon [41, 47] and has not been observed in PMAA (although increasing polymer size does slow the kinetics of polymer reconfiguration [27]). Figure 3. g Polyacylamide chemical structures. Polyacylamide chemical structures. important factor in flocculation kinetics [53]. Following the adhesion of polymer chain of sufficient length for a loop or tail to extend into the solvent, secondary attachment to secondary particles can occur in a process known as ‘polymer bridg- ing’. Polymer chains adsorbed on the particle surfaces via only a few points of attachment leave the majority of the chain in solution whilst increasing adsorption onto the particle to saturation reduces the flocculation potential of the polymer. Bridging can be impacted by both the charge density and the molecular weight of the polymer [55] (Figure 4). important factor in flocculation kinetics [53]. Following the adhesion of polymer chain of sufficient length for a loop or tail to extend into the solvent, secondary attachment to secondary particles can occur in a process known as ‘polymer bridg- ing’. Polymer chains adsorbed on the particle surfaces via only a few points of attachment leave the majority of the chain in solution whilst increasing adsorption onto the particle to saturation reduces the flocculation potential of the polymer. Bridging can be impacted by both the charge density and the molecular weight of the polymer [55] (Figure 4). p y g Polyacrylamide is used in a range of other scenarios including erosion con- trol [51, 57–59], medical implants [60–63], and reduction of water seepage via increasing aqueous viscosity to both stabilise soil and dust prevention [51]. g q y p Poly(acrylamide) was one of the first polymers used to reduce soil losses in furrow irrigation [64] and the polymer has been sold commercially for this purpose since at least 1995 [51]. Large quantities of this material are therefore escaping into the environment [59, 65] and a body of research is being built up regarding its effect on the ecosystem [66]. Generally, the polymer is considered non-toxic, with most concerns around its use arising due to its close association from the potent neu- rotoxin monomer from which it is formed. Since Swedish researchers discovered that acrylamide can be found in heated foodstuffs [67–72], there has been low level public concern about the use of polyacrylamides in a range of industries. However, studies of polyelectrolyte flocculants of all types have been carried out and consistently poly(acrylamide) is identified as being the primary ‘toxicant’ [66]. 3. Poly(acrylamides) Not all polyacrylates demonstrate electrolyte properties, and one of the most common non-ionisable acrylate materials produced today is acrylamide copolymers. This chapter concerns itself specifically with two specific materials of particular interest with divergent properties, although there are a range of further examples. These polymers are poly(acrylamide) (PAM) and the hydrophobically modified poly(N-isopropylacrylamide) (PNIPAM), whose properties are driven by the additional hydrophobic groups along the polymer side chain. As such one is widely used as an inexpensive, mass-market commodity whilst the other is a very heavily investigated [40, 49], high value material with particular interest in its biomedical applications [50] (Figure 3). pp g Random copolymers of acrylamide (both anionic, cationic and neutrally charged) have been extensively used in the water industry for many years [51–54]. They are extensively employed to remove dissolved organic matter (DOM) for water clarification purposes. Flocculation of fine particles can occur via several mechanisms including polymer bridging, charge neutralisation, polymer-particle complex formation and depletion flocculation; often a combination of several of these processes [55]. Binding in poly(acrylamide) is primarily by hydrogen bonding [56], although copolymerised sections may also assist with electrostatic interac- tion or ion binding. In a sufficiently long polymer chain, there are many potential binding sites, and once sufficient repeat units along a single polymer chain have adhered to a particle surface, the adsorption is often considered irreversible despite the fact each individual binding site is acting in an equilibrium [53]. Once a polymer has adhered to a particle, it can be divided into three segments: trains (adhered to the particle surface), loops (that extend from the surface) and tails (which project into the solution). The speed by which the polymer shifts is difficult to assess but an 5 Acrylate Polymers for Advanced Applications Figure 3. Polyacylamide chemical structures. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 Testing of poly(acrylamide) interaction with the gill tissues of several aquatic species including fish [66, 73, 75–77], crustaceans [66, 73, 77], algae [66, 77] and insects [77] have been carried out. In many studies of adult fish, the anionic and non-ionic form of the poly(acrylamide) cause only low levels of damage to the fish, with effects increasing at higher concentrations [75]. However, sustained exposure of organisms over a 40 day period has shown that low levels of these polymers are intrinsically toxic to almost all aquatic fauna [73]. Environmental exposure is unlikely to be sustained over long periods due to the polymer desorption to organic matter but few studies have been undertaken into the metabolic rate at which they are removed from living organisms. Even in tests where fish survival was not impacted, the general activity and swimming behaviour of the fish were sub normal [76]. Conversely the cationic form of the polymer is known to be far more toxic, causing pathological issues at sub mg ml−1 concentrations, as the polymer builds up on negatively charged gill surfaces [75, 77]. Reduced gill functional- ity impairs oxygen uptake in the fish and results in death. Further studies have shown that polyelectrolytes can cause adverse changes in fish organ cells (liver and kidneys) [73], decrease animal locomotion and greatly increase respiratory rate. This suggests that the presence of dissolved flocculants may not be lethally toxic but suggests it is capable of causing the fish elevated levels of distress. In inverte- brates, their mechanical action was reduced as polyelectrolytes adsorbed onto their body surfaces, reducing their vital functions [73], and again the cationic form of the polymer is far more toxic than the anionic form. [77] In microcosms tests, it has been shown that high polyelectrolyte concentration can reduce algal growth [66, 77]. This in turn can increase the potential toxicity of the polymer as the algae acts as a neutralising agent towards the polymer. To algae, even the anionic and non-ionic polymer is toxic, negatively affecting both cell growth and O2 produc- tion [73]. It has been observed that addition of combinations of both anionic and cationic polymer can reduce toxicity [77] and several patents have been issued suggesting that anionic polymers can be used to detoxify cationic polyelectrolytes [78, 79]. Figure 3. Within much of the poly(acrylamide) literature, there has been a lot of emphasis placed on the toxicity of the monomer, resulting in studies discounting the effects of the polymer and only focusing on residual monomer spread [65]. However, some studies have shown that poly(acrylamide) is unlikely to degrade into residual monomers, or any other toxic compounds [73], and this has only been observed under specific harsh conditions [74]. Therefore, a complete study of the environmental impact of these polymers should include the raw polymeric product. Figure 4. Increasing polymer adsorption to surfaces. Figure 4. g 4 Increasing polymer adsorption to surfaces. 6 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 Figure 5. T l h Figure 5. Typical thermoresponsive properties of polymers [83]: (temperature (T) vs. volume fraction (Ø)). The LCST of PNIPAM can be affected by the addition of hydrophobic or hydro- philic end groups [84], or the molecular weight and concentration of the sample [85]. Due to the LCST is reasonably close to body temperature, there has been much work to manipulate PNIPAM to act as a drug-delivery agent or trigger or apply it in other bio-engineering circumstances [86, 87]. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 In conclusion, the discrepancy between anionic and cationic polymers in regard to aquatic toxicity must be considered in the application of these polymers [75, 77]. The cationic form of the polymer is regarded as generally more toxic but the anionic form has also been shown to cause chronic, sub-lethal responses even at low concentrations [66]. l h h h l l d d l d Although it has some larger applications, PNIPAM is not produced or utilised in such great quantities. It is mainly of interest due to a thermally induced conforma- tion the polymer exhibits at 32°C, caused by the hydrophobic isopropyl groups [49]. This ‘smart’ response has led to great interest in the polymer, both to understand its properties and apply them in a range of fields, specifically in Bioengineering [50]. In essence, the polymer has a lower critical solution temperature (LCST), a conformational change that occurs via a two stage process. Firstly, the polymer has an intramolecular collapse, where individual chains contract in upon themselves as they break hydrogen bonds with the aqueous solution, followed by a second- ary event of intramolecular aggregation of the collapsed coils [80]. This event is triggered by the increasing entropic cost at high temperature of the restricted water that solubilises the dissolved polymer chains below the LCST. The event has some hysteresis between heating and cooling radii of gyration [81], governed by two intermediate states that give PNIPAM four potential conformations: globule, molten globule crumpled coil and coil [82]. During the collapse the globular state dispenses approximately 34% of the water molecules [82], meaning that although this is a desolvation event leading to an insoluble material, collapsed PNIPAM can never be described as a hydrophobic system (Figure 5) [49]. 7 7 Acrylate Polymers for Advanced Applications Figure 5. Typical thermoresponsive properties of polymers [83]: (temperature (T) vs. volume fraction (Ø)). 4. Acrylate interpolymer complexes Interactions between multiple polymers in a formulation are almost inevitable, and there has been plenty of studies of specific driving factors undertaken over the last 50 years to build a strong picture of inter-polymer interactions. This phase separation phenomena is observed in even the most dilute solutions, as it is driven by a mixture of electrostatic, hydrogen bonding and hydrophobic interactions, all dependent on pH, salt concentration and temperature [88, 89]. To our knowledge this type of complex was first patented in 1966 [90], with much of the following fundamental measurements carried out over the following decades [14, 91–94]. Since then, the system has been described as a laddered sequences of bonds between the molecules, occasionally interrupted with loop defects [95], an evolution similar to the model previously described of polymer adhesion to surfaces. This theory originally posed that the polymers will form rigid, static structures due to repeated hydrogen-bonding across molecules. More recent studies have put less emphasis on the polymer rigidity and have given an alternative description of these repeated labile interactions more as ribbons (i.e., two flexible materials that can slide over each other). The interactions between PAA and PAM are one of the more studied systems of interpolymer complex formation (IPC) [93, 96–101], and in both solution and solid state the interaction has been shown to be pH dependant [14, 96]. Mixed solutions of PAA and PAM form a turbid solution that precipitates when cooled [97]. This phase separation follows the formation of complexes between PAA and PAM that varies in structure depending on the concentration, medium and the ionisation constant [97]. For complexes between PAA and a proton-acceptor polymer it has been shown that IPCs will only form below a critical value of pH (pHcrit) [14, 99], the structure dependent point above which any partial neutralisation of the poly- acid inhibits complex formation [14, 100]. Early work within this field required high molecular weight materials to detect complex formation [99, 102], however, modern instrumentation has facilitated detection of smaller complexes down at the parts per million loading level [103]. 4. Acrylate interpolymer complexes The structure of the resultant IPC (whether in a gel or a compact solvated complex) 8 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 depends on the relative molecular weight of complexing partners, [104] but as this is a multivalent effect of repeated binding sites, larger molecular weight materials demonstrate stronger interactions. Furthermore, it has been indicated that very large molecular weight polyacids have been seen to raise pHcrit [93]. g g p y p When dissolved in high ionisation solutions, both polymers have rapid seg- mental motion, existing as random polymeric coils. If the solution ionisation is decreased, this deprotonates the acidic polyelectrolytes and reduces its affinity for inter-polymer complexation. This occurs as can now form both intramolecular H-bonds internally across the chain backbone or intermolecularly forming H-bonds with other polymers [105], leading to a rigid polymer mixture with restricted chain motions. PAA forms stronger complexes to PAM than some other polymers (i.e., poly(ethylene oxide) or poly(vinyl acetate)) due to additional ion-dipole inter- action of the partially protonated amide groups and the C═O dipoles of PAA [106]. In ambient conditions the peak aqueous interaction between PAA and PAM occurs ≈ pH 2.69 [107] but this is affected by many environmental factors including tem- perature [89, 101], ionic strength [93, 107, 108] and the addition of inorganic binders [99]. The complex polymer/polymer/solvent ratio of interactions is temperature sensitive causing PAM-PAA copolymers to become upper-critical solution tempera- ture materials (an inversion of the LCST seen in PNIPAM where they become only soluble above a specific temperature [87]). These combined external factors dete- riorate the thermodynamic quality of the solvent, strengthening polymer-polymer interactions by weakening polymer-water solvation [109]. However, below pHcrit, only small portions of the PAM form into ‘multimacroion clusters’, indicating that in an equivalent system with 1:1 acid/acrylamide repeat units, a large percentage of acrylamide will be free in solution unbound to PAA [110]. This was the first result of several which have cast doubt on the ladder model, and computational modelling software of polymer/polymer ionic interactions has proposed a range of complexing structures ranging from ladders to scrambled egg structures [111]. Figure 6. Figure 6. Three conformational states of PAA-PAM in mixed solutions. along the chain is deprotonated whilst more hydrophobically modified polymers are more tolerant to partial ionisation when complexing with PAA. Computational modelling of the solvation energy of each repeat unit shows a clear correlation between solvation potential and pHcrit [14]. along the chain is deprotonated whilst more hydrophobically modified polymers are more tolerant to partial ionisation when complexing with PAA. Computational modelling of the solvation energy of each repeat unit shows a clear correlation between solvation potential and pHcrit [14]. Clearly the polyacid dictates the potential of IPC with receptive polymers [96], and in this mind, it is worth revisiting the PAA-PAM IPC structure. As the pKa of the PAA is higher than pHcrit, it will be ‘non-swollen’ before it encounters a complexing partner. Early literature in the subject, comparing the supposed ‘non- response’ of PAA compared to PMAA cited, its only-slight alteration to solution viscosity and inability to solubilise hydrophobic dyes as evidence it had no confor- mational response. However, more recent studies with more sensitive techniques have shown that this is not the case and PAA does indeed go through a lesser swelling-contraction event. As such, it is proposed that the PAA can exist in three potential conformations in the presence of a polymeric bonding partner (Figure 6). h d h h l l We suggest that compacted PAA has no entropic or enthalpic reason to uncoil or swell prior to complexation. Given the combined evidence from two separate sources that (1) most PAM chains are not binding to partners and (2) PAA chains do not swell further apart on PAM binding (in fact there is some evidence of contrac- tion), it seems reasonable to propose that the PAA-PAM complex is not amorphous in nature, and certainly not an extended ribbon/ladder structure. 4. Acrylate interpolymer complexes Further experi- mental evidence has shown that a PAA coil does not unwind or swell on addition of a PAM polymer but potentially contracts into a smaller co-globule [112], and an explanation for this can be found when considering the difference between the pHcrit of IPC formation and pKa of PAA conformational change. p p g Other acrylate materials, such as PNIPAM, demonstrate similar responses to polyacids, and exhibit their own IPC potential [113]. Whilst PAM-PAA interactions are dominant at lower temperatures, PAA-PNIPAM show increased interactions at high temperatures, indicating that the complex formation is driven by hydro- phobic interactions not seen in the base acrylamide structure [114]. Studies using dissolved pyrene indicated these lead to stronger interactions between PAA and PNIPAM than PAA-PAM [115, 116]. Furthermore, the hydrophobic isopropyl side chain causes PNIPAM to alter its response to ionic strength. Whereas PAM-PAA complexes are strengthened by increasing ionic strength, PNIPAM complexes show decreased critical pH reducing their bond forming potential [109]. As the initial critical pH for IPC formation was larger than 3, Khutoryanskiy theorised that the increasing ionic strength partially dissociates the polyacid. As only non-ionised carboxylic groups are able to form hydrogen bonds, this impedes IPC formation and reduces the pKa. For PAA-PAM, pHcrit was found to be 2.7, whilst for increasing hydrophobic additions to the acrylamide unit (poly(ethyl acrylamide), poly(dimethyl acryl- amide), poly(diethyl acrylamide), the pHcrit was found to increase to 3, 4, 5 respec- tively [14]. It is interesting to note that the most hydrophilic acrylamide polymers (including a hydrophilic-functionalised poly(hydroxyl ethyl acrylamide)) show lower pHcrit, indicating that their IPC with the polyacid are less tolerant of deprot- onation. The PAA-PAM complex appears to separate when the first acid repeat unit 9 Acrylate Polymers for Advanced Applications Figure 6. Three conformational states of PAA-PAM in mixed solutions. 5. Conclusion This chapter reviews some of the recent developments in polyacrylate properties and interactions, and delves deeply into their industrial applications to provide both further context and understanding. During the early study many assumptions were made due to the difficulty to analyse these large macromolecules, particularly in dilute solutions, and our understanding of these systems has slowly evolved as more advanced technology with greater sensitivity has facilitated deeper interrogation of these systems [117]. This chapter only touches on a few choice themes of polymer-responsiveness and ignored many of the more challenging aspects of the field. The state of ionisation of all of these polymers has clearly been shown to have an effect on their solution properties, and although the field is still under development after several decades of work, common themes can be seen across the subject dictating macromolecular conformational changes. 10 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties DOI: http://dx.doi.org/10.5772/intechopen.82569 Acknowledgements This book chapter is both an update, summation of, and substantial revision to my Ph.D., work area carried out at the University of Sheffield, originally under Dr. Linda Swanson, who has published extensively on the photophysical analytical techniques described in this paper. For further reference on the application of those techniques in these systems, particularly in the study of polyelectrolytes, please see her book chapter ‘Optical Properties of Polyelectrolytes’ [26]. Conflict of interest There are no conflicts of interest to declare. Abbreviations Abbreviations IPC interpolymer complex formation DNA deoxyribonucleic acid DOM dissolved organic matter PAA poly(acrylic acid) PAM poly(acrylamide) PMAA poly(methacrylic acid) PNIPAM poly(N-isopropylacrylamide) Thanks Written following the arrival of, and dedicated to, Jonathan Swift, born in 2017. Author details 11 Acrylate Polymers for Advanced Applications [17] Barone G, Crescenzi V, Liquori AM, Quadrifoglio F. Solubilization of polycyclic aromatic hydrocarbons in poly(methacrylic acid) aqueous solutions. The Journal of Physical Chemistry. 1967;71:2341-2345 References [1] Polyacrylate Market - Segmented by Product Type, Application, and Geography - Trends and Forecast (2018-2023). 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Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity of Viking and Jakmania’s Supporters in the Indonesian Football Industry
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Suggested Citation: Wibowo, Andry. (2018). “Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity of Viking and Jakmania’s Supporters in the Indonesian Football Industry” in MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September, pp.149-168. Bandung, Indonesia: UPI [Indonesia University of Education] Press, ISSN 2527-3868 (print) and 2503-457X (online). Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity of Viking and Jakmania’s Supporters in the Indonesian Football Industry ABSTRACT: Football is most popular sport in the world community. Football is also one form of pop culture itself, where football is no longer interpreted as a game or a way to exercise alone, but also become something that can be interpreted as a culture that bind various communities in the world. This research, by using the qualitative approach with case studies on conflict, will explore deeply about how social identity is present in football, especially in the context of football supporters in Indonesia as part of the development of the football industry in the world. This study has a focus on researching the phenomenon of cultural and local identity in the “Viking” of PERSIB (Bandung Indonesian Football Association) and “Jakmania” of PERSIJA (Jakarta Indonesian Football Association) supporter groups in football matches that are played in national competitions. The formation of identity transforming become an identity conflict in a crowd of football supporters to become the hallmark of football as part of the pop culture in Indonesia. KEY WORD: Pop Culture; Conflict; Identity; Crowds; Football in Indonesia. KEY WORD: Pop Culture; Conflict; Identity; Crowds; Football in Indonesia. ABSTRAKSI: “Budaya Pop, Konflik Identitas, dan Kerusuhan: Studi tentang Budaya dan Identitas Suporter Viking dan Jakmania dalam Industri Sepakbola Indonesia”. Sepakbola merupakan olahraga yang sangat populer pada masyarakat dunia. Sepakbola adalah juga salah satu bentuk dari budaya pop itu sendiri, dimana sepakbola tidak lagi dimaknai sebagai sebauh permainan atau cara untuk berolahraga saja, melainkan menjadi sesuatu yang dapat dimaknai sebagai sebuah kebudayaan yang mengikat beragam masyarakat di dunia. Penelitian ini, dengan menggunakan pendekatan kualitatif tentang studi kasus konflik, akan mengkaji secara mendalam mengenai bagiamana pembentutakan identitas sosial hadir dalam sepakbola, terutama dalam konteks suporter sepakbola di Indonesia sebagai bagian dari perkembangan industri sepakbola di dunia. Penelitian ini memfokuskan kajian tentang fenomena identitas budaya dan lokalitas pada kelompok suporter “Viking” dari PERSIB (Persatuan Sepakbola Indonesia Bandung) dan “Jakmania” dari PERSIJA (Persatuan Sepakbola Indonesia Jakarta) dalam pertandingan sepakbola yang berlangsung melalui kompetisi nasional. Pembentukan identitas hingga kemudian bertansformasi menjadi suatu konflik identitas dalam kerumunan berbentuk aksi kerusuhan dan konflik menjadi ciri khas sepakbola sebagai bagian dari budaya pop di Indonesia. KATA KUNCI: Budaya Pop; Konflik; Identitas; Kerumunan; Sepakbola di Indonesia. About the Author: Andry Wibowo, M.H., M.Si. is a Student of Doctoral Program at the STIK-PTIK (Indonesian Police Science Institute – Indonesian Police Science University), Jalan Tirtayasa Raya No.6, Kebayoran Baru, Jakarta Selatan, Jakarta, Indonesia. About the Author: Andry Wibowo, M.H., M.Si. is a Student of Doctoral Program at the STIK-PTIK (Indonesian Police Science Institute – Indonesian Police Science University), Jalan Tirtayasa Raya No.6, Kebayoran Baru, Jakarta Selatan, Jakarta, Indonesia. E-mail: wibowoandry1993@gmail.coml MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, V l 3(2) S b 2018 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, V l 3(2) S b 2018 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 esia untuk Kajian Pendidikan, Volume 3(2), September 2018 © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik Article Timeline: Accepted (July 1, 2018); Revised (August 17, 2018); and Published (September 30, 2018). INTRODUCTION part of the life in the world community. This condition encourages the form of industrialization of football in the world, which can be illustrated by an indication of the massive amount of jersey sales (clothing used in football) by sports equipment industries, such as Nike and Adidas. This example can indicate a massive football’s industrialization by using an indication of the role of media in viewing football as a new culture that is regarded as something that has a very high capital value. The implications for the emergence of football industry is like media, such as Bein Sport, Fox Sport, and ESPN or Entertainment and Sports Programming Network (Blair, 2011; Clark, 2011; Billings & Ruihley, 2013; Miles, 2015; and Eyong, 2018). The concept of pop culture is something interesting to be studied since 1950s (Rushkoff, 1994; Sifaki, 2003; and Loyo, 2012). The uniqueness in researching about pop culture is driven by the emergence of our awareness that the world has shown the reality of social life that depends on the cultures that are the way of life for the world community (Swingewood, 1977 and 1998; and Borg et al., 2005). The phenomenon of cultural industrialization encourages academics to look back on pop culture as part of the life of modern society. It is characterized by the inclusive nature of culture in a social class, which transforming into an exclusive one without distinction of social class (Kellner, 2003; Adorno & Bernstein, 2004; Borg et al., 2005; and Ritzer, 2009). Football as a phenomenon of pop culture in Indonesia can also be seen from the great enthusiasm of society towards football in Indonesia. Indonesia, as a country with the fourth largest population in the world, also ogled by the world football industry as one of the countries that have the largest market for football industry. The indication can be seen from the large number of football spectators in Indonesia, who watch through the medium of technology, such as television and internet; or who watch directly in various stadiums to watch the local or international competition (Clark, 2011; Jones, 2015; Gadiraju, 2016; and Duerden, 2017). A simple example of the existence of pop culture in today’s society can be seen by the life of urban society today is highly dependent on gadgets, such as smartphones that no longer serve as a means of communication but as part of a lifestyle. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik 150 Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity of Viking and Jakmania’s Supporters in the Indonesian Football Industry E-mail: wibowoandry1993@gmail.coml Suggested Citation: Wibowo, Andry. (2018). “Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity of Viking and Jakmania’s Supporters in the Indonesian Football Industry” in MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September, pp.149-168. Bandung, Indonesia: UPI [Indonesia University of Education] Press, ISSN 2527-3868 (print) and 2503-457X (online). 149 149 149 © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos INTRODUCTION In the world of sports, there is also a phenomenon similar to the phenomenon of the smartphone in urban society. Football, which at the beginning of the 20th century, has a function not only as a game as well and as a way to exercise for the community, but football can be a part of bussiness and entertainment instruments (Sullivan, 1979; Blair, 2011; Billings & Ruihley, 2013; and Jones, 2015). The meaning of football has changed a lot in modern times, where football has become part of lifestyle and even a determinant value ​​in society. Football in Indonesia is also used as a pop culture industry. This is marked by the presence of national competitions, such as Go- Jek Traveloka Competition in 2017 ago. The warm atmosphere of the local competition also affects the enthusiasm of the public towards the effort to support the football club based on regional identity, for example Jakarta residents tend to favor and support the PERSIJA (Persatuan Sepakbola Indonesia Jakarta or Jakarta Indonesian Football Association)’s Club; West Java residents or Sundanese ethnic groups tend to support the club of PERSIB Football is most popular sport in the world community. Almost two-thirds of the world’s population witnessed the World Cup Championship’s event through the help of technological developments, such as radio, television, and internet. This illustrates that football is no longer a way for doing body’s exercise but also become an important 150 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 (Persatuan Sepakbola Indonesia Bandung or Bandung Indonesian Football Association); Minangkabau ethnic community or West Sumatrans tend to support the SPFC (Semen Padang Football Club); and so forth (Ayati, 2010; Hanifan & Herfiyana, 2014; Kristanto, 2016; Amirio, 2017; Sufiyanto, 2017; and Wirayudha, 2018). also capitalizes on the crowd of football supporters and their behavior inside and outside the game. The identity of Vikings and Jakmanias, for example, are two very strong identity supporters in Indonesia, who often clash these two groups in football matches that have caused riots, security disturbances, and public order disturbances. The uniqueness of the Viking and Jakmania as a crowd identity is the identity of values, which ​​formed from the culture of football and cultural identities which is produced based on regional similarity, the value of the struggle, and so forth (Berlonghi, 1995; Kari, 2009; Conigliaro et al., 2015; Huda, 2016; Hadi, 2017; and Wirayudha, 2018). INTRODUCTION The combination of pop culture and football industry in Indonesia is also inseparable with the efforts to capitalize on the issue of identity in the aspect of football. Regional or ethnic distinctiveness is attached to the football club itself. For examples, fans club named themselves to make self-identification of the crowds with the same intention of supporting their respective football clubs. Identification of this identity can be exemplified by naming Viking supporters for club supporters of PERSIB; Jakmania for supporters of PERSIJA; and so on (Brown, 1998; Dobson & Goddard, 2001; Gibbons, 2012; Miles, 2015; Hadi, 2017; and Wirayudha, 2018). This study will look at the conditions of Viking and Jakmania as an identity and as a culture characterized by Viking and Jakmania as a part of pop culture and as a group that produces a “riot” identity in Indonesia’s football industry. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik RESEARCH METHODS y ) Identity is also highly visible when football matches based on jersey use, attribute use, and their own behavior. The use of jersey as a symbol of identity has always been a major determinant of self- identification, which later became the identity identification among supporters. Orange color, for example, to be the basic color for Jakmania’s identity, blue color for Viking, and so forth. The use of flag attributes is also a form that symbolizes the existence of the supporter group, especially when the game is held. Behavior can also be a symbol that depicts identity within a crowd of football supporters. This can be identified in the form of riots occurring in the identity clashes between supporters in football games (Ritzer, 2009; Fuller, 2017; Hadi, 2017; and Wirayudha, 2018). Data collection process in this study is using qualitative approach with case studies on conflict between Viking and Jakmania supporters (Creswell, 1998; Auerbach & Silverstein, 2003; Yazid, 2015; and Wibowo, 2017). The conflict took place inside and outside the PERSIB (Persatuan Sepakbola Indonesia Bandung or Bandung Indonesian Football Association) and PERSIJA (Persatuan Sepakbola Indonesia Jakarta or Jakarta Indonesian Football Association) football matches that took place over the last five years. The data collected are data generated through in-depth interviews, direct observation, focus group discussions, expert panel discussions, and document studies over the past five years. The data generated will be analyzed with some references, and information from Police institution in Indonesia (Creswell, 1998; Auerbach & Silverstein, 2003; Yazid, 2015; and Wibowo, 2017). Riots in the football crowd became one of the hallmarks of football in Indonesia. The football industry in Indonesia not only shows football as a pop culture, but 151 151 ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos Jakmania and Viking, as a supporter group, are a synthetic identity that have a particular sub-culture. Sub-cultures of good and bad supporters as a sub-culture identity are found in Jakmania and Viking in their daily behavior. The spirit of regionalism, through the principles of conquering and awakening, also marks as the sub-culture of Jakmania and Viking. Both supporters also use violent symbols on the club’s symbolic status or supporters. FINDINGS AND DISCUSSION The Establishment of Viking and Jakmania Identities as a Crowded Football Supporters. As a synthetic identity (Hoofnagle, 2007; ID Analytics, 2014; and Voris, 2015), Viking is a metamorphosis of Bobotoh, which became a common icon of PERSIB (Persatuan Sepakbola Indonesia Bandung or Bandung Indonesian Football Association) supporters. Bobotoh is a symbol of the continuity of the greatness of the Padjadjaran kingdom in West Java at this time (Ryza, 2011; Kristanto, 2016; Hadi, 2017; and Kaka, 2017). The naming of Viking is based on Ayi Beutik Suparman’s imagination, on the values ​​that existed in the Vikings who once lived in Scandinavia (Ryza, 2011; Hanifan & Herfiyana, 2014; and Haryadi, 2014). Meanwhile, Jakmania is a popular name used on the basis of intention to attract as many members as the term “Jak” symbolizes the identity of the names “Jakarta” and “mania” as maniac. This statement is supported by interviews conducted on Gugun Gondrong and former Governor of DKI (Daerah Khusus Ibukota or Special Capital Region) Jakarta, Sutiyoso (Ayati, 2010; Sufiyanto, 2017; Wirayudha, 2018; interview with Respondent C, 17/7/2017; and interview with Respondent D, 24/7/2017). ; , ) Jakmania and Viking have an exploratory and expansive character. This character is formed naturally and fabricated in times of growth Jakmania and Viking as a community of football’s biggest supporters in Indonesia. The growth of character in Viking and Jakmania is also in line with the development of football as an industry. This is coherent with the number of football fans as the most popular sport in Indonesia. Family, peers, mass media, social media, and club achievements have also been influential as a trigger for the growing number of Jakmania and Viking’s members. The expansion culture contained in the Vikings and Jakmanias can be seen with the pattern of expansion of members in one region, and one ethnic group, which evolves into different regions and to various ethnic groups. In addition, its cultural expansion is also influenced by the role of social media as part of the information revolution, which also enriches both positive and negative impact in Jakmania and Viking’s membership (Brown, 1998; Castells, 2000; Ayati, 2010; and Eko, 2012).f Jakmania has a close relationship with the character of poor people, who live in Jakarta. RESEARCH METHODS PERSIJA (Persatuan Sepakbola Indonesia Jakarta or Jakarta Indonesian Football Association) uses the meaning of Macan Kemayoran (Kemayoran Tiger) as an imaginary value to the supporters; and PERSIB uses the Maung Bandung (Bandung Tiger) or Viking imagination as an wild’s value on their identity (Brown, 1998; Castells, 2000; Ayati, 2010; and Eko, 2012). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION Several factors that influencing the dynamics of Jakmania’s identity are: firstly, Jakarta as a city with multicultural population, which causes the value of being adopted would not could be in uniform. Secondly, the formation of impure Jakmania comes from lower society as marked by the intervention of the management and Governor Sutiyoso in the Jakmania organization (Brown, 1998; Huda, 2016; Sufiyanto, 2017; and Wirayudha, 2018). The condition which influencing the value in the organic process in the Viking body is their condition in their community. The community will continue to hold the value of culture and togetherness in establishing various activities. Together, PERSIB in West Java community put together and form a new community entity. As a cultural binder, this condition is also vulnerable to polluted inappropriate notions, such as enmity with Jakmania that continues to be produced without finding a way out, and conflict without ending which is characteristic of a cultural-based, and conflict based on history and identity (Eko, 2012; Kristanto, 2016; and Wirayudha, 2018). In the perspective of the second relation, the Vikings against Jakmanias, or otherwise, have dual theoretical perspectives, namely: Jakmania and Viking in the process of self- looking can be regarded as an entity that has solidity and solidarity, so transformed into a big power (Canetti, 1973; Kennedy, 2014; Kristanto, 2016; and Wirayudha, 2018). Jakmania in the process of viewing Viking or reciprocally is regarded as a rivalry of Jakmania and Viking, where they regard each other as something monstrous (Le Bon, 2002; Kristanto, 2016; Fuller, 2017; and Wirayudha, 2018). f Jakmania has a more dynamic group identity. Several factors that influencing the dynamics of Jakmania’s identity are: firstly, Jakarta as a city with multicultural population, which causes the value of being adopted would not could be in uniform. Secondly, the formation of impure Jakmania comes from lower society as marked by the intervention of the management and Governor Sutiyoso in the Jakmania organization (Brown, 1998; Huda, 2016; Sufiyanto, 2017; and Wirayudha, 2018). Jakmania and Viking have different historical paths. Viking is a PERSIB supporters who have been born first with a background of fanaticism for PERSIB. The love of the tribal base in West Java further strengthens in the Viking itself, which implies the emergence of a “hooliganistic” infiltration that begins to enter into the Viking self-worth. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION This identity is represented in the form of Jakmania as illustrated by Clarke McPhail (1997), which shows that members of Jakmania tend to be lower class society and people living in Jakarta (cf McPhail, 1997; Sufiyanto, 2017; and Wirayudha, 2018). This view is reinforced by sociologists, such as George Ritzer (2009) and Kenny M. John et al. (2001), who claim that Jakmania is part of the symbol of the lower middle class who living in Jakarta (cf Ritzer, 2009; John et al., 2001; Fuller, 2017; Sufiyanto, 2017; and Wirayudha, 2018). Theory of crowds offered by Elias Theory of crowds offered by Elias © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik 2 152 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 Canetti (1973); Gustav Le Bon (2002); and others can look Jakmania and Viking as the same groups, which have cultural properties that are characterized by the character of expansiveness in reaching the goal (Canetti, 1973; Le Bon, 2002; and Ritzer, 2009). Jakmania and Viking have similarities in the value of irrational violence based on criminogenic factors that are viewed through the perspective of police officers in Indonesia (Oliver ed., 2000; Le Bon, 2002; Kari, 2009; Ayati, 2010; and Eko, 2012). conditions of Viking behavior until now. The solidarity of the power of the mass grew. The implication of this is indirectly the frequent politicians in West Java, who are using Vikings in their campaign to attract numbers of vote (Stott & Pearson, 2007; Haryadi, 2014; Kristanto, 2016; Fuller, 2017; and Wirayudha, 2018).l 2017; and Wirayudha, 2018). The condition which influencing the value in the organic process in the Viking body is their condition in their community. The community will continue to hold the value of culture and togetherness in establishing various activities. Together, PERSIB in West Java community put together and form a new community entity. As a cultural binder, this condition is also vulnerable to polluted inappropriate notions, such as enmity with Jakmania that continues to be produced without finding a way out, and conflict without ending which is characteristic of a cultural-based, and conflict based on history and identity (Eko, 2012; Kristanto, 2016; and Wirayudha, 2018). Jakmania has a more dynamic group identity. FINDINGS AND DISCUSSION This has greatly affected their behavior, while in the stadium to spectating matches (Conigliaro et al., 2015; McLean, 2015; Kristanto, 2016; Rogers, Woodhouse & Politowski, 2016; and Wirayudha, 2018). Ethnic originality and birth places also do not affect the internal segregation of Jakmania’s membership identity. This is shown by the existence of a Jakmania named Bung Ferry, which is a figure who admired Jakmania, but he was born in Bandung, and originally was the Commandos, a Pelita Jaya’s Fans Club in West Java (Ismunanto, 2017; Sufiyanto, 2017; and Wirayudha, 2018). Hooliganistic conditions are always in reproduction by a figure who admired as a protector, i.e. Ayi Beutik Suparman. It further reinforces the value that is in the Vikings with its hooliganism practices that are considered as proud/cool/militant people. The culture continues to shape the The value of militancy and holiganism 153 ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos adopted by Viking is more rooted than Jakmania. In conflicts among supoorters, Viking will be hostile to Jakmania with more descending traits. This condition is evidenced by the explanation of one of the Vikings that use the nuanced attribute of racist: “Let this Enmity Stay Eternal” and “Jak is Dog” (Ayati, 2010; Eko, 2012; Huda, 2016; and Rahayu, 2016). According to a Viking’s informant, the use of the attribute only applies what Ayi Beutik Suparman used to say that the power of Padjadjaran kingdom as a priority seed. Ayi Beutik Suparman is considered by Viking’s members as a respected figure and as a binding cultural identity other than the Sundanese from the majority of the population of West Java (Hanifan & Herfiyana, 2014; Haryadi, 2014; Hadi, 2017; and interview with Respondent A, 3/7/2017). Executive of Jakmania stated, that is not part of Jakmania. To anticipate the widespread influence of Rojali, Chairman of Jakmania formed various campaigns, one of the campaigns is by campaigning 7 characteristics of Jak astray. The campaign is to identify Rojali’s behavior that often used undermines, vandalism, and anarchy activities. Rojali became one of the causes of the reproduction of the conflict, especially with Viking. In the context of a conflict involving Jakmania organizers, “Rojali” is always placed as a scapegoat without understanding the root cause of the conflict itself (Huda, 2016; Wibowo, 2017; Wirayudha, 2018; and interview with Respondent B, 10/7/2017). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik 154 FINDINGS AND DISCUSSION Rojali, according to the claim of the Chairman of Central 154 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 driving factors, such as the decision of the referee, and the provocation of the audience. This incident also resulted in one Policeman from the Provost unit injured his nose hit by a throw (cf Goldstein, 1993; Le Bon, 2002; Chrysnanda, 2011; Rahayu, 2016; Fuller, 2017; and Ismunanto, 2017). at the venue of stadium (Abbot & Geddie, 2001; and Wibowo, 2017). Vikings that are present dominated by young males and are rarely seen by many children and women, in addition to their difficult access to reach, a great risk is also a consideration of the infrequent presence of children and women in every game (Ryza, 2011; Hadi, 2017; and Kaka, 2017). The crowds, in the view of Gustav Le Bon (2002), are (crowd’s) life is the transformation of though (Le Bon, 2002). Another view represented by Elias Canetti (1973), who sees crowds as transformation of power. This shows that crowds have the ability to influence and be influenced in positions as objects or subjects (Canetti, 1973). In this context, the football audiences can be said to be crowds which in popular terms the football audiences are referred as the twelfth player (Canetti, 1973; Le Bon, 2002; Kari, 2009; and Wibowo, 2017). Solo case in Central Java, in 2017, indicated that the match between PERSIJA versus PERSIB should be held in Bekasi, West Java, but they do not get permission and must be transferred to Solo by PERSIJA’s management. The reasons are based on security aspect; and because of the fact that on the same day, there will be a massive demonstration in Jakarta. The security forces use risky action by presenting two large masses at a point that are at risk of direct clashes. The transfer of the venue became controversial, because it happened in a fast tempo where within three days the decision of the match in Bekasi, West Java was canceled and had to move to Solo in Central Java. Officials did sudden preparations for the match between PERSIJA dan PERSIB. FINDINGS AND DISCUSSION p ) Characteristics of spectators from the matches between PERSIB and PERSIJA can be identified depending on where the game is held. If the match held in Bandung, it was seen that the Vikings, who attended the game at the GBLA (Gelora [Gelanggang Olahraga] Bandung Lautan Api or Bandung a Sea of Flames’ Sports Arena) stadium, were clustered irregularly. This is due to a number of factors, firstly the factors of crowd’s management and crowd’s control (cf Berlonghi, 1995; Abbot & Geddie, 2001; Ayati, 2010; and Duerden, 2017) that are not working well; and secondly is the irregularity of parking lot, the escape of the bottles that enter the stadium, the dense access road to and exit the stadium, to plotting officers as ineffective security forces (McLean, 2015; Ayati, 2010; and Duerden, 2017). In the phenomenon of Viking and Jakmania, there are also other groups of Viking’s border groups. Characters from the border region that are part of Viking members often looking for conflict with Jakmania in areas such as Depok and Bekasi in West Java. This group is also in the Viking – Jakmania’s conflict, which always making violence to the Jakmanias group. Viking’s borders continue to reproduce existing conflicts with Jakmania. The settlement of prejudices arising from Viking, who think on unfinished borders, will continue to be a physical conflict even outside the stadium. The last case is the beating of Ricko (Viking) by the Viking border that strikes Ricko as Jakmania in the July 22, 2017’s match (Eko, 2012; Hanifan & Herfiyana, 2014; Huda, 2016; Hadi, 2017; and Wirayudha, 2018). The result of this irregularity is the casualties in the Vikings. They are often to be the victims of wrong targeting. KAPOLRESTABES (Kepala Kepolisian Resort Kota Besar or Head of Big City Police) Bandung has banned Jakmania from attending the GBLA stadium, but there are still beatings. This condition is driven by Bobotoh (PERSIB supporters)’s disappointment over the game, so the Bobotoh acts irrational because of their In Jakmania, there is a group called the Rojali group. This group is often to be the main cause of Jakmania’s involvement in conflicts, including Vikings. The central Jakmania committee admitted the difficulty in dealing with this group. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION The selection of Solo as the venue is based on the cooperation between officials of PERSIJA with local officials from PERSIS (Persatuan Sepakbola Indonesia Solo or Solo Indonesian Football Association) in Solo, Central Java (Rahayu, 2016; Saudale & Ganesha, 2016; Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). On the other hand, the phenomenon of conflict within the crowds also still uses the concept of prejudice (Brown, 2011; and Wibowo, 2017). It can be described in the form of a Viking who always feels that Jakmania will be a means of vengeance. This evident can be seen from the statements of the perpetrators, who explain the motive is hatred against Jakmania. If match’s management can do well-organized mechanism, the riots should be avoided by using the crowd control concept that focuses on crowds organizing crowd’s action process (Berlonghi, 1994; Tamara & Knutsson, 2011; Rahayu, 2016; Fuller, 2017; Ismunanto, 2017; and Wibowo, 2017). Police in the Solo case, in 2017, used silahturahmi (good relationship) approach before the game. The direction of this gathering is done by KABAG OPS POLRESTA (Kepala Bagian Operasi Kepolisian Resort Kota or Head of City Police Operation Division) Surakarta in Central Java to minimize friction and asking for cooperation between Pasoepati (PERSIS supporters) with Jakmania (PERSIJA supporters) in securing the match. Police The interesting thing about the characteristics of Viking is the variety of processes used in the assembling process; see, for example, the presence of supporters coming from different directions (cf John et al., 2001; Redhead, 2009; and Hadi, 2017). Vikings, who go to the venue of the game, are coordinated by their respective districts. the assembling process is accompanied by a gathering process (gathering at one point) that is associated with crowd’s management 155 ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos irregularities that will occur in Solo. The small Solo area will have 17,250 people coming from Jakarta; and if not done a good crowds management process will be a big potential riot (Berlonghi, 1994; Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). It should also be strengthened by crowds control conducted by Surakarta Police by inspecting Jakmania entourage, who arrived in Solo. This inspection resulted in a group of supporters who were caught possessing or consuming liquor. The perpetrator was held in Surakarta Police until the match was over (Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik 156 FINDINGS AND DISCUSSION always maintaining security by calling for a ban on the consumption of local liquor, which is quite famous in Solo to the fans. It aims to strengthen the coordination and security preparations that need to be done (cf Parks, 1981; Braiden, 1992; Oliver ed., 2000; Chrysnanda, 2011; Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). Due to the displacement venue of this match, the assembling process did not run with massive force. Tickets sold for 17,250 tickets, which is the result of ticket sales for 4 days. Jakmania, in this context, succeeded in mobilizing mass in large numbers in preparation for a short time. This assembling process (cf Berlonghi, 1994; John et al., 2001; and Prpic et al., 2017) was escorted by police to Jakmania’s entourage (Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). A similar phenomenon that occurs in Bandung, West Java, and also occurs in Solo, Central Java, is the action of raiding the opposing supporters who are identified as people who are not part of the supporters. Counted 6 times assault by Jakmania to groups identified as Viking. This action is not only triggered by prejudice as happened in Bandung (Brown, 2011; Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). However, it was also triggered by conditions in the field of a match considered to be detrimental to PERSIB, ranging from injury to Kim Kurniawan, disallowed goal, until a penalty decision to nourish the anger and disappointment of the supporters (cf Berlonghi, 1994; Brown, 2011; Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). ; , ; y , ) This process is done with a form of cooperation between the POLDA METRO JAYA (Kepolisian Daerah Metropolititan Jakarta Raya or the Great Jakarta Metropolitan Regional Police); POLDA JABAR (Kepolisian Daerah Jawa Barat or West Java Regional Police); and POLDA JATENG (Kepolisian Daerah Jawa Tengah or Central Java Regional Police) who escort a relay. To secure this process, Polices also carried out the communication process between the police and the community. Communications formed between the police and the community are effective enough to build mutual trust (Parks, 1981; Braiden, 1992; Goldstein, 1993; and Prpic et al., 2017). A similar process is done when traveling back from Solo to Jakarta or dispersal process (Oliver ed., 2000; John et al., 2001; Chrysnanda, 2011; Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). FINDINGS AND DISCUSSION J k i fi i d i This process is done with a form of cooperation between the POLDA METRO JAYA (Kepolisian Daerah Metropolititan Jakarta Raya or the Great Jakarta Chaos Tradition in Supporters as Football Culture and Character in Indonesia. Against the law and acting aggressively to the Police becomes a distinct culture in the lives of the hooligans (Pearson, 1983; Poulton, 2008; and Wibowo, 2017). The culture of the crowd of football supporters becomes a new culture that is formed and produced in certain forms. Resistance to the Police, which seeks to maintain security and order, is the hallmark of the culture. The local ethnic and cultural character is also a matter of encouraging Viking and Jakmania groups to do things Jakmania entourage first arrived in Solo at 02.00 AM (Ante Meridiem). They were dominated by a group of 17-25 year-old boys, who will be channeled in the velodrome area at Manahan Stadium in Solo, Central Java, Indonesia. This canalization aims to anticipate the MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 Indonesia, in addition to being shown in the form of cultural identity and local identity, is also indicated by the meaning of the value of each victory achieved by each football club in every game involving both clubs. The club’s victory becomes an important marker of the level of dominance possessed by each of the crowd’s identities. This makes it very important to look at such aspects as the influence of the referee’s decision as that is considered to affect the win or the defeat of a club. The implication is that masculine aggressiveness can be triggered by referee decisions in the game. This is shown by the many riot football supporters triggered by goal scorer, yellow/red card given by referee, and so forth (Williams & Dunning, 1990; Stone, 2007; Dick & Kernick, 2016; Duerden, 2017; and Fuller, 2017).l that are contrary to the law, such as throwing bottles and taking action against security in certain ways (David et al., 2008; Hadi, 2017; Wibowo, 2017; and Wirayudha, 2018). Culture supporters like this also happen in other countries, like England for example. Eric Dunning (2006) shows how the football industry has also spawned a violent industry. National characters within the context of the nationalist issues of Wales, Ireland, and Scotland are symbolized and applied in support actions to their respective football clubs. FINDINGS AND DISCUSSION The character of violence arises, when these values are contained by football matches that cause a pattern of masculine aggressiveness to emerge as the root of violence in football hooliganism (Dunning, 2006; Stott & Pearson, 2007; Rookwood & Pearson, 2010; Lo, 2011; dos Reis, Lopes & Martins, 2015; and Rogers, Woodhouse & Politowski, 2016). Conflict in Matches as Cultural Identity and Conflicts between Viking and Jakmania. To understand the Vikings and Jakmanias’ conflict, as a rivalry of PERSIB (Persatuan Sepakbola Indonesia Bandung or Bandung Indonesian Football Association) and PERSIJA (Persatuan Sepakbola Indonesia Jakarta or Jakarta Indonesian Football Association), must begin with the understanding that football is a tool of Indonesian nation’s identity struggle against the Dutch colonial. From that history, football in Indonesia is a legacy sport of colonial culture (transnational sport) introduced by the Dutch. And, not yet known the term football supporters. There are clubs and players. Football also became a portrait of social class (indigenous, non- indigenous, and Dutch). It shows that since the birth of football in Indonesia, the values of social segregation, based on ethnicity (Dutch, Chinese, and Native) have grown as differentiators between clubs (Colombijn, 2000; Ayati, 2010; Kristanto, 2016; Rahayu, 2016; Sufiyanto, 2017; and Wirayudha, 2018). Violence became a concept that has a relationship with the identity of football supporters and became part of a popular culture among certain fans in the national football industry. This is as shown in the Richard Guilianotti, Norman Bonney & Mike Hepworth (1994)’s study which stated that violence is capitalized with certain interests such as political interests and economic interests. Their research shows that political interests in violence supporters in Italy are used to distort aggression based on reality, such as poverty and social welfare that occurs in most supporter populations. Economic interests also affect chaos culture reproduction in rioting crowds of football supporters, where such influence could be the destruction of trademark image and capitalization of certain products that become the main attribute in the identity symbol of football supporters (Williams & Dunning, 1990; Guilianotti, Bonney & Hepworth, 1994; Dunning, 2006; dos Reis, Lopes & Martins, 2015; and Wann et al., 2015). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION The Natives (Indonesia) raised their identity through football clubs, such as the BIVB (Bandoeng Inlandsche Voetbal The Natives (Indonesia) raised their identity through football clubs, such as the BIVB (Bandoeng Inlandsche Voetbal The mass culture element contained in the phenomenon of football fanatical riots in 157 ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos of Sundanese moving widely (Utami, 2011; Hanifan & Herfiyana, 2014; Kristanto, 2016; Hadi, 2017; and Wibowo, 2017). In cyberspace, Bobotoh fans are also monitored, there are settlers abroad such as Malaysia, Singapore, Saudi Arabia, Britain, USA (United States of America), and others (Kristanto, 2016; Bahri, 2017; and Putri, 2018). Bond) during the Dutch colonial period as well as through the formation of other club clubs based on regional names. It is a manifestation of the Indonesian nationalist attitude, to show a sense of coincidence with the clubs in created by the colonialist. It shows the character of the egalitarian concept, which is shown by Indonesians or indigenous (Colombijn, 2000; Hanifan & Herfiyana, 2014; Kristanto, 2016; and Hadi, 2017). The role of media, as identity formers, also encourages in “how identity is formed” (Croteau & Hoyness, 2003; and Effendy, 2003). As Manuel Castells (2000) states that humans are passing through the industrial age to the information age. With the era of globalization and communication and information technology, Manuel Castells also stated that the information age will give birth to a new social, industry, and cultural network (Castells, 2000). The Japanese occupation government (1942-1945) saw the important position of football club formed by Indonesians (indigenous) as a medium in showing identity as Indonesian as well as a symbol of resistance. Consequently, in the concept of occupation (relations of domination), colonial nation and colonized nation make indigenous football club is considered very dangerous for the Japanese nation. This has implications for the banning policy of Indonesian football club by the Japanese government (Colombijn, 2000; Beck, 2015; and Colombijn & Cote, 2015). ( , ) This also happened to Jakmania and Viking football supporter groups. Cyberspace becomes the arena of communication, contestation (rivalitas), as well as hostility supporters. This is exacerbated by the capitalization of Jakmania and Viking conflicts through online and social media. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik 8 FINDINGS AND DISCUSSION This is shown by several things: firstly, that Bobotoh as the parent of the synthetic culture of delays has metamorphosed naturally into several other support groups. The group’s naming is imaginary from portraits of European supporters, such as the Italian Ultras, the FCC (Football Club Cincinnati) of Ohio, Vikings like Scandinavia, and Orientalism that show Asian identity. Secondly, social and class segregation does not only happen to football clubs in the Dutch colonial, but also happens to football fans in the era of development. This means that social segregation is the background of SARA (Suku, Agama, Ras, dan Antargolongan or Ethnic, Religion, Race, and Inter-Groups) that gave birth to synthetic identity naturally or through fabrication, which always grows in society (cf Canetti, 1973; Voris, 2015; Kristanto, 2016; Hadi, 2017; Sufiyanto, 2017; and Wirayudha, 2018). p ) The emergence of fanatic football supporters, in line with the era of national football competition, is considered as part of national development in the field of sports during the New Order era (1966- 1998). Bobotoh grows in various regions in West Java. In fact, its development grew in other areas, inside and outside the country. The number was from dozens transformed into hundreds of thousands and even millions. Bobotoh is an imagine community (Anderson, 1983), which is based on value of ke-Sunda-an (Sundanese) in line with PERSIB (Hanifan & Herfiyana, 2014; Kristanto, 2016; Hadi, 2017). y , ) Vikings are symbols of conquering values that are the product of Ayi Beutik Suparman’s imagination of the legendary name of the Scandinavian nation. Viking identic with the values as a strong nation, savage, brave, never give up, and like to attack. Nonetheless, Viking is a metamorphosis of Bobotoh, because of its natural nature, the Vikings do not recognize the semi-bureaucratic organizing system (Haryadi, 2014; and interview with Respondent A, 3/7/2017). Like the crowds, Viking also develops naturally, through the hereditary mechanisms of the family. Viking also develops to other regions and is no longer confined to the Cibangkong and Pasirluyu regions in West Java as the Viking homebirth (cf Canetti, 1973; Hanifan & Herfiyana, 2014; Kristanto, 2016; Hadi, 2017; and interview with Respondent A, 3/7/2017). According to John Voris (2015), Bobotoh can be categorized as a synthetic identity from ke-Sunda-an (Sundanese mattres). As part of Bobotoh, there is a group of supporters called Viking PERSIB Fans Club. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION From social media network, the researchers can know that most of Bobotoh or Viking comes from Cianjur with 637,263 people; Garut with 627,240 people; Bandung in third place with 506,774 people; fourth is Jakarta with 260,056 people; and Bobotoh in Surabaya, East Java is in fifth position with 93,965 people (Hanifan & Herfiyana, 2014; Kristanto, 2016; Rahayu, 2016; Sufiyanto, 2017; Wirayudha, 2018). From the historical description, PERSIB as pride of Bandung’s citizens, especially among Sundanese in the era of independence is also a symbol of continuity or symbolic metamorphosis of the Padjadjaran kingdom in West Java. This spurred the formation of a new sub-culture of identity of delays or identities into Padjadjaran. PERSIB is a symbol of a new identity formed in Sundanese society that is identical with Sundanese culture (Hanifan & Herfiyana, 2014; Kristanto, 2016; and Hadi, 2017). Bobotoh does not operate as limited in Bandung or West Java. Although PERSIB is headquartered in Bandung, but Bobotoh supporters are widespread from Banjar, Ciamis, Tasikmalaya, Bogor, Cirebon, Kuningan, Karawang, and Bekasi in West Java. Bobotoh also located outside the Province of West Java, such as Jakarta, Tangerang, and Serang in Banten Province. Widespread membership of Bobotoh in accordance with the characteristic identity Based on the data of 5.3 million Bobotoh in West Java, 26 percent are women. Approximately 1,378,000 this female Bobotoh has demographic status with age ranging from 21-40 years old, married, and tend to like PERSIB due to influence of husband, and child (cited in Hanifan & Herfiyana, 2014; Kristanto, 2016; and Hadi, 2017). In the match, Bobotoh will always meet the stadium, either if PERSIB compete 158 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 in Bandung or Jakarta, with always bring attributes. Bobotoh also performs watching together in front of the television for those who cannot go to another city if PERSIB perform away games as a form of their commitment to the club (Utami, 2011; Hanifan & Herfiyana, 2014; Kristanto, 2016; and Hadi, 2017). The Bobotoh generally have two major groups, namely: Viking PERSIB Fans Club and Bobotoh Maung Bandung Bersatu or the BOMBER. There are also Bobotoh type based on the characteristics consist of: Bandung Ultras, Flower City Casuals, Oriental Bobotoh, and Singapore Bobotoh (Hanifan & Herfiyana, 2014; Kristanto, 2016; Hadi, 2017; and interview with Respondent A, 3/7/2017). multiculturalism of football supporters. FINDINGS AND DISCUSSION Viking can be identified as a synthetic identity of Bobotoh that indicates the strength of transcultural or mix culture as affecting the authentic culture of the Sundanese. It also co-stars with football as an imitation culture from European football’s origin culture (cf Voris, 2015; and Hadi, 2017). Vikings also remain familiar with group values as the requirement of an identity, and called Vikingnisme as a naturally formed From the naming of the Viking and Bobotoh, it is true to show the 159 ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos society. The acculturation of values between the Vikings as the imagine norm and the delay as part of Bobotoh of PERSIB is seen from value, loyalty (to the group), solidarity (loyal friends), and values in urban society (style) symbols, authentic identity brought from the birth of each individual and social environment, in which he lived to the Viking group (Hanifan & Herfiyana, 2014; Kristanto, 2016; and Hadi, 2017). is a determinant factor of the Viking metamorphosis process. Even, if we imagine the Vikings are a group of beliefs, then, in the Vikings are also born rituals that distinguish between members of Viking with other Bobotoh. Ayi Beutik Suparman becomes the object of a ritual for Viking members, who are believed to be able to give “strength” in the form of courage (cf Canetti, 1973; Fathurrohman, 2014; Haryadi, 2014; and interview with Respondent A, 3/7/2017). The growing number of Viking members also boosts business growth related to accessories and attributes of football supporters. That supporters also have an important role in an economic activity, especially the football industry. In the Viking group, there are symbolic figures that influence the Viking development up to now. The figure is Ayi Beutik Suparman, who can be regarded as the central figure in the Indonesian archipelago, like General Soedirman for the military, Sukarno for Marhaen nationalist, or R.A. (Raden Ajeng) Kartini for women’s emancipation in Indonesia (cf Stone, 2007; Fathurrohman, 2014; Hanifan & Herfiyana, 2014; Kennedy, 2014; Kristanto, 2016; Hadi, 2017; Kaka, 2017; and Eyong, 2018). Meanwhile, PERSIJA in Jakarta as a club, that was born since the Dutch colonial era, has a historical similarity with PERSIB in Bandung. PERSIJA, previously named VIJ (Voetbal Indonesische Jacatra), is a symbol of the existence of indigenous people of Betawi. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik 0 FINDINGS AND DISCUSSION In addition, Gugun Gondrong as icon Jakmania is an artist who uses his popularity as a magnet to attract new members (Hamley, 2001; Sufiyanto, 2017; Wirayudha, 2018; and interview with Respondent C, 17/7/2017). As a synthetic identity of a new group, Jakmania members are bound by the rules, which are mentioned by AD/ART. From the beginning Jakmania in form, in support of the development of PERSIJA club from the social and economic, such as ticket entry, merchandise, etc. (Sufiyanto, 2017; and Wirayudha, 2018). Jakmania is a group of supporters, whose development is heavily influenced by bureaucratic policies. This is in line with the common concern among Jakarta youngsters, who love to watch PERSIJA with the management and the Provincial Government of DKI Jakarta under the leadership of Governor Sutiyoso for the lack of audience when the club Persija Jakarta compete. This feeling of alienation and marginalization is a major consideration of Jakmania’s establishment as a new synthetic identity (cf Colombijn, 2000; Voris, 2015; Sufiyanto, 2017; and Wirayudha, 2018). As the early history of football in Indonesia as a means of identity struggle against the Dutch colonial, Viking and Jakmania relations as part of PERSIB and PERSIJA competitiveness cannot be separated from the form of harmony metamorphosis relation to a relationship that is conflict. In the history of relations, they can be described as the crowd at the Siliwangi Stadium was influenced by the situation of the matches that took place in the field, the emotions of the audience were strongly influenced by the field conditions, such as the outcome of the match (cf Berlonghi, 1994; Colombijn, 2000; Abbot & Geddie, 2001; Hanifan & Herfiyana, 2014; Conigliaro et al., 2015; Kristanto, 2016; Sufiyanto, 2017; Wibowo, 2017; and Wirayudha, 2018). The pattern of Jakmania formation differs from the pattern of formation that occurs in Viking. If Viking is based on natural, bottom-up, fluid, cultural values, although familiar with simple organizational systems, then Viking is a continuation of the greatness of the Padjadjaran kingdom or PERSIB. Jakmania is a tool or instrument fabricated through the collaboration of the provincial government and the will of the Jakarta children, so that the underlying spirit of Jakmania’s presence is the resurrection values of the Jakarta community to host its own region. FINDINGS AND DISCUSSION PERSIJA as a symbol of the spirit of equality and resistance to colonial symbols and class distinctions or VBO (Voetbalbond Batavia Omstaken) as the Dutch formation. The difference with PERSIB is PERSIJA originally named VIJ has had a group of fans or fans known as VIJers (Sufiyanto, 2017; and Wirayudha, 2018). In post-independence era, PERSIJA and PERSIB are the founding initiators of PSSI (Persatuan Sepakbola Seluruh Indonesia or All Indonesian Football Association). PERSIJA later named itself as “Kemayoran’s Tiger”, which has the meaning of a beast that in imagination as the King of Forest, which has the characteristics of ferocious, brave, in respect, and the ruler. This is reinforced by the use of red on club uniforms (Kristanto, 2016; Sufiyanto, 2017; and Wirayudha, 2018). Ayi Beutik Suparman is the founder and patron of the entire sub-culture of the Viking members. He was a charismatic figure, who provided the first normative fundamentals of the Vikings. His boldness became the most basic reference value of Viking members. As a leader, from a group of natural and cultural, willing to sacrifice for other members has become the value that makes him very much in love with the Viking groups (Fathurrohman, 2014; and Haryadi, 2014). The existence of PERSIJA until 1994, or the Indonesian league, is identified with a club that does not have many supporters; and this is indicated by the minimal number of spectators. This affects the club’s internal conditions. This situation prompted the role of DKI (Daerah Khusus Ibukota or Special Capital Region) Jakarta local government to intervene to help PERSIJA as the club pride As a group that goes on naturally and culturally, Ayi Beutik Suparman’s leadership character becomes the main magnet affecting the rapid growth of Viking members, not only in Bandung but also in other regions. If the analogy, with the process of formation of butterflies, it can be said that Ayi Beutik Suparman 160 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 of the capital. From it can be interpreted that PERSIJA is a club that has a historical value, in the dynamics of Indonesia, the symbol of the pride of the capital, both people and government (cf Berlonghi, 1995; Colombijn, 2000; Sufiyanto, 2017; Wibowo, 2017; and Wirayudha, 2018). moves to invite the people of Jakarta to join Jakmania as part of the exploration process (Utami, 2011; Sufiyanto, 2017; and Wirayudha, 2018). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION Jakmania was formed based on the norms of the formation of a social organization, one of which is by arranging AD/ART (Anggaran Dasar/ Anggaran Rumah Tangga or Organizational Constitution). Recruitment involves the village, to socialize as well as encourage the local people to watch the game conducted by PERSIJA (Hanifan & Herfiyana, 2014; Kristanto, 2016; Sufiyanto, 2017; and Wirayudha, 2018). Crowds management failed to build in Jakmania’s body, since the 2000s in the process come to Bandung. The crowds control operated by the POLDA METRO JAYA (Kepolisian Daerah Metropolititan Jakarta Raya or the Great Jakarta Metropolitan Regional Police) on the road trip agenda to the Siliwangi Stadium shows a failure that can be identified through the crowd’s stage based on Kenny M. John et al. (2001)’s research. At the stage of assembling Jakmania, miscalculation Crystal crowds Jakmania, totaling 40 people into a community group that 161 ANDRY WIBOWO, Pop Culture, Identity Conflict, and Chaos occurred which of the quota of 400 people to 1,000 people (John, 2001; Wibowo, 2017; and Wirayudha, 2018). f anytime and anywhere (Williams & Dunning, 1990; Berlonghi, 1994; Hanifan & Herfiyana, 2014; Kristanto, 2016; Fuller, 2017; Sufiyanto, 2017; Wibowo, 2017; and Wirayudha, 2018). l This condition affects the next process of gathering at the Siliwangi Stadium in Bandung, the capacity of the stadium is not large with the match allocation of the tickets with the number of spectators in attendance resulting in mass accumulation outside the stadium. In this mass build up, there was a clash between the Vikings and Jakmanias. In the process of dispersal shown by a group of Jakmania, who will return to Jakarta, involving clash activity with Vikings. From the description, it can be interpreted that the Police and match management is not ready in the face of overcrowding situation, which is always identic with Jakmania (Abbot & Geddie, 2001; Kristanto, 2016; Fuller, 2017; Wibowo, 2017; and Wirayudha, 2018). Conflicts are also exacerbated by the interpretation of cases occurring, perpetrated by both parties, both through interaction with the direct victims/perpetrators as well as the modified interpretations made by Jakmania and Viking members. This extends with the increasing number of Jakmania and Viking members; and the use of social media. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik 162 FINDINGS AND DISCUSSION The protracted conflict as well as the modified interpretation of each of the sides that resulted in the acute prejudice, not only to individuals involved in the conflict but extended to group members are generative or hereditary (Blumer, 1968; Castells, 2000; Sunarto, 2000; Brown, 2011; Hanifan & Herfiyana, 2014; Kristanto, 2016; Fuller, 2017; Sufiyanto, 2017; Wibowo, 2017; and Wirayudha, 2018). The prejudices held by Jakmania on Viking came after the events that took place at the Siliwangi Stadium since the 2000s. Jakmania feels like a loser, because they are on the agenda to attend their away match, which is not in line with expectations. This action was responded by beatings to a group of Vikings who attended a quiz show at one of the TV stations in Jakarta. This conflict is repeated and reproduced by the Vikings as well as Jakmania as the basis for efforts of conflict reproduction. This then produces a prejudice that gave birth to a stereotype of the Viking group’s rogue by the Jakmania (cf Sears, Freedman & Peplau, 1994; CONCLUSION The characteristic of crowds in the football industry in Indonesia is strongly influenced by issues of cultural and local identity. In addition, the character of football supporters in Indonesia is strongly influenced by social identity, such as socio- economic level. This causes the level of aggression in the football industry in Indonesia seen in the form of riots and competition of identity symbols, such as jersey, colors, flags, and so forth. Then also if drawn further, the formation of identity is correlated with the phenomenon of football as a pop culture, that is that football into a culture that can be enjoyed universally, so it can involve anyone and anywhere. ( f p Kristanto, 2016; Fuller, 2017; Wibowo, 2017; Wirayudha, 2018; and interview with Respondent B, 10/7/2017). This illustrates the metamorphosis of conflict between Jakmania and Viking. Conflict continues to occur involving Vikings and Jakmanias, during matches and beyond football matches. This illustrates the metamorphosis of conflict with the encouragement of precipitant factors, such as bad crowd management to conflicts related to group identity, which can happen The implications of looking at the phenomenon of football in Indonesia as a pop culture phenomenon also provide help to illustrate that the level of masculine aggression in riots between Viking in West Java and Jakmania in Jakarta cannot be 162 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan, Volume 3(2), September 2018 released as a culture of its own produced continuously to achieve certain interests. The phenomenon of behavior and attitude of hooliganism in the body of Viking and Jakmania as well as in the phenomenon of hooliganism in England. l homogeneity of Sundanese culture. The spirit of cultural homogeneity has been acculturated with values and passion drawn from the imagination of the “Vikings” characterized by brave values, conquerors, and loyalty; thus encouraging Bobotoh to join the growing, growing, and growing Viking supporters from the grassroots. The phenomenon of conflict involving the two supporter groups has crystallized into an identity conflict based on prejudice between the two groups. Subsequent forms of prejudice that also grew out of the state and the public have given birth to stereotypes that see Jakmania and Viking as monstrous that have the potential to pose a threat to security and order disorders, especially if the group is present in the arena of matches. CONCLUSION The root cause of the riots can be summed up because the internalized “prejudices” in both members of both supporters’ camps are continuously produced into a popular culture in the Viking and Jakmania’s areas. The effort to build trust in the level of awareness, peace, and mutual respect is not easy, so it requires a sustainable effort to build a culture of sportsmanship in the football competition. However, cultural cultivation is not easy to implement, because the riot culture is more popular than the sprotif culture, peace, and so forth. In accordance with the historical, philosophical, cultural, and socio-economic background, that inspired the formation and founding of Jakmania in Jakarta and Viking in Bandung, it can also be concluded that initially both organizations were born to provide support and love for PERSIJA (Persatuan Sepakbola Indonesia Jakarta or Jakarta Indonesian Football Association) and PERSIB (Persatuan Sepakbola Indonesia Bandung or Bandung Indonesian Football Association)’s football clubs. But in its journey, organization and management, especially Jakmania, is more formal with hierarchical organizational structure and division of organizational tasks more structured. Such forms and organizational and management characteristics of course have an influence on the behavior and attitudes of members of Jakmania. The youth empowerment of marginal and highly heterogeneous community groups in Jakarta has aroused the spirit of militant Jakmania’s members coincidentally inspired by community leaders, who are considered to have high fighting spirit. Jakmania and Viking if they want to clean up must carry out the members’ mental reform that involves the reconstruction of values. This is done considering the unrest between the two members of the organization, not only occurred in the arena of matches between PERSIJA and PERSIB, but also elsewhere such as Europe. 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Developmental Trajectories of Internally and Externally Driven Temporal Prediction
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RESEARCH ARTICLE a11111 OPEN ACCESS OPEN ACCESS Citation: Mento G, Tarantino V (2015) Developmental Trajectories of Internally and Externally Driven Temporal Prediction. PLoS ONE 10(8): e0135098. doi:10.1371/journal.pone.0135098 OPEN ACCESS Citation: Mento G, Tarantino V (2015) Developmental Trajectories of Internally and Externally Driven Temporal Prediction. PLoS ONE 10(8): e0135098. doi:10.1371/journal.pone.0135098 OPEN ACCESS Citation: Mento G, Tarantino V (2015) Developmental Trajectories of Internally and Externally Driven Temporal Prediction. PLoS ONE 10(8): e0135098. doi:10.1371/journal.pone.0135098 Editor: Hedderik van Rijn, University of Groningen, NETHERLANDS Editor: Hedderik van Rijn, University of Groningen, NETHERLANDS Received: February 25, 2015 Accepted: July 16, 2015 Published: August 11, 2015 Copyright: © 2015 Mento, Tarantino. 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. Received: February 25, 2015 Accepted: July 16, 2015 Published: August 11, 2015 Copyright: © 2015 Mento, Tarantino. 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. Received: February 25, 2015 Accepted: July 16, 2015 Published: August 11, 2015 Copyright: © 2015 Mento, Tarantino. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Mento, Tarantino. 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. Developmental Trajectories of Internally and Externally Driven Temporal Prediction Giovanni Mento1*, Vincenza Tarantino1,2 1 Department of General Psychology, University of Padua, Via Venezia 8, 35131, Padua, Italy, 2 Department of Neurosciences: SNPSRR, University of Padua, Via Giustiniani 5, 35128, Padova (PD), Italy Giovanni Mento1*, Vincenza Tarantino1,2 1 Department of General Psychology, University of Padua, Via Venezia 8, 35131, Padua, Italy, 2 Department of Neurosciences: SNPSRR, University of Padua, Via Giustiniani 5, 35128, Padova (PD), Italy * giovanni.mento@unipd.it Abstract The ability to generate temporal prediction (TP) is fundamental to our survival since it allows us to selectively orient our attention in time in order to prioritize relevant environmental infor- mation. Studies on adult participants showed that externally and internally driven mecha- nisms can be engaged to establish TP, both resulting in better behavioural performance. However, few studies on children have investigated the ability to engage internally and externally driven TP, especially in relation to how these mechanisms change across devel- opment. In this study, 111 participants (88 children between six and eleven years of age, and 23 adults) were tested by means of a simple reaction time paradigm, in which temporal cueing and neutral conditions were orthogonally manipulated to induce externally and inter- nally driven TP mechanisms, as well as an interaction between the two. Sequential effects (SEs) relative to both tasks were also investigated. Results showed that all children partici- pating in the study were able to implement both external and internal TP in an independent fashion. However, children younger than eight years were not able to combine both strate- gies. Furthermore, in the temporal cueing blocks they did not show the typically-observed asymmetric SE pattern. These results suggest that children can flexibly use both external and internal TP mechanisms to optimise their behaviour, although their successful com- bined use develops only after eight years of age. a1111 Introduction The ability to temporally predict relevant events (temporal prediction, or TP) is a fundamental skill allowing us to reduce uncertainty about the future and to optimise our behaviour [1–3]. Crossing a busy street at the rush hour, or deciding whether to accelerate or to stop when our car arrives at an amber traffic light are just two among thousands of daily situations in which a bad prediction of the correct time to act may threaten our survival. In spite of this, the ontogen- esis of the cognitive mechanisms enabling human beings to develop a ‘predictive brain’ is still a challenging topic. In this study, we tried to address this issue by investigating the developmen- tal trajectory of different types of TP. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the grant ‘Progetto Giovani Studiosi’ from the University of Padua (prot. 1682/2012) to GM. http://www.unipd.it/ en/home-page. Competing Interests: The authors have declared that no competing interests exist. 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction It has been demonstrated that TP can be externally generated by the temporal properties of stimuli. More specifically, externally driven TP (E–TP) may be established by exploiting the temporal information provided either by regular sensorial structures (i.e., auditory rhythms) or discrete signals cueing the onset of task-relevant stimuli [4–15]. In both cases, the possibility of using external information to predict the onset of upcoming events induces the orienting of attention toward specific points in time. This ability is defined as temporal orienting (TO) [1,3,4]. By contrast, in the absence of external cues providing information about incoming events, TP must be deployed over time by using an internally driven process (I–TP). A classic example of I–TP is the well-known variable foreperiod (FP) effect. When FPs of different dura- tions are randomly presented in a block of trials with the same a priori probability of occur- rence, the mean reaction time (RT) to targets decreases with increasing FPs [16–19]. The specific benefit of long vs. short FP trials is mainly explained by the unidirectional flow of time itself, which intrinsically and conditionally biases target predictability [6,16,20–23]. The proba- bility of an event occurring at a given time is conditioned by the accumulating evidence that it has not yet occurred, a phenomenon also known as hazard function [16,20–22,24]. An addi- tional source of TP is reflected in the so-called sequential effects (SEs) [17,18,25–27]. Sequen- tial effects rely on the observation that the temporal structure of trials itself can induce TP by biasing the allocation of cognitive and motor resources over time. Specifically, a participant’s ability to predict the onset of forthcoming events will depend on the temporal rule experienced in the preceding trial. Behaviourally, this translates into slower RTs in the current trial, when this is preceded by a longer FP in the previous trial, than when preceded by an equally long or shorter one. Notably, SEs are asymmetric, as they are mainly present with the shortest FP in a block of trials, but tend to vanish as FP increases. PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Participants One hundred and twenty participants were initially enrolled. Eight children were excluded since they did not complete the experimental task. One subject was excluded because he exceeded 2 SD from the age group RT average. The final sample was composed of 111 partici- pants (88 children and 23 adults). Children were recruited from a primary school in the Vene- tian Region of Italy and were allocated into one of three groups: 6–7, 8–9 and 10–11 years. Adults were the children’s parents or undergraduate psychology students at the University of Padua. The demographic characteristics of the four groups are described in Table 1. Ethics statement. All children's parents and adult participants signed a written consent form. The director of the school also signed an agreement that formally allowed the children to be tested in the school. All experimental procedures were approved by the Ethics Committee of the School of Psychology of the University of Padua (protocol n° 1362) and were conducted according to the principles expressed in the Declaration of Helsinki. Development of Temporal Prediction on internal sources of information; this mechanism translates into behavioural advantage in terms of motor preparation. Concerning the ability to use E–TP, there is evidence that preverbal infants are already able to learn the temporal regularities as well as the durations associated with external sensorial events [38–42]. This suggests that the ability to exogenously orient attention in time, making use of external information, should also be operating at 6 years of age. The ability of using external, discrete cues to endogenously orient attention in time has been much less investigated in children. In a recent study, Johnson and colleagues [43] employed a cueing paradigm to dis- entangle the automatic and voluntary mechanisms underlying spatial and temporal orienting in children. Interestingly, they found that, although children were able to automatically orient their attention in space and time, they showed voluntary orienting of attention in space but not in time. More specifically, in the temporal cueing blocks children showed FP and SEs, but not TO effects. As discussed by the authors, the lack of significant TO in their study may be accounted for by the use of lateralised rather than central targets, which could have introduced a spatial uncertainty bias that possibly reduced the behavioural benefit provided by the tempo- ral cues. Moreover, Johnson and colleagues did not directly test the developmental trajectories of cueing effects, given that they tested a single group of children ranging from six to sixteen years of age. Hence, the questions are still open as to whether E–TP, generated by discrete tem- poral cues, may emerge ontogenetically and whether and how it interacts with other forms of implicit timing involving I–TP (i.e., FP and SEs) across development. To address the above questions, in this cross-sectional study we compared the developmen- tal trajectories of E–TP and I–TP in six to eleven year-old children and adults according to a paradigm in which a TO and a FP task were factorially combined to respectively induce E–TP and I–TP; we also investigated their interplay. We adopted a child-friendly approach including the presentation of central targets and a fixed 100% cue validity to facilitate children in the deliberate use of cues to orient their attention in time, as well as to enhance the benefits of tem- poral cueing by keeping fixed spatial target predictability. Sequential effects in both TP condi- tions were also measured and analysed. Data Availability Statement: All relevant data are within the paper. According to the ‘dual-process’ model pro- posed by Vallesi and collaborators [28–30], while SEs per se are automatic in nature, their observed asymmetry, depending on the relative durations, reflect a voluntary re-preparation process triggered by the conditional probability of target appearance over time. Indeed, condi- tional probability for target onset at the long FP is always equal to 1, irrespective of the previous FP duration. While the cognitive mechanisms and the underlying neural bases of E–TP and I–TP in adults are relatively known [1,3,6,10,11,17,18], how they emerge and interact ontogenetically is less clear. In a pioneering study, Elliot [31] tested a large sample of children aged 5–13 years, and adults performing a simple auditory FP task, including several preparatory intervals. Unlike many following studies (see below), he only reported a slight and non-significant FP effect. However, this discrepancy may be attributed to methodological issues, including the wide ranges of intervals used (1–16 s), making it harder for children to maintain sustained attention over time. In a later study, Czudner and Rourke [32] tested children with brain-dam- age, subdivided into two groups, ‘young’ (6 to 9 years) and ‘old’ (10 to 13 years), by using both regular and irregular FPs. Interestingly, they found that young control, old control, and old brain-damaged groups performed better than the young brain-damaged group, suggesting that, with advancing years, brain-damaged children may adapt to and/or recover from deficit (s) in their ability to develop and maintain a state of readiness to respond. A FP effect in chil- dren between around 7 and 11 years was also reported by Ozmun et al. [33]. More recently, Vallesi and Shallice [34] investigated both FP and sequential effects in 4 to 11 year-old children, showing a developmental dissociation between the two effects; while SEs were already present at 4–5 years of age, FP appeared gradually from 6–7 years on. These findings suggest that, although being both implicit forms of temporal preparation, SEs reflect a more automatic pro- cess than variable FP effects. This suggestion is supported by the observation that only the FP, but not the SEs, are affected by dual-task interference [35–37]. In summary, the available stud- ies provide consistent evidence that school-age children can implicitly generate TP by relying 2 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Stimuli and tasks Stimuli were presented on a 17–inch monitor at a resolution of 1,280 × 1,024 pixels. Partici- pants were seated comfortably in a chair at a viewing distance of 60 cm from the monitor. All participants performed two TP tasks within the same experimental session. The trial structure was the same in both tasks. Each trial began with the display of a visual cue, followed by the presentation of a target stimulus that stayed on the screen for a maximum of 3,000 ms. The visual cue consisted of a picture of a black camera lens surrounded by a circle (total size of the stimulus: 840 × 840 pix- els, 144 dpi, 10.62° × 10.54° of visual angle). The target stimulus consisted of a picture of an animal, which was displayed centrally within the camera lens. The cue-target stimulus-onset- asynchrony (SOA) was manipulated (either 600 or 1,400 ms). The inter-trial-interval (ITI) was randomly manipulated between 600 and 1,500 ms. The task consisted of speeded target detec- tion; participants were required to press the space bar with the index finger of the dominant hand as quickly as possible at target occurrence. To encourage participants to produce a good performance, they were given the following instruction: ‘Imagine that you are at the zoo and you have a camera. Your task is to take a picture of the animals appearing within the camera lens as soon as possible. To do that, you have to press the space bar with your index finger. Please, be careful to click as quickly as possible once you see the animals otherwise they will dis- appear. Please, also be careful not to click before the animals appear!’ The experimental task included temporal and neutral cueing blocks, which were adminis- tered block-wise rather than trial-by-trial in order to reduce top-down control required to switch continuously from a predictive to a non-predictive setting [36]; this may have caused additional difficulty for the children. As detailed below, children were also instructed block-by- block that they could or not predict in advance the animals’ temporal onset. Temporal cueing blocks. In the temporal cueing blocks (Fig 1a) the visual cue provided fixed temporal information concerning the SOA duration. In particular, the outer circle of the camera lens was coloured either blue or orange. Each of the two colours was associated with a SOA duration (600 or 1,400 ms). Development of Temporal Prediction collected all behavioural data. Children reported as having learning disabilities or neurological or psychiatric disorders were excluded. All had normal or corrected-to-normal vision. Table 1. Experimental groups. Main demographic characteristics of the participants to the study. SD = Standard Deviation. Gender Handedness Group Mean Age ± SD (range) Female Male Left Right n 6–7 77 ± 4 months (69–84) 16 14 2 28 30 8–9 90 ± 4 months (84–96) 18 16 4 30 34 10–11 105 ± 4 months (97–127) 12 12 3 21 24 Adults 35.6 ± 15 years (18–59) 11 12 4 19 23 doi:10.1371/journal.pone.0135098.t001 Table 1. Experimental groups. Main demographic characteristics of the participants to the study. SD = Stand collected all behavioural data. Children reported as having learning disabilities or neurological or psychiatric disorders were excluded. All had normal or corrected-to-normal vision. collected all behavioural data. Children reported as having learning disabilities or neurological or psychiatric disorders were excluded. All had normal or corrected-to-normal vision. Experimental procedure The children underwent the experimental task individually in a quiet room at their school, while the adults completed the experimental task at the University. The same experimenter 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Stimuli and tasks In other words, the colour of the visual cue provided fixed information about the SOA duration, leading to a temporal short (T–Short) and a temporal long (T–Long) SOA condition. The association between the colour of the outer circle and the SOA duration was counterbalanced between subjects. In order to maximise the E–TP magni- tude, the cue was 100% valid (the association between the colour and the SOA remained fixed throughout the task), no catch trials were included (the target stimulus always appeared after the SOA) and the target was always presented centrally. Neutral cueing blocks. In the neutral cueing blocks (Fig 1b), the outer circle surrounding the camera lens was always black, providing no information about SOA duration; the cue 4 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction Fig 1. Experimental paradigm. The Temporal Cueing task (a) was purposely designed to induce externally driven temporal prediction (E–TP). The visual cue provided fixed temporal information concerning the SOA duration, which could be short (left panel) or long (right panel), according to the colour of the cue. By contrast, the Neutral Cueing task (b) was specifically designed to induce internally driven temporal prediction (I–TP). In this case participants never knew in advance the duration of the SOA, which could nevertheless have the same short or long duration than in the Temporal Cueing task. Fig 1. Experimental paradigm. The Temporal Cueing task (a) was purposely designed to induce externally driven temporal prediction (E–TP). The visual cue provided fixed temporal information concerning the SOA duration, which could be short (left panel) or long (right panel), according to the colour of the cue. By contrast, the Neutral Cueing task (b) was specifically designed to induce internally driven temporal prediction (I–TP). In this case participants never knew in advance the duration of the SOA, which could nevertheless have the same short or long duration than in the Temporal Cueing task. doi:10.1371/journal.pone.0135098.g001 doi:10.1371/journal.pone.0135098.g001 simply acted as a warning signal, which prepared participants for the forthcoming target with- out furnishing temporal information about it. Nevertheless, regarding the temporal cueing blocks, the SOA was also manipulated to create a neutral short (N–Short) and a neutral long (N–Long) SOA condition (600 and 1,400 ms, respectively). To maximise the magnitude of the I–TP effect, we used an ‘aging’ probability distribution [16,44], with an equal a priori odds ratio for each SOA duration. Stimuli and tasks This probability distribution is known to obey a hazard function [6,16,20]; that is, the subjective probability of target onset will increase over time, given that it has not yet occurred. Experimental design As illustrated in Fig 2, the experimental manipulation yielded a factorial design in which the CUE (temporal vs. neutral) and the SOA (short vs. long) factors were orthogonally contrasted to calculate E–TP, I–TP and their combined effect (E/I–TP) on behavioural performance. Spe- cifically, the E–TP effect was measured by comparing mean RTs between N–Short and T– Short trials (TO–Short SOA) and between N–Long and T–Long trials (TO–Long SOA). The effect of I–TP was measured by comparing mean RTs in N-Short trials to mean RTs in N– Long trials (Uncued–FP Effect). Finally, the combined effect of E- and I–TP was measured by comparing mean RTs in T–Short trials to mean RTs in T–Long trials (Cued–FP Effect). In the PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 5 / 18 Development of Temporal Prediction Fig 2. Experimental Design. The cue (Temporal vs. Neutral) and the SOA (Short vs. Long) were factorially combined to obtain a measure of E–TP (revealed as the TO effect at short and long SOA), I–TP (revealed as the Uncued–FP effect) and E/I–TP (revealed as the Cued–FP effect). doi:10.1371/journal.pone.0135098.g002 Fig 2. Experimental Design. The cue (Temporal vs. Neutral) and the SOA (Short vs. Long) were factorially combined to obtain a measure of E–TP (revealed as the TO effect at short and long SOA), I–TP (revealed as the Uncued–FP effect) and E/I–TP (revealed as the Cued–FP effect). doi:10.1371/journal.pone.0135098.g002 doi:10.1371/journal.pone.0135098.g002 doi:10.1371/journal.pone.0135098.g002 latter case, the performance of participants is supposed to be influenced by both the temporal information of the cue (always valid) and the hazard function (i.e., the elapsing of time itself), resulting in an interaction of external and internal TP (E/I–TP). Notably, all conditions were matched for sensorimotor requirements, since the visual stimuli and the required response were always the same across the experiment; the only difference between conditions was the level of target predictability. Before starting the experimental session, participants were presented with a block of 20 training trials for each condition, to ensure they understood task instructions and had learnt the temporal association between cue–type and SOA in the temporal cueing task. After train- ing, the participants were asked to identify the temporal interval associated with each of the cues to ensure understanding of the cues, and were reminded to detect the targets as quickly as possible. All the children were able to correctly identify the cue–SOA association before start- ing the experimental blocks. Experimental design After concluding the training, each child underwent a total of four experimental blocks, including two temporal cueing and two neutral cueing blocks. Each block lasted approximately four minutes and participants were able to take breaks between the blocks. For all conditions, a total of 40 trials were delivered. The experiment was block-wise randomised. The order of the tasks was counterbalanced between subjects. E-prime 2 software (Psychology Software Tools, Pittsburgh, USA) was used to create and administer the stimuli. Development of Temporal Prediction were considered correct. Moreover, participants showing mean RTs deviating 2SD or more from the group average were excluded. The Kolmogorov-Smirnov test revealed that accuracy scores (percentages of correct responses) were not normally distributed. Hence, non–paramet- ric tests were applied for accuracy analyses. Linear mixed models were conducted to test for between– and within–subject effects on RTs since these were normally distributed. To reliably control for the possible age–related effect on behavioural performance, RT raw data were loga- rithmically transformed. As suggested by Puccioni and Vallesi [45], this allows the reduction of intra– and inter–subject variability by partialling out the age-related effect across conditions. Hence, significant statistical effects resisting logarithmic transformations can be considered as being due to reliable condition–specific effects rather than to the effects of general factors, such as age–related motor response speed [46]. To explore the developmental trajectories of E–TP and I–TP and their combined effect (E/ I–TP) on behavioural performance, a 2 (Cue) × 2 (SOA) × 4 (Group) mixed ANOVA was per- formed. Specifically, the behavioural benefit conferred by E–TP is revealed by the main effect of Cue while its developmental trajectory was measured by the Cue × Group interaction. The behavioural benefit conferred by I–TP is revealed by the main effect of SOA, and its develop- mental trajectory by the SOA × Group interaction. Finally, E/I–TP was tested by the Cue × SOA interaction, and its developmental trajectory by the 3-way Cue × SOA × Group interaction. To evaluate the difference in the magnitude of the single TP effects across groups, planned comparisons (one-way ANOVAs) were performed contrasting RTs on T–Short vs. N– Short; T–Long vs. N–Long; N–Short vs. N–Long; and T–Short vs. T–Long. In addition, Pear- son’s correlations were performed only on the children’s data to assess the relationships between age and RTs for all TP effects (TO–Short, TO–Long, Uncued–FP and Cued–FP). Sequential effects were tested by comparing mean RTs in the current trial (SOAn) to those obtained in the preceding trial (SOAn-1) across groups for both temporal and neutral cueing tasks. More specifically, a 2 (Cue) × 2 (SOAn) × 2 (SOAn-1) × 4 (Group) mixed ANOVA was performed. The overall behavioural benefit of SEs was revealed as the behavioural benefit con- ferred by preceding trials on the current ones (main effect of SOAn-1), and its developmental trajectory by the SOAn-1 × Group interaction. The Cue × SOAn-1 interaction tested SEs in rela- tion to the task (temporal vs. neutral cueing), while its developmental trajectory was revealed by the Cue × SOAn-1 × Group interaction. The SE asymmetry was globally tested by the SOAn × SOAn-1 interaction, and its developmental trajectory by the 3–way SOAn × SOAn-1 × Group interaction. Finally, the SE asymmetry in relation to the task (temporal vs. neutral cueing) was revealed by the 3–way Cue × SOAn × SOAn-1 interaction, and how it is influenced by age by the 4–way Cue × SOAn × SOAn-1 × Group interaction. For all analyses, an alpha level of 0.05 was used. Post-hoc Fisher's least significant difference (LSD) comparisons were performed to evaluate pairwise differences among the means. The effect size was estimated by the partial eta square measure (ηp 2). When appropriate, critical values were adjusted using the Greenhouse– Geisser correction for violation of the assumption of sphericity. The raw response time data from the experiment are available in S1 Dataset. Data analysis As independent variables, age group (6–7 years vs. 8–9 vs. years vs. 10–11 years vs. adults), cue (temporal vs. neutral), and SOA duration (short vs. long) were considered. Mean accuracy and mean RTs were the dependent variables. Omissions, anticipated responses (within the cue and 100 ms after target onset), and delayed responses (1,500 ms after target onset) were considered errors and excluded from analysis. Response times exceeding 2 SD from the individual average for each dependent variable and for each participant were excluded. The remaining responses 6 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction Table 2. Behavioural measures. Mean and standard deviation (in parentheses) measures of accuracy (percentage of correct responses) and speed (reac- tion times) for all groups and conditions. Accuracy (%) Mean RTs (ms) Group T-Short T-Long N-Short N-Long T-Short T-Long N-Short N-Long 6–7 93.4(1.1) 91.5(1.7) 95.7(1) 92.2(1.4) 490(12) 491(12) 562(15) 501(13) 8–9 94.5(1.8) 96.7(0.9) 98.7(2) 96.5(0.9) 433(13) 401(10) 481(12) 422(11) 10–11 95.2(0.9) 96.6(0.9) 98.6(0.5) 96.8(0.7) 390(11) 359(11) 433(13) 379(12) Adults 95.7(1.1) 95.6(1.7) 99(0.5) 94.5(1.8) 313(12) 291(8) 355(10) 306(10) Note that the analyses of variance reported in the text were performed using RT log-transformed data whereas the in the table we report raw RT data for reasons of clarity. d i 10 1371/j l 0135098 t002 Table 2. Behavioural measures. Mean and standard deviation (in parentheses) measures of accuracy (perce tion times) for all groups and conditions. Note that the analyses of variance reported in the text were performed using RT log-transformed data whereas the in the table we report raw RT data for reasons of clarity. Note that the analyses of variance reported in the text were performed using RT log-transformed data whereas th reasons of clarity. doi:10.1371/journal.pone.0135098.t002 Mean RTs for each TP effect and Group are plotted in Fig 3. The 2 × 2 × 4 mixed ANOVA revealed an expected Group effect (F(3,107) = 51.1, p < 0.0001, ηp 2 = 0.59). Post-hoc tests con- firmed that RTs were progressively faster with age (6–7 years > 8–9 years > 10–11 years > adult RTs, all ps  0.04). The ANOVA also revealed the presence of the TO effect (main effect of Cue; F(1, 107) = 92.33, p < 0.0001, ηp 2 = 0.46), with participants being faster in detecting temporally than neutrally cued targets (Fig 3a and 3b), as well as the presence of the FP effect (main effect of SOA; F(1, 107) = 60.89; p < 0.001, ηp 2 = 0.36), with RTs significantly shorter in long than short SOA trials (Fig 3c). The Cue factor did not interact with Group ((F(3, 107) = 0.82; p > 0.95; ηp 2 = 0.02), while a significant two-way Cue × SOA interaction emerged (F(1, 107) = 60.89; p < 0.001; ηp 2 = 0.36). Results Accuracy and RT scores are reported in Table 2. Overall, delayed and null responses decreased as a function of age; however, significant dif- ferences emerged only in the T–Long condition (Kruskal-Wallis test H (3, N = 111) = 13.2, p < 0.01), where the 6 to 7 year-old children showed a significantly lower accuracy level (M = 91.5, SD = 1.34) compared to older children and adults. 7 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction Fig 3. Mean reaction times. Panels show the pair-wise comparison of the mean reaction times for the TO–Short (a), TO–Long (b), Uncued–FP (c) and Cued–FP (d) effects for each age group. Error bars indicate standard errors of the mean. doi:10.1371/journal.pone.0135098.g003 Fig 3. Mean reaction times. Panels show the pair-wise comparison of the mean reaction times for the TO–Short (a), TO–Long (b), Uncued–FP (c) and Cued–FP (d) effects for each age group. Error bars indicate standard errors of the mean. doi:10.1371/journal.pone.0135098.g003 Fig 3. Mean reaction times. Panels show the pair-wise comparison of the mean reaction times for the TO–Short (a), TO–Long (b), Uncued–FP (c) and Cued–FP (d) effects for each age group. Error bars indicate standard errors of the mean. doi:10.1371/journal.pone.0135098.g003 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Post-hoc tests revealed all comparisons to be significant, although the behavioural benefit con- ferred from temporal cues was greater in short (mean TO effect = 51 ms; Cohen’s d = 0.53) than long trials (mean TO effect = 16 ms; Cohen’s d = 0.17) SOAs (p < 0.001). This asymmet- ric effect was explained by the mean RTs in the T-Long trials being significantly faster than the mean RTs in the T-Short trials (t(110) = -5.44; p < 0.001) and in N-Long (t(110) = -4.74; p < 0.001). A significant interaction between Group and SOA was also found (F(3, 107) = 3.93; p < 0.02, ηp 2 = 0.09). Planned comparisons confirmed that, when considering together tempo- ral and neutral cueing conditions, the SOA effect (faster RTs for long than short SOAs) emerged gradually, with the younger children showing a smaller effect (mean SOA effect = 29 ms; Cohen’s d = 0.22) than older children and adults (all mean SOA effects  35 ms; all Cohen’s ds  0.37). Most importantly, we found a significant 3-way Cue × SOA × Group interaction (F(3, 107) = 2.79; p < 0.05, ηp 2 = 0.07; Fig 3d). To better identify the source of this interaction, planned comparisons across age groups (one-way ANOVAs) were performed. These allowed to statisti- cally test for the presence of possible developmental dissociations for each TP effect. One-way ANOVAs revealed no group effects on the TO–Short SOA, the TO–Long SOA or the Uncued– FP effects (all Fs(3, 107) < 1.18; all ps > 0.32). By contrast, the Cued–FP effect was significantly affected by age (F(3, 107) = 4.64; p < 0.005, ηp 2 = 0.14). The post-hoc analysis revealed that 6 to 7 year-old children significantly differed from older groups (all ps  0.02). More specifically, as depicted in Fig 4, the Cued-FP effect was absent (around zero) in the youngest group, but present and stable at later ages. In other words, the combined effect of internally and externally driven TP (E/I–TP) emerged after 8 years of age. 8 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Sequential effects The 4 × 2 × 2 × 2 mixed ANOVA yielded a main SOAn-1 effect (F(1, 107) = 194.31; p < 0.0001, ηp 2 = 0.64), confirming that RTs in the current trials were affected by the preceding ones (SEs). This effect was further influenced by development, as shown by the SOAn-1 × Group interac- tion (F(3, 107) = 2.92; p < 0.05, ηp 2 = 0.07). This interaction was explained by the observation that the advantage provided by the preceding trial on the current one diminished as age increased, being greatest for the youngest group (43 ms on average) and smallest for the oldest one (15 ms on average). We also found a SOAn × SOAn-1 interaction (F(1, 107) = 64.7; p < 0.001, ηp 2 = 0.38). This interaction was explained by the fact that SEs were asymmetric, that is, RTs were overall We also found a SOAn × SOAn-1 interaction (F(1, 107) = 64.7; p < 0.001, ηp 2 = 0.38). This interaction was explained by the fact that SEs were asymmetric, that is, RTs were overall shorter on the current trials when these were preceded by short rather than long SOA trials. The ANOVA further revealed a 3–way Cue × SOAn × SOAn-1 interaction (F(3, 107) = 14.91; shorter on the current trials when these were preceded by short rather than long SOA trials. The ANOVA further revealed a 3–way Cue × SOAn × SOAn-1 interaction (F(3, 107) = 14.91; 9 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction Fig 4. Developmental trajectories of each TP effect. The upper panel shows the developmental trajectories of each single TP effects derived by pair-wise comparisons. In particular, the upper panel shows TO–Short (T–Short minus N–Short) and TO–Long (T–Long minus N–Long) effects, both measures of E–TP. The lower panel displays the developmental trajectories of the Uncued–FP (N–Long minus N–Short) effect as a measure of I–TP and of the Cued–FP (T–Long minus T–Short) effect as a E/I–TP combined measure. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the text were performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. doi:10 1371/journal pone 0135098 g004 Fig 4. Developmental trajectories of each TP effect. The upper panel shows the developmental trajectories of each single TP effects derived by pair-wise comparisons. doi:10.1371/journal.pone.0135098.g004 Development of Temporal Prediction Fig 5. Sequential Effects. Mean RTs are plotted as a function of the current (SOAn; x axis) and preceding (SOAn-1; lines) cue-target intervals for each age group and for Temporal (a) and Neutral (b) cueing Tasks. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the text were performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. Fig 5. Sequential Effects. Mean RTs are plotted as a function of the current (SOAn; x axis) and preceding (SOAn-1; lines) cue-target intervals for each age group and for Temporal (a) and Neutral (b) cueing Tasks. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the text were performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. doi:10.1371/journal.pone.0135098.g005 shown by 6 to 7 year-olds in the temporal cueing task were symmetrical as compared to those seen in the neutral cueing task and older children. Following Vallesi and Shallice [34] statistical approach, to corroborate this observation sub- sequent 2 (Cue) × 2 (SOAn) × 2 (SOAn-1) repeated-measures ANOVAs were performed sepa- rately on each group, with SOAn and SOAn-1 as the within-subjects variables. It emerged that the Cue × SOAn × SOAn-1 interaction was significant only in the 6 to 7 year–old group (F(1, 29) = 4.72, p < 0.05) but not in the older ones (F(1, 33) = 2.26 for 8–9 year olds; F(1, 23) = 3.63 for 10–11 year olds; F(1, 22) = 3.44 for adults). Post-hoc tests conducted on the 6–7 year-old group confirmed that, in the temporal cueing task RTs in the current trials were equally affected when preceded by short or long SOAn-1 intervals (both p < 0.01). Whereas, in the neu- tral cueing task the SE was present in short SOAn (p < 0.001) but not in long SOAn trials (p > 0.07). In other words, they showed symmetrical SEs. By contrast, in the neutral cueing task SEs were present only in short SOAn trials, with all participants being faster when these were preceded by short rather than long SOAn-1 trials (symmetrical SEs). Sequential effects In particular, the upper panel shows TO–Short (T–Short minus N–Short) and TO–Long (T–Long minus N–Long) effects, both measures of E–TP. The lower panel displays the developmental trajectories of the Uncued–FP (N–Long minus N–Short) effect as a measure of I–TP and of the Cued–FP (T–Long minus T–Short) effect as a E/I–TP combined measure. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the text were performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. doi:10.1371/journal.pone.0135098.g004 p < 0.001, ηp 2 = 0.17). This interaction revealed that, in the temporal cueing condition, the SEs were less asymmetric than in the neutral cueing condition. The 4–way Cue × SOAn × SOAn-1 × Group interaction was not significant (F(3, 107) = 0.13; p > 0.94, ηp 2 = 0.01). It should be noted that a lack of significant interaction between SE asym- metry and age group was previously reported by Vallesi and Shallice [34], who tested 4 to 12 year-old children with a variable FP paradigm. As discussed by these authors, this null interac- tion effect may be accounted for by the high intra–and inter–subject variability of RTs in chil- dren that could be only reduced, but not completely eliminated, by log-transformation [46]. Although the 4-way interaction was not significant, the visual inspection of the developmental trajectory of each TP effect plotted separately for cueing conditions (Fig 5) suggests that SEs PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 10 / 18 Discussion The main aim of the present study was to investigate the developmental trajectories of different types of temporal prediction (TP) mechanisms in typically developing school-aged children. To our knowledge, this study is the first purposely designed to delineate the developmental trends of internal (i.e., the information provided by the passage of time itself) vs. external (i.e., the temporal information provided by discrete cues) TP mechanisms in such population. Over- all, the results demonstrate that all children participating at the study (from six to eleven years) were able to implement internally (E–TP) and externally (I–TP) driven TP mechanisms, show- ing an efficient ability to orient attention in time and, consequently, to optimise their beha- vioural performance. The ability to implement E–TP was demonstrated by the observation that children benefitted from the information provided by external discrete temporal cues to improve their performance in terms of target detection speed (i.e., TO effect). Furthermore, findings highlighted two noteworthy issues. First, the TO effect did not interact with age, fol- lowing a stable developmental trajectory. Indeed, while RTs showed a linear shortening with increasing age, the RT difference between temporally vs. neutrally cued targets remained nearly constant across age. Second, the presence of this effect was independent from the SOA dura- tion, although its magnitude was larger for short than long SOAs. In other words, all partici- pants exhibited faster RTs in temporal cueing as compared to neutral cueing blocks for both short and long SOA durations, suggesting that the cognitive mechanisms underlying E-TP gen- erated by discrete cues are fully established as early as six years of age and do not show signifi- cant changes over time. A recent study by Johnson and colleagues [43] employed a cueing paradigm to test the mechanisms underlying the ability to orient attention in space and time in children. Interest- ingly, they found that children can voluntarily and automatically orient attention in space, as revealed by the presence of both cueing and sequential effects on children’s behavioural perfor- mance. However, in contrast to our findings, they found only automatic, but not voluntary, temporal orienting in children. That is, children did not benefit from the temporal information provided by symbolic cues (TO effect) although their performance was affected by the condi- tional probability of target occurrence in time (FP effect) as well as the sequential temporal structure of trials (SEs). Correlational analyses The correlational analyses conducted between the age and each single TP effect revealed that the only index showing a significant correlation with age was the Cued–FP effect (r = 0.43, 95% BCa CI [.27, .59], Bonferroni adjusted-p < 0.02), while the other effects were not affected by age (all rs < 0.17; all ps > 0.07). That is, while the behavioural benefit conveyed separately by E–TP and I–TP was developmentally stable, the effect conferred by their combined use increased significantly with age. 11 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction event-related design including both temporal and neutral cues in the same block, children failed to show TO effect, even if the target was centrally presented. We suggest that memory demands associated with maintaining, retrieving and updating the cue meaning from trial to trial, as well as the necessity to switch from a predictive to a non predictive context, may have selectively impaired temporal, but not spatial, voluntary orienting. As suggested by the authors, it should be taken into account that using cues to orient attention in time may be conceptually more demanding than using spatial cues. Finally, we cannot completely exclude the possibility that the different kinds of stimuli employed in the two studies may have further affected the possibility of eliciting the TO effect in children. Indeed, in our study, we used child-friendly stimuli (i.e., coloured circles and pic- tures of animals for the cue and the target, respectively) while Johnson and colleagues pre- sented children with symbolic stimuli representing different parts of a clock-face for the cue and the ‘+’ or ‘x’ signs for the target stimuli, which may be more difficult to discriminate. Most importantly, Johnson and colleagues tested a single group of six to sixteen year-old children, whereas in our study children were younger than eleven years. From eleven to sixteen years, neurodevelopmental outcomes substantially change. In summary, in the light of the above con- siderations it is difficult to draw reliable conclusions from the comparisons of the two studies. A second finding of our study was that children were able to use internal information (i.e., the passage of time itself) to predict target occurrence and consequently to orient attention in time (I–TP). According to our experimental paradigm, the I–TP could be measured as the ben- efit in RT speeding up, conferred by long vs. short SOAs in the context of either the temporal (Cued–FP) or the neutral (Uncued–FP) cueing task. All participants showed a global shorten- ing of RTs as well as a reduction of E–TP effect in long SOAs due to the variable FP effect [13– 17]. The FP effect is generally explained by the hazard function [6,16,26], which is defined as the conditional probability of an event occurring at a given time, given that it has not occurred yet. Discussion There are several reasons possibly accounting for the lack of consis- tence between the results of Johnson and colleagues and those we report in the present study. Firstly, as discussed by the authors, the target presentation in their paradigm was lateralised rather than central; this experimental choice may have disrupted the cueing effect in children. Indeed, it has been shown that the benefit of temporal cues is enhanced when the target posi- tion is fixed and therefore spatially predictable [47]. Hence, it is possible that in our study the central presentation of target stimuli and the consequent fixed spatial focus of attention made it easier for children to benefit from temporal cues. A second possible explanation may rely on the difference in the degree of temporal certainty experienced by children in the two studies. While Johnson and colleagues manipulated the cue validity (i.e., 80% of the cues provided valid, and 20% invalid, information), we kept the validity of the cue constant to 100%. It follows that, in order to optimise behaviour, in our study children could benefit both from the tempo- ral cue and from the maximum temporal certainty that the target would have appeared always at the time predicted by the cue. By contrast, the invalid conditions used by Johnson and col- leagues may have introduced an additional difficulty given that, in such a case, children could not completely trust the temporal information provided by the cue. It is also possible that the mixing of temporal and neutral cueing conditions within the same experimental block in John- son et al.’s study may have made the task too challenging for children (especially for the youn- ger ones). In line with this, in a pilot study (unpublished data), we found that when using an PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 12 / 18 As suggested by Vallesi and Shallice [34], the lack of significant interaction between age, task and the emerging of the typical SE asymmetry can be accounted for by the high inter– and intra–subject variability, that can be only reduced, but not completely eliminated, after applying a logarithmic transformation on raw RTs [46]. Although the null 4-way interaction represents a limitation of the present study, we nevertheless tried to provide further statistical evidence in support of the observed data. To this aim, we conducted additional 3-way ANOVAs separately for the temporal and neutral cue- ing task. These confirmed that, in the youngest children, the SE pattern was symmetrical only in the temporal cueing task, given that their behavioural performance in SOAn trials was equally affected irrespective of the duration of the preceding SOAn-1 trials. Remarkably, in the neutral cueing task all groups, including the youngest one, exhibited the typical asymmetric pattern described in the literature, with RTs in the current short, but not long, trials being affected by SOAn-1 durations. The apparent presence of a developmental dissociation including either the Cued–FP effect and the symmetrical SEs demonstrates that, while from six years of age onwards children can successfully and independently make use of external and internal TP mechanisms to orient attention in time, the ability to combine these two different sources of information emerges only from eight years of age. A possible explanation for this is that, before eight years of age, their limited available cognitive resources do not allow children to make a combined use of external and internal strategies. From a theoretical perspective, according to the dual process view [18] a controlled monitoring of the change of conditional probability would explain both the FP effect and the asymmetry of SEs. In the first case, the subjective target predictability will be highest in the longest SOAs due to the hazard function. In the second case, in any trial the participant implicitly expects a repetition of the previous SOA, hence, the maximum prepared- ness is biased toward the same SOA as that of the previous trial. If SOAn is shorter than SOAn- 1, then participants will be not fully prepared when the imperative stimulus occurs, resulting in relatively slower RTs. In other words, the boost of expectation conditionally induced from the passage of time heightened the probability of target occurrence, inducing faster responses in long cue–target intervals. Similarly to TO, in the neutral cueing task the Uncued–FP effect did not show any interaction with age, suggesting that the mechanisms underlying I–TP are already developed and functioning at six years of age. This evidence is in line with most of previous studies report- ing FP effects in children [32–34,43, 48–49]. As a third important finding, we found that, although even young children were able to independently use internal and external mechanisms to generate TP, and consequently to ori- ent their attention in time, their ability to combine these two sources of information emerged only after eight years of age. According to our experimental design, the Cued–FP effect mea- sures the combined use of external and internal TP processes. Namely, in the temporal cueing blocks, the occurrence of targets is highly predictable based on the full validity of the cue (TO effect) as well as on the hazard function that conditionally biases target predictability (FP effect). Hence, the combined effect of both these sources of information on target occurrence leads to the highest level of target expectancy, and therefore to the lowest RTs in T–Long trials. The evidence that in the temporal cueing blocks the FP effect (Cued–FP effect) emerged only after eight years of age demonstrates that the ability to make a combined use of E–TP and I–TP is developmentally constrained. We found a developmental dissociation also in SEs; in all age groups the behavioural perfor- mance in the current trials (SOAn) was overall affected by the duration of the preceding ones (SOAn-1). Yet, the magnitude of this effect depended on the SOAn duration; that is, the SEs were greater in short rather than long SOAn, confirming the typical asymmetrical pattern reported in the literature [17,18,26]. Critically, as suggested by the visual inspection of mean RTs (Fig 5), the SE asymmetrical pattern seemed to be further affected by the interplay between 13 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 Development of Temporal Prediction age group and task. Namely, in the temporal cueing task, the behavioural advantage conferred by the preceding trials on the current ones appeared to be comparable for short and long SOAn (i.e., SE symmetrical pattern) only in the six to seven-year old children, while this difference became biased toward the short SOAn (i.e., asymmetrical SE pattern) at later ages. By contrast, in the neutral cueing task, the magnitude of the SE asymmetry appeared to be constant across all groups. Yet, we did not find an ominibus statistical validation of this effect, since the ANOVA did not yield a significant 4-way interaction. As suggested by Vallesi and Shallice [34], the lack of significant interaction between age, task and the emerging of the typical SE asymmetry can be accounted for by the high inter– and intra–subject variability, that can be only reduced, but not completely eliminated, after applying a logarithmic transformation on raw RTs [46]. Although the null 4-way interaction represents a limitation of the present study, we nevertheless tried to provide further statistical evidence in support of the observed data. To this aim, we conducted additional 3-way ANOVAs separately for the temporal and neutral cue- ing task. These confirmed that, in the youngest children, the SE pattern was symmetrical only in the temporal cueing task, given that their behavioural performance in SOAn trials was equally affected irrespective of the duration of the preceding SOAn-1 trials. Remarkably, in the neutral cueing task all groups, including the youngest one, exhibited the typical asymmetric pattern described in the literature, with RTs in the current short, but not long, trials being affected by SOAn-1 durations. age group and task. Namely, in the temporal cueing task, the behavioural advantage conferred by the preceding trials on the current ones appeared to be comparable for short and long SOAn (i.e., SE symmetrical pattern) only in the six to seven-year old children, while this difference became biased toward the short SOAn (i.e., asymmetrical SE pattern) at later ages. By contrast, in the neutral cueing task, the magnitude of the SE asymmetry appeared to be constant across all groups. Yet, we did not find an ominibus statistical validation of this effect, since the ANOVA did not yield a significant 4-way interaction. However, when SOAn is longer than SOAn-1, it is assumed that partici- pants can update and re-orient their attention over time and consequently re-prepare for opti- mal preparation, resulting in SE abolishment for long intervals. Within this theoretical framework, we speculated that, for young children, it may be difficult to efficiently monitor the time–based conditional probability of target onset while concurrently using the temporal infor- mation provided by the cues, which implies that the Cue–SOA associations has to be learnt, maintained and retrieved trial-by-trial. This hypothesis is corroborated by the observation that only the SE asymmetry, but not the presence of SEs per se (which is assumed to be more auto- matic by the ‘dual process’ view), was disrupted in young children. In other words, the effort made in using external, discrete cues to generate predictions over time may have disrupted the monitoring of the conditional probability of target occurrence in young children. As an alternative explanation, we propose that six to seven year-old children may not yet have developed the capacity to disengage attention from externally to internally driven information when the two have to be simultaneously processed, requiring considerable effort to re-orient atten- tion over time in long SOAn trials. In fact, younger children can effortlessly disengage and re-ori- ent attention in the current trials (FP effect) as well as from the duration of the preceding trial (SE asymmetry) when they only need to use I-TP. However, when they must make a combined use of PLOS ONE | DOI:10.1371/journal.pone.0135098 August 11, 2015 14 / 18 Development of Temporal Prediction I–TP and E–TP, the attentional disengagement may result more effortful for them, as revealed by the absence of both the Cued–FP effect and by the SE asymmetry only in temporal cueing blocks. In support of this hypothesis, we also found that the Cued–FP effect, which is assumed to depend on attentional resources more than other TP measures, is the only index showing a positive corre- lation with age. That is, the older the child, the stronger the Cued–FP. A further, simpler hypothesis explaining why younger children did not show FP effect and SE asymmetry in the temporal cueing task, is also that at this age children invest considerable atten- tional resources on externally available cues, given that this is the first useful information always providing valid target predictability. Acknowledgments The authors desire to thank all children and their families who participated to the study. A thank goes to Federica De Osti for helping in collecting data as well as to Dr. Paola Bortoletto and to all the staff of the ‘Istituto Comprensivo di Spresiano’, Treviso (Italy) for making possi- ble this study. We would also like to thank Dr. Prematurata for inspiring the present study. Supporting Information S1 Dataset. Mean accuracy and response time for each participant. (XLSX) In other words, they can immediately and completely use the cues to speed up their RTs to targets. By contrast, in the case of I–TP, children are only required to perceive the elapsing of time, given that the cues do not provide useful information to generate predictions. Then, they have to use the internal information to conditionally prepare for the next time in which targets are likely to occur. Even if young children can effortlessly build up a mental representation of durations, as indirectly shown by the presence of Uncued–FP effect, this operation could be more demanding for them when it must be combined with the processing of information externally provided by discrete cues, which do not require per se a dynamic and metric temporal representation. Though, further studies are needed to better disentangle the above different accounts. A possible way to better unravel the developmental mechanisms under- lying dissociations in TP effects may come from neurophysiological studies depicting the timing of the neural mechanisms underlying I–TP and E–TP across development. Although our present data do not allow neurophysiological hypotheses to be tested directly, a possible hypothesis to be addressed may be that while the single cortical networks underlying E–TP and I–TP are already established at six years of age, they cannot be simultaneously recruited before the age of eight. It may be also possible that their functional connectivity may not be yet developed until this time. In support of this view, structural neuroimaging studies using diffusion tensor imaging (DTI) demonstrated a developmental increase in grey matter activation in frontal and parietal cortices which would be in turn the direct consequence of the maturation of white matter connection [50]. More specifically, the inferior parietal cortices, which have been shown to play a key role in temporal orienting [3,4,23,51–54] and the lateral pre-frontal cortices, which are implied in the monitoring of the hazard function [5,6,18,21] are among the last areas to myelinate, a change that would result in more stable fronto-parietal activity. Although the neuroimaging approach promises to provide a useful key for a deeper understanding of the developmental mechanisms underlying TP phenomena in children, much work has yet to be done in this field. References 1. Correa A. 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Complementing the US Food and Drug Administration Adverse Event Reporting System With Adverse Drug Reaction Reporting From Social Media: Comparative Analysis (Preprint)
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Abstract Background: Adverse drug reactions (ADRs) can occur any time someone uses a medication. ADRs are systematically tracked and cataloged, with varying degrees of success, in order to better understand their etiology and develop methods of prevention. The US Food and Drug Administration (FDA) has developed the FDA Adverse Event Reporting System (FAERS) for this purpose. FAERS collects information from myriad sources, but the primary reporters have traditionally been medical professionals and pharmacovigilance data from manufacturers. Recent studies suggest that information shared publicly on social media platforms related to medication use could be of benefit in complementing FAERS data in order to have a richer picture of how medications are actually being used and the experiences people are having across large populations. Objective: The aim of this study is to validate the accuracy and precision of social media methodology and conduct evaluations of Twitter ADR reporting for commonly used pharmaceutical agents. Methods: ADR data from the 10 most prescribed medications according to pharmacy claims data were collected from both FAERS and Twitter. In order to obtain data from FAERS, the SafeRx database, a curated collection of FAERS data, was used to collect data from March 1, 2016, to March 31, 2017. Twitter data were manually scraped during the same time period to extract similar data using an algorithm designed to minimize noise and false signals in social media data. Results: A total of 40,539 FAERS ADR reports were obtained via SafeRx and more than 40,000 tweets containing the drug names were obtained from Twitter’s Advanced Search engine. While the FAERS data were specific to ADRs, the Twitter data were more limited. Only hydrocodone/acetaminophen, prednisone, amoxicillin, gabapentin, and metformin had a sufficient volume of ADR content for review and comparison. For metformin, diarrhea was the side effect that resulted in no difference between the two platforms (P=.30). For hydrocodone/acetaminophen, ineffectiveness as an ADR that resulted in no difference (P=.60). For gabapentin, there were no differences in terms of the ADRs ineffectiveness and fatigue (P=.15 and P=.67, respectively). For amoxicillin, hypersensitivity, nausea, and rash shared similar profiles between platforms (P=.35, P=.05, and P=.31, respectively). Conclusions: FAERS and Twitter shared similarities in types of data reported and a few unique items to each data set as well. Complementing the US Food and Drug Administration Adverse Event Reporting System With Adverse Drug Reaction Reporting From Social Media: Comparative Analysis Complementing the US Food and Drug Administration Adverse Event Reporting System With Adverse Drug Reaction Reporting From Social Media: Comparative Analysis Zeyun Zhou*, PharmD, MPH; Kyle Emerson Hultgren*, PharmD College of Pharmacy, Purdue University, West Lafayette, IN, United States *all authors contributed equally Corresponding Author: Kyle Emerson Hultgren, PharmD College of Pharmacy Purdue University 575 W Stadium Ave West Lafayette, IN United States Phone: 1 765 494 5820 Email: khultgre@purdue.edu Corresponding Author: Kyle Emerson Hultgren, PharmD College of Pharmacy Purdue University 575 W Stadium Ave West Lafayette, IN United States Phone: 1 765 494 5820 Email: khultgre@purdue.edu Abstract The use of Twitter as an ADR pharmacovigilance platform should continue to be studied as a unique and complementary source of information rather than a validation tool of existing ADR databases. (JMIR Public Health Surveill 2020;6(3):e19266) doi: 10.2196/19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 1 (page number not for citation purposes) JMIR PUBLIC HEALTH AND SURVEILLANCE JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren Original Paper KEYWORDS adverse drug reactions; FAERS; social media reporting; pharmacovigilance https://publichealth.jmir.org/2020/3/e19266 XSL•FO RenderX XSL•FO RenderX JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren social media and FAERS [10,11]. These studies have shed light on the role of social media in ADR reporting. However, many studies only examined one or two less commonly used pharmaceutical agents, and some included more than 1000 drugs. While the inclusion tested a general scheme of social media reporting, it overlooked the role of social media reporting for common drugs. Background Adverse drug reactions (ADRs) are the unintended effect of medicine at doses used for prophylaxis, diagnosis, or treatment [1]. ADRs can occur anytime when a patient takes a medication. Factors including drug and food interactions, medication errors, allergies, and metabolism contribute to the occurrence of ADRs. ADRs have been identified as one of the leading causes of death in the United States. ADRs resulted in more deaths than the pulmonary diseases, diabetes, HIV/AIDS, and pneumonia [2,3]. A systematic review on ADR-induced hospital admissions found that 5.3% of hospital admissions were associated with ADRs [4]. New drug therapies, the aging population, and polypharmacy expose the population to increased risks of ADRs [5]. The burden of ADRs necessitates appropriate detection and assessment, and reporting is fundamental to successful pharmacovigilance systems. The Center for Medication Safety Advancement (CMSA) at Purdue University College of Pharmacy aims to adopt previous research strategies and compare ADR reports in social media and FAERS. Twitter was selected as the social media for evaluation thanks to its simplicity and timeliness in information sharing and access. Twitter users can report an ADR in one tweet pursuant to the FDA guideline, which requires as a minimum dataset to constitute a viable report an identifiable patient, an identifiable reporter, a product exposure, and an adverse event [12]. Additionally, the FDA does not require reports to demonstrate causation or to be specific regarding the type of error. All suspected medication errors, ADRs, or adverse events are accepted as reports. Given the advantage of the Twitter database, the objective of this study is to validate the accuracy and precision of the research methodology and conduct evaluations of social media ADR reporting via tweets for commonly used pharmaceutical agents. The US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) is a database for reports of adverse events, medication errors, and product quality complaints [6]. Although FAERS serves as a valuable data source for postmarket pharmacovigilance, only drug manufacturers are required to send reports received from health care professionals and consumers to the FDA. Health care professionals and consumers may voluntarily submit reports, which may lead to incomplete data in FAERS. In order to obtain more comprehensive information on drug products, multiple data sources should be used to fill the information gap. Ethics Statement All social media data used in data collection and analysis were extracted from public sources. Example tweets were paraphrased and edited to prevent unmasking through a reverse search on Twitter. FAERS reports on SafeRx were also anonymized. As data used in this study were publicly available, no institutional review board approval was sought. Social media has been proposed as a potential data source as it allows an easily accessible information sharing platform with almost no chronological and geographical constraints. A systematic review of 51 studies compared ADR reports on social media and other pharmacovigilance systems, and the review noted that the prevalence of all ADR reports ranged from 0.2% to 8% and social media contained more reports of mild ADRs than severe ADRs [7]. Previous studies showed that ADRs were underrepresented in clinical trial data, and less severe ADRs were more frequently reported on social media. Social media ADR reports reflected the ADRs reported on FAERS on average 11 months earlier [8,9]. Comparative studies suggested the practicality of using social media as a complementary resource and demonstrated a moderate agreement on ADR data between JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 2 (page number not for citation purposes) Overview This study was divided into 3 sections: drug selection, FAERS data collection, and Twitter data collection. Collecting FAERS data included searching for ADR reports of a pharmaceutical agent and calculating relative frequencies of the 5 most frequently reported ADRs, whereas Twitter data collection required an additional step to identify relevant tweets according to inclusion and exclusion criteria. Figure 1 demonstrates the overall scheme for the methodology of this study. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 2 (page number not for citation purposes) https://publichealth.jmir.org/2020/3/e19266 XSL•FO RenderX Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE cheme. ADR: adverse drug reaction; FAERS: FDA Adverse Event Reporting System. Figure 1. Methodology scheme. ADR: adverse drug reaction; FAERS: FDA Adverse Event Reporting System. brand names [15,16]. Accordingly, this study included common brand names in the searching criteria as Twitter users could be discussing ADRs using common brand names. For the data collection purpose of this study, the most commonly used brand name for each selected drug was identified according to Micromedex: Norco for hydrocodone/acetaminophen, Synthroid for levothyroxine, Deltasone for prednisone, Prinivil for lisinopril, Amoxil for amoxicillin, Neurontin for gabapentin, Glucophage for metformin, Lipitor for atorvastatin, Xanax for alprazolam, and Norvasc for amlodipine. brand names [15,16]. Accordingly, this study included common brand names in the searching criteria as Twitter users could be discussing ADRs using common brand names. For the data collection purpose of this study, the most commonly used brand name for each selected drug was identified according to Micromedex: Norco for hydrocodone/acetaminophen, Synthroid for levothyroxine, Deltasone for prednisone, Prinivil for lisinopril, Amoxil for amoxicillin, Neurontin for gabapentin, Glucophage for metformin, Lipitor for atorvastatin, Xanax for alprazolam, and Norvasc for amlodipine. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 3 (page number not for citation purposes) Pharmaceutical Agents To identify the 10 most popular prescribed medications, prescription data were used from GoodRx, a health care company that operates a telemedicine platform. GoodRx generates a list of the top 10 drugs from monthly claims submitted by pharmacies in the United States; in November 2017, those drugs were hydrocodone/acetaminophen, levothyroxine, prednisone, lisinopril, amoxicillin, gabapentin, metformin, atorvastatin, alprazolam, and amlodipine [13]. Previous studies included both brand and generic names in data collection to expand the data that could be obtained [10,14]. Some studies further suggested that patients tended to use the most common brand name in social media if a drug had multiple XSL•FO RenderX JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren Statistical Analysis The analysis of ADR data from SafeRx and Twitter included the following components: calculation of relative frequencies, examination of ADR distribution, and test for association and independence. A chi-square test was used to statistically quantify the difference in ADRs between the FAERS data and Twitter data. It was appropriate to use the chi-square test as no cell in the cross-tabulation contained an expected value of 5 or below. The sample size required to achieve an a priori α<.01 was 96, and samples from both sources exceeded the threshold. The null hypothesis (H0) was “there is no significant difference between FAERS data and Twitter data on common ADRs.” The failure aADR: adverse drug reaction. to reject H0 would signify that Twitter data were similar to and independent from the FAERS data. The statistical analysis in this study was conducted using SAS version 9.4 (SAS Institute Inc). US Food and Drug Administration Adverse Event Reporting System Data hydrocodone/acetaminophen, prednisone, levothyroxine could be prescribed to individuals from all age groups regardless of gender and geographic areas, Twitter’s population represented a robust data source for generalizability. Purdue University College of Pharmacy’s CMSA designed and maintained a searchable database for all published FAERS reports since 2012 under SafeRx. SafeRx enables large-scale studies to improve prescription medication safety as the database contains a collection of 4,935,048 ADRs, representing 294,652 different drugs from the fourth quarter of 2012 through December 2016. ADR reports were obtained via the FAERS Data Explore function in SafeRx. The search criteria were set to display data from March 1, 2016, to March 31, 2017, and the data included both brand and generic names of selected drugs as the primary suspect and the secondary suspect drug. After obtaining all ADR reports from SafeRx, the 5 most reported ADRs for each selected drug were recorded for data analysis. Tweets were obtained from the Advanced Search webpage on Twitter’s website [18]. Both generic and brand names of the selected medication were entered as keywords into the “any of these words” field in the Advanced Search engine. To exclude tweets containing advertisements, hyperlinks to external webpages, and retweets, characters including “rt” for retweets, “http,” and “.com” were entered into the “none of these words” field. By eliminating tweets from pharmaceutical companies, health care marketers, and agencies, Twitter data became more comparative to the FAERS data. Table 1 describes additional exclusion criteria in the selection of tweets. The “written in” field was set so that only tweets in the English language would be displayed. The time frame was chosen to be from March 2016 to March 2017 in order to correspond with the FAERS data obtained from CMSA’s SafeRx database. All tweets displayed were subsequently reviewed to include only those that described ADRs after consuming the medication. Those tweets served as the final source for data recording, which included the username, offending medication, content of the tweet, and types of ADRs. At the time of data collection, the number of tweets was benchmarked at 100 for analysis. Twitter Data Searchability and generalizability were the main factors in selecting Twitter as the social media platform. Twitter’s search engine enabled keyword-based searching within a predetermined time frame, and all public tweets containing the keyword could be displayed. According to the Pew Research Center, Twitter users were diverse in terms of age distribution and well balanced in terms of gender and geographic areas at the time of study in 2016 [17]. As medications including Table 1. Additional exclusion criteria in the collection of tweets. aADR: adverse drug reaction. JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 4 (page number not for citation purposes) JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 5 (page number not for citation purposes) JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 5 https://publichealth.jmir.org/2020/3/e19266 (page number not for citation purposes) L•FO US Food and Drug Administration Adverse Event Reporting System Data Result A total of 40,539 FAERS ADR reports from March 1, 2016, to March 31, 2017, were obtained via SafeRx. Table 2 summarizes the 5 most reported ADRs for each of the 10 drugs. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 4 (page number not for citation purposes) XSL•FO RenderX Zhou & Hultgren Drug and the top 5 adverse drug reactions Xanax resulted in 35 relevant tweets, lisinopril and Prinivil were found in 33 relevant tweets, and only 3 relevant tweets were found for atorvastatin and Lipitor. No relevant tweets were found for keywords amlodipine and Norvasc. Due to the insufficiency of relevant tweets to meet the benchmark, the final Twitter data analysis did not include levothyroxine, alprazolam, lisinopril, atorvastatin, and amlodipine. Table 3 presents the ADRs reported for the remaining 5 drugs. JMIR PUBLIC HEALTH AND SURVEILLANCE JMIR PUBLIC HEALTH AND SURVEILLANCE Table 2. Five most frequently reported FDA Adverse Event Reporting System adverse drug reactions from March 1, 2016, to March 31, 2017, for ea selected drug on SafeRx. n (%) Drug and the top 5 adverse drug reactions Hydrocodone/acetaminophen (Norco, n=1765) 429 (24.31) Ineffectiveness 371 (21.02) Nausea 353 (20.00) Fatigue 345 (19.55) Pain 267 (15.13) Headache Levothyroxine (Synthroid, n=3728) 881 (23.63) Fatigue 828 (22.21) Ineffectiveness 733 (19.66) Nausea 664 (17.81) Headache 622 (16.68) Diarrhea Prednisone (Deltasone, n=5689) 1423 (25.01) Ineffectiveness 1332 (23.41) Fatigue 1067 (18.76) Dyspnea 976 (17.16) Nausea 900 (15.82) Diarrhea Lisinopril (Prinivil, n=5386) 1243 (23.08) Ineffectiveness 1172 (21.76) Fatigue 1136 (21.09) Diarrhea 1062 (19.72) Nausea 773 (14.35) Dyspnea Amoxicillin (Amoxil, n=797) 328 (41.15) Hypersensitivity 126 (15.81) Fatigue 123 (15.43) Diarrhea 121 (15.18) Nausea 99 (12.42) Rash Gabapentin (Neurontin, n=5734) 1637 (28.55) Ineffectiveness 1220 (21.28) Fatigue 997 (17.40) Nausea 966 (16.85) Pain 914 (15.94) Diarrhea Metformin (Glucophage, n=5109) 1311 (25.66) Hyperglycemia 1111 (21 75) N Zhou & Hultgre JMIR PUBLIC HEALTH AND SURVEILLANCE n (%) Ineffectiveness Nausea Fatigue Pain Headache Levothyroxine (Synthroid, n=3728) Fatigue Ineffectiveness Nausea Headache Diarrhea Prednisone (Deltasone, n=5689) Ineffectiveness Fatigue Dyspnea Nausea Diarrhea Lisinopril (Prinivil, n=5386) Ineffectiveness Fatigue Diarrhea Nausea Dyspnea Amoxicillin (Amoxil, n=797) Hypersensitivity Fatigue Diarrhea Nausea Rash Gabapentin (Neurontin, n=5734) Ineffectiveness Fatigue Nausea Pain Diarrhea Metformin (Glucophage, n=5109) Hyperglycemia Nausea Ineffectiveness Diarrhea https://publichealth.jmir.org/2020/3/e19266 n (%) Drug and the top 5 adverse drug reactions 795 (15.56) Fatigue Atorvastatin (Lipitor, n=6588) 4601 (69.84) Type 2 diabetes 586 (8.89) Hypersensitivity 537 (8.15) Fatigue 445 (6.75) Ineffectiveness 419 (6.36) Nausea Alprazolam (Xanax, n=2551) 561 (21.99) Ineffectiveness 548 (21.48) Fatigue 547 (21.44) Nausea 451 (17.68) Anxiety 444 (17.40) Headache Amlodipine (Norvasc, n=3192) 696 (21.80) Diarrhea 682 (21.37) Fatigue 636 (19.92) Ineffectiveness 611 (19.14) Nausea 567 (17.76) Dyspnea Twitter Data Result More than 40,000 tweets containing the drug names as keywords from March 1, 2016, to March 31, 2017, were obtained from Twitter’s Advanced Search engine. Although searching on Twitter yielded an overall large quantity of tweets, ADRs of some drugs were simply not mentioned in enough tweets. Within the study period, searching keywords levothyroxine and Synthroid yielded 50 relevant tweets, keywords alprazolam and Xanax resulted in 35 relevant tweets, lisinopril and Prinivil were found in 33 relevant tweets, and only 3 relevant tweets were found for atorvastatin and Lipitor. No relevant tweets were found for keywords amlodipine and Norvasc. JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 6 (page number not for citation purposes) JMIR PUBLIC HEALTH AND SURVEILLANCE Due to the insufficiency of relevant tweets to meet the benchmark, the final Twitter data analysis did not include levothyroxine, alprazolam, lisinopril, atorvastatin, and amlodipine. Table 3 presents the ADRs reported for the remaining 5 drugs. Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE n (%) Drug and the top 5 adverse drug reactions 795 (15.56) Fatigue Atorvastatin (Lipitor, n=6588) 4601 (69.84) Type 2 diabetes 586 (8.89) Hypersensitivity 537 (8.15) Fatigue 445 (6.75) Ineffectiveness 419 (6.36) Nausea Alprazolam (Xanax, n=2551) 561 (21.99) Ineffectiveness 548 (21.48) Fatigue 547 (21.44) Nausea 451 (17.68) Anxiety 444 (17.40) Headache Amlodipine (Norvasc, n=3192) 696 (21.80) Diarrhea 682 (21.37) Fatigue 636 (19.92) Ineffectiveness 611 (19.14) Nausea 567 (17.76) Dyspnea Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 7 (page number not for citation purposes) Twitter Data Result More than 40,000 tweets containing the drug names as keywords from March 1, 2016, to March 31, 2017, were obtained from Twitter’s Advanced Search engine. Although searching on Twitter yielded an overall large quantity of tweets, ADRs of some drugs were simply not mentioned in enough tweets. Within the study period, searching keywords levothyroxine and Synthroid yielded 50 relevant tweets, keywords alprazolam and https://publichealth.jmir.org/2020/3/e19266 XSL•FO RenderX Table 3. Reported adverse drug reactions on Twitter from March 1, 2016, to March 31, 2017, for 5 drugs. Value % Drugs and adverse drug reactions Hydrocodone/acetaminophen 36 Fatigue 22 Ineffectiveness 10 Pruritus 9 Nausea 5 Mood changes 3 Vivid dreams 3 Insomnia 2 Headache 2 Constipation 2 Dizziness 1 Chest tightness 1 Delusion 1 Hallucination 1 Singultus 1 Inattention 1 Short-term amnesia 1 Sweating 1 Vomiting Prednisone 25 Insomnia 23 Increased appetite 10 Mood changes 8 Moon face 8 Weight gain 5 Fatigue 4 Muscle weakness 3 Jitteriness 2 Diaphoresis 2 Tachycardia 2 Anxiety 1 Bradycardia 1 Cataracts 1 Xerostomia 1 Dyspnea 1 Heartburn Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Table 3. Reported adverse drug reactions on Twitter from March 1, 2016, to March 31, 2017, for 5 drugs. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 8 (page number not for citation purposes) JMIR PUBLIC HEALTH AND SURVEILLANCE Value % Drugs and adverse drug reactions 16 Rash 15 Ineffectiveness 8 Nausea 5 Diarrhea 3 Fatigue 3 Pruritus 3 Vomiting 1 Stomachache Gabapentin 31 Drowsiness 24 Fatigue 23 Ineffectiveness 8 Weight gain 5 Dizziness 2 Nausea 1 Blurred vision 1 Dysphasia 1 Confusion 1 Headache 1 Jitteriness 1 Mood changes 1 Vivid dreams Metformin 57 Nausea 22 Diarrhea 5 Ineffectiveness 3 Fatigue 3 Renal dysfunction 2 Bloating 2 Headache 1 Hypersensitivity 1 Heartburn 1 Hypoglycemia 1 Mood changes 1 Vomiting Drug and Adverse Drug Reaction Matching The process was completed through consolidating the ADRs reported in the Twitter dataset to match the top 5 ADRs from frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile with that of FAERS, one should fail to reject H0 according to th P l f th hi t t T bl 4 h t h d Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Value % Drugs and adverse drug reactions 16 Rash 15 Ineffectiveness 8 Nausea 5 Diarrhea 3 Fatigue 3 Pruritus 3 Vomiting 1 Stomachache Gabapentin 31 Drowsiness 24 Fatigue 23 Ineffectiveness 8 Weight gain 5 Dizziness 2 Nausea 1 Blurred vision 1 Dysphasia 1 Confusion 1 Headache 1 Jitteriness 1 Mood changes 1 Vivid dreams Metformin 57 Nausea 22 Diarrhea 5 Ineffectiveness 3 Fatigue 3 Renal dysfunction 2 Bloating 2 Headache 1 Hypersensitivity 1 Heartburn 1 Hypoglycemia 1 Mood changes 1 Vomiting Drug and Adverse Drug Reaction Matching The process was completed through consolidating the ADRs reported in the Twitter dataset to match the top 5 ADRs from SafeRx. Following the matching, a chi-square test was performed to test nonsignificant differences in the relative frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile with that of FAERS, one should fail to reject H0 according to the P value from the chi-square test. Table 4 shows matched ADRs between the two data sources, relative frequencies of ADRs of each drug, and the results of chi-square test. Drugs and adverse drug reactions frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile with that of FAERS, one should fail to reject H0 according to the P value from the chi-square test. Table 4 shows matched ADRs between the two data sources, relative frequencies of ADRs of each drug, and the results of chi-square test. frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile with that of FAERS, one should fail to reject H0 according to the P value from the chi-square test. Table 4 shows matched ADRs between the two data sources, relative frequencies of ADRs of each drug, and the results of chi-square test. Hydrocodone/acetaminophen 9 Nausea 5 Mood changes 3 Vivid dreams 3 Insomnia 2 Headache 2 Constipation 2 Dizziness 1 Chest tightness 1 Delusion 1 Hallucination 1 Singultus 1 Inattention 1 Short-term amnesia 1 Sweating 1 Vomiting Prednisone 25 Insomnia 23 Increased appetite 10 Mood changes 8 Moon face 8 Weight gain 5 Fatigue 4 Muscle weakness 3 Jitteriness 2 Diaphoresis 2 Tachycardia 2 Anxiety 1 Bradycardia 1 Cataracts 1 Xerostomia 1 Dyspnea 1 Heartburn 1 Osteoporosis 1 Stomachache 1 Visual hallucination 1 Thirst Amoxicillin 46 Hypersensitivity JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 7 https://publichealth.jmir.org/2020/3/e19266 (page number not for citation purposes) FO XSL•FO RenderX Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Drug and Adverse Drug Reaction Matching Drug and Adverse Drug Reaction Matching The process was completed through consolidating the ADRs reported in the Twitter dataset to match the top 5 ADRs from SafeRx. Following the matching, a chi-square test was performed to test nonsignificant differences in the relative https://publichealth.jmir.org/2020/3/e19266 https://publichealth.jmir.org/2020/3/e19266 XSL•FO RenderX XSL•FO RenderX JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren Zhou & Hultgren Table 4. Matched adverse drug reactions and chi-square test results for 5 drugs. P value Chi-square Relative frequencies, Twitter data (%) Relative frequencies, FAERSa data (%) Drug and adverse drug events Hydrocodone/acetaminophen .60b 0.3 22.00 24.31 Ineffectiveness .02 5.3 9.00 21.02 Nausea <.001 14.7 36.00 20.00 Fatigue <.001 13.2 2.00 15.13 Headache Prednisone <.001 18.8 5.00 23.41 Fatigue <.001 47.0 1.00 18.76 Dyspnea Amoxicillin .35b 0.9 46.00 41.15 Hypersensitivity .005 7.9 5.00 15.43 Diarrhea .05b 3.8 8.00 15.18 Nausea <.001 11.8 3.00 15.81 Fatigue .31b 1.0 16.00 12.42 Rash Gabapentin .15b 2.1 22.00 28.55 Ineffectiveness .68b 0.2 23.00 21.28 Fatigue <.001 16.4 2.00 17.40 Nausea Metformin <.001 70.1 57.00 21.75 Nausea <.001 12.7 5.00 19.04 Ineffectiveness .30b 1.1 22.00 18.00 Diarrhea <.001 11.9 3.00 15.56 Fatigue aFAERS: US Food and Drug Administration Adverse Event Reporting System. bIndicates a P value above .05, leading to the failure of rejecting the null hypothesis and indicating that there is no difference in ADR frequency reported Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Table 4. Matched adverse drug reactions and chi-square test results for 5 drugs. adverse drug reactions and chi-square test results for 5 drugs. bIndicates a P value above .05, leading to the failure of rejecting the null hypothesis and indicating that there is no difference in ADR frequency reported between FAERS and Twitter. bIndicates a P value above .05, leading to the failure of rejecting the null hypothesis and indicating that there is no difference between FAERS and Twitter. ADRs remain one of the leading causes for preventable hospital admissions, reduced quality of life, increased financial burdens in the society, and mortality [19]. Prevention relies on adherence to evidence-based medicine, monitoring, medication therapy management, and pharmacogenomic testing [20]. Management of ADRs should emphasize effective prevention and timely detection, yet the current ADR reporting mechanism has shown delays in detection [21]. The cause for delays is multifactorial. Consumers might not know about such a reporting system, and the reporting steps could be troublesome. Drug and Adverse Drug Reaction Matching Further, as clinicians and patients are not required to report ADRs, many could be underreported. Social media and online resources have been proposed as additional resources for pharmacovigilance. In 2017, MacKinlay et al [22] evaluated ADRs of 3055 drugs on Twitter and found that Twitter had up to 72% precision of ADR detection. By extracting ADRs of erlotinib, nivolumab, and pembrolizumab through social health networks, Nikfarjam et al [23] detected that social media ADRs were comparable and 7 months ahead of ADRs from literature reports. Along with JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 9 (page number not for citation purposes) Limitations This study does have two prominent limitations: sample size and search methodology. Among multiple social media platforms, only Twitter was selected as the data source. Despite Twitter’s users being from multiple age groups, patients may choose to share their ADR experiences on other sites such as Facebook, Instagram, Reddit, and online forums, which prevented this study from examining social media data across different platforms. Additionally, due to Twitter’s privacy setting, private tweets are not searchable, which can reduce the number of tweets for data collection. The sample size of tweets obtained for the drugs was relatively small compared with that of FAERS reports from March 1, 2016, to March 31, 2017. The sample size could be largely increased in future studies as Twitter contains a large collection of tweets. During the search process, the keywords hydrocodone/acetaminophen and Norco yielded more than 100 tweets in the time period, which could potentially improve the accuracy of Twitter ADR data. However, there was a lack of relevant tweets for 4 of the 10 drugs, even with the benchmark of 100 tweets. This situation could potentially be resolved by extending the time frame to more than 1 year; however, the extent of sample size improvement might not be significant given the low number of social media users when studying specific drugs such as atorvastatin and amlodipine. Regarding the search mechanism, only one common brand name per drug was used to search for tweets, yet many drugs have multiple brand names. Lisinopril is sold under the brand names Prinivil and Zestril, and levothyroxine has brand names Synthroid, Levoxyl, and Thyrax. Using only one brand name in the study could limit the number of tweets obtained in this study, as patients might have shared their ADRs by using the brand names that were not included in this study. Other challenges to gathering all tweets through keywords include typographical errors, abbreviations, and unstructured lexicons. Furthermore, social media intrinsically bears a limitation in terms of patient follow-up. So far, research methodology involving social media pharmacovigilance has yet to be capable of investigating the causes of ADRs, the consequences of ADRs, and the actions taken to resolve ADRs. Some challenges are being tackled by computational technologies. For example, text normalization and classification through machine learning have been investigated by Sarker et al [27], and they offered insights into processing text data on social media. Principal Findings Among the 5 drugs in the final analysis, a number of Twitter ADR relative frequencies were not significantly different from those of FAERS ADRs. For metformin, diarrhea was one of the side effects. As no significant difference was detected between FAERS and Twitter data on diarrhea (P=.30), it showed that Twitter ADR reports could be further studied for their use as a complementary ADR dataset. In the hydrocodone/acetaminophen group, there were no significant differences in ineffectiveness between sources (P=.60). Gabapentin was shown to comparatively result in ineffectiveness and fatigue according to FAERS and Twitter (P=.15 and P=.67, respectively). Three ADRs of amoxicillin, hypersensitivity, nausea, and rash, shared similar profiles on FAERS and Twitter (P=.35, P=.05, and P=.31, respectively). https://publichealth.jmir.org/2020/3/e19266 Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE the medication and seeing its effect. This study result should also prompt patient education on regular monitoring and diet adjustment when managing diabetes, as ineffectiveness for an antidiabetic drug, metformin, was 19.04% and 5.00% of all ADRs on FAERS and Twitter, respectively. Data mining to track ineffectiveness for hydrocodone/acetaminophen may offer a potential avenue for regulatory bodies in examining opioid use patterns. the medication and seeing its effect. This study result should also prompt patient education on regular monitoring and diet adjustment when managing diabetes, as ineffectiveness for an antidiabetic drug, metformin, was 19.04% and 5.00% of all ADRs on FAERS and Twitter, respectively. Data mining to track ineffectiveness for hydrocodone/acetaminophen may offer a potential avenue for regulatory bodies in examining opioid use patterns. numerous major publications on validating ADR reports across different social media platforms, Hoang et al [24] took a step further and incorporated content authenticity and user credibility to improve ADR detection on Twitter. With more advanced technology for data mining and ADR detection, social media can serve as an additional channel for monitoring ADRs. In this study, 10 drugs were identified, and ADR reports of these drugs on Twitter were retrospectively obtained by searching for tweets containing the drug names that mentioned ADR experiences. While adopting comparative methods used in previous studies, this study specifically focused on the 10 most commonly prescribed drugs to investigate if discrepancies existed pursuant to different drugs. Based on the results of this study, FAERS data and Twitter data showed some similar ADR profiles for hydrocodone/acetaminophen, amoxicillin, gabapentin, and metformin. Principal Findings In the data collection process, levothyroxine, alprazolam, lisinopril, and atorvastatin did not appear as keywords in sufficient tweets from March 1, 2016, to March 31, 2017. A possible explanation of the low number of tweets is the demographics of patients taking these medications. Atorvastatin, a lipid-lowering agent, is usually initiated for elderly patients, as are the antihypertensive agents lisinopril and amlodipine. Individuals aged 50 to 64 years and those older than 65 years represented 21% and 10% of all Twitter users, respectively [16]. Fewer Twitter users in these age ranges could potentially explain the low number of tweets for those drugs. The number of reports of these 3 drugs on FAERS further demonstrates that the lack of tweets was due to fewer users, as atorvastatin, lisinopril, and amlodipine had 6588, 5386, and 3192 reports on FAERS. Other social media–based studies have also experienced this challenge and achieved opposite conclusions due to inactivity for most of the drugs studied on social media [25,26]. Nevertheless, data from the remaining drugs indicates the potential role of Twitter as a complementary source of ADR reporting to FAERS. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 10 (page number not for citation purposes) Conflicts of Interest None declared. Acknowledgments We acknowledge the team that developed the SafeRx database and the CMSA staff for administrative support for this study. Publication of this article was funded in part by Purdue University Libraries Open Access Publishing Fund. We acknowledge the team that developed the SafeRx database and the CMSA staff for administrative support for this study. Publication of this article was funded in part by Purdue University Libraries Open Access Publishing Fund. Conclusion early detection and a reporting system for mild symptoms. To demonstrate the accuracy and usability of social media ADR data in complementing FAERS, future studies may benefit by using a larger sample of data, including specific drugs, and assessing multiple social media platforms. It is also important to apply technology, along with structured reporting systems, to avoid arbitrary entries to better provide health care professionals, regulatory bodies, patients, and pharmaceutical companies with robust ADR data. While the use of Twitter as an ADR reporting platform has limitations, should be considered as a unique and complementary source of information rather than a validation tool of an existing ADR database. Future research should focus on validating Twitter and other social media platforms using involving larger sample sizes and different medications. Additionally, evaluating the types of ADRs on social media that share the most similarity with those on FAERS would be helpful to promote effective use of this source of information. Limitations Other challenges of social media ADR reporting may continue to be barriers for taking full advantage of this data source. The similarities observed for some ADRs between Twitter and FAERS data were disparate across the individual drugs studied. This variability further suggests that patients’actual experiences with medications are not being shared with their providers or that providers have not reported these experiences to national ADR repositories at a similar rate. Moreover, the insufficiency of tweets for some drugs may indicate that social media ADR reporting should consider drug classes and the demographics of patients taking them. One recommendation is to further investigate social media ADR reporting for drugs that are consumed by a population that represents a large share of social media users and drugs that require early ADR detection. In addition to being a supplementary data source for pharmacovigilance services, social media can also serve as a resource for pharmaceutical companies, regulatory bodies, researchers, health care professionals, patients, and policymakers. In this study, ineffectiveness appeared as an ADR for hydrocodone/acetaminophen, gabapentin, and metformin on both data sources. Gabapentin, for example, takes time to exert its full effect in controlling neurological pain. As 23.00% of Twitter ADRs and 28.55% of FAERS ADRs for gabapentin were ineffectiveness, it should encourage prescribers and pharmacists to consult patients on the time lag between taking Although social media cannot replace professional reporting systems such as FAERS at this stage, studies including this analysis have indicated the role of social media as a tool for XSL•FO RenderX Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren References Methods to compare adverse events in Twitter to FAERS, drug information databases, and systematic reviews: proof of concept with adalimumab. Drug Saf 2018 D 41(12) 1397 1410 [FREE F ll t t] [d i 10 1007/ 40264 018 0707 6] [M dli 30167992] 11. Smith K, Golder S, Sarker A, Loke Y, O'Connor K, Gonzalez-Hernandez G. 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Drug manufacturers' delayed disclosure of serious and unexpected adverse events to the US Food and Drug Administration. JAMA Intern Med 2015 Sep;175(9):1565-1566. https://publichealth.jmir.org/2020/3/e19266 References Drug Saf 2019 Dec;42(12):1393-1407 [FREE Full text] [doi: 10.1007/s40264-019-00858-7] [Medline: 31446567] 27. Sarker A, Belousov M, Friedrichs J, Hakala K, Kiritchenko S, Mehryary F, et al. Data and systems for medication-related text classification and concept normalization from Twitter: insights from the Social Media Mining for Health (SMM4H)-2017 shared task. J Am Med Inform Assoc 2018 Oct 01;25(10):1274-1283 [FREE Full text] [doi: 10.1093/jamia/ocy114] [Medline: 30272184] 27. Sarker A, Belousov M, Friedrichs J, Hakala K, Kiritchenko S, Mehryary F, et al. Data and systems for medication-related text classification and concept normalization from Twitter: insights from the Social Media Mining for Health (SMM4H)-2017 shared task. J Am Med Inform Assoc 2018 Oct 01;25(10):1274-1283 [FREE Full text] [doi: 10.1093/jamia/ocy114] [Medline: 30272184] Edited by G Eysenbach; submitted 10.04.20; peer-reviewed by D Degnan, KNB Nor Aripin; comments to author 04.05.20; revised version received 09.06.20; accepted 25.06.20; published 30.09.20 Please cite as: Zhou Z, Hultgren KE Complementing the US Food and Drug Administration Adverse Event Reporting System With Adverse Drug Reaction Reporting From Social Media: Comparative Analysis JMIR Public Health Surveill 2020;6(3):e19266 URL: https://publichealth.jmir.org/2020/3/e19266 doi: 10.2196/19266 PMID: 32996889 ©Zeyun Zhou, Kyle Emerson Hultgren. Originally published in JMIR Public Health and Surveillance (http://publichealth.jmir.org), 30.09.2020. 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Organizational and technological cycle of life of a construction object
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* Corresponding author: aljurgaitis@gmail.com, fayzdim@mail.ru Organizational and technological cycle of life of a construction object Muhammet Fakhratov1,*, Vitaly Chulkov1, Dmitry Fayzullin1, and Salavat Zaidullin1 1Moscow State University of Civil Engineering, 129337, 26, Yaroslavskoye Shosse, Moscow, Russia Abstract. During the life cycle, the state of an object is modified. The information for stepwise and phased study of innovation processes is characterized as local and torn in time, while the life cycle approach regards the process of creating and developing technological innovations as a dynamically synchronized system. The development of organizational and technological systems is being implemented in two directions: the improvement of basic and the creation of fundamentally new technologies. The life cycles of all objects, processes and systems are built on one info graphic model: any life cycle begins with the birth, passes through the stages of growth, maturity, decay and decline. Therefore, it is advisable to consider the innovative investment and construction life cycle of an object as a combination of a series of successive stages (cycles). They are sub- cycles during the period from the beginning of the idea of the initial design and construction of an object until the demolition, complete disassembly, disposal of construction and demolition waste (CDW) and non-recyclable parts of them. https://doi.org/10.1051/e3sconf /201911002161 https://doi.org/10.1051/e3sconf /201911002161 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2161 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2 Methods The development of organizational and technological systems is being implemented in two directions: the improvement of basic and the creation of fundamentally new technologies. With the improvement and modernization of technologies, their transition to the stage of maturity and saturation of the market with this commodity, further technological develop- ment within the framework of the old paradigm becomes unprofitable, volume of sales and profits fall. In the depths of the existing technological structures, fundamentally new solu- tions, "breakthrough" technologies emerge, which lays the foundation for new technologi- cal structures, industries and branches [2]. The life cycles of all objects, processes and systems are built according to a single info- graphic model: any life cycle begins with the birth, passes through the growth, maturity, wilt and decline stages (Figure 1). Fig. 1. The life cycle of an object, process or system. I - the stage of origin and the beginning of growth; II - rapid growth; III - growth retardation, maturity; IV - stabilization; V - wilting, aging, decay, end of life cycle. Fig. 1. The life cycle of an object, process or system. I - the stage of origin and the beginning of growth; II - rapid growth; III - growth retardation, maturity; IV - stabilization; V - wilting, aging, decay, end of life cycle. Fig. 1. The life cycle of an object, process or system. I - the stage of origin and the beginning of growth; II - rapid growth; III - growth retardation, maturity; IV - stabilization; V - wilting, aging, decay, end of life cycle. The infographic model indicated in Figure 1 adequately sets the dynamics and structure of the life cycle of numerous objects of innovation management: innovative enterprises, new equipment and technology, new market goods (norms, products and services). The concept of the life cycle and its model (Fig. 1) provides: accounting for the use of various resources (including the time factor, material, information and financial flows); identifica- tion of the central trend of the process; visualization of the dynamics of transformations; process deployment logic; the possibility of using alternative forecasting methods; identifi- cation of the relationship of different objects. The concept of the life cycle requires the study of its constituent objects and systems from the standpoint of their self-development and improvement. It is closely connected to the system analysis that introduces regularity, completeness and complexity in the process of innovation management. 1 Introduction Temporary, resource and organizational synchronization of all processes and production stages is possible on the basis of life cycles of innovation processes, products and systems. Local information torn in time is characteristic for stepwise and phased study of innovation processes, while the life cycle approach regards the process of creating and developing technological innovations as a dynamically synchronized system. Innovation activity consists of a series of events combined into one logical chain. Each component of this chain (the innovation cycle stage) has its own development logic, its own laws and features. Connecting together, research, development and technological develop- ment, investment and financial, marketing activities, production facilities and organization- al structures are subordinated to one main goal - the creation of innovation [1]. Originating in the depths of marketing research, the life cycles of market goods (norms, products and services), demand and technology in a short time have occupied a dominant position in the study of economic objects, processes and systems. The concepts of the life cycles of organizations, branches of industrial production, construction sites and structures, machines and mechanisms, processes and procedures have gained significant development. , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2161 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2161 https://doi.org/10.1051/e3sconf /201911002161 For the study of innovation, the life cycles of a new product, a new technique, technolo- gy and innovative organizations as open systems are most important. The most productive modern idea of life cycles is the concept of large technological systems, including the evolution and transformation of technologies as economic objects. The study of the life cycles of large technological systems leads to the theory of generations of technics and technology, developing in the framework of both the traditional and the new technological paradigm. 2 Methods Even if several innovations bring the same production effect, they have unequal eco- nomic efficiency; differently affect changes in the production apparatus and the materials used, and the organization of production. The urgency of the problem of choosing the object of innovation is that the correct ini- tial choice predetermines the entire course of the subsequent innovation activity and makes the development of production irreversible. 2 Methods In other words, innovation processes of different scale and level form the basis for the development of activity systems. Studying the features of innovation processes in industri- al, scientific, technical and organizational systems is the basis for increasing the effective- ness of innovation management. 2 2 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2161 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2161 https://doi.org/10.1051/e3sconf /201911002161 In the dynamic system of the innovation process, subsystems (stages) of the creation, production, and consumption of innovation are distinguished. The stage of creating innova- tion requires the emergence of a new idea, new knowledge, the use of new equipment, new technology, new materials to produce the idea into a prototype model [3]. The stage of sci- entific and technical implementation of innovations is the result of engineering develop- ments, laboratory tests, creation of design documentation, production of experimental and prototypes, and technological preparation of production. Distribution, diffusion and com- mercialization of innovations in the market conditions allow obtaining of the expected ef- fect (Fig. 2). Fig. 2. Generalized scheme of life cycle of innovation. Stimulating the use of innovations in production is associated with the problem of their correct choice. The variety of possible impact of each of the innovative solutions offered by science and technology creates the complexity of their selection for manufacturing applica- tion. Even if several innovations bring the same production effect, they have unequal eco- nomic efficiency; differently affect changes in the production apparatus and the materials used and the organization of production Fig. 2. Generalized scheme of life cycle of innovation. Stimulating the use of innovations in production is associated with the problem of their correct choice. The variety of possible impact of each of the innovative solutions offered by science and technology creates the complexity of their selection for manufacturing applica- tion. Even if several innovations bring the same production effect, they have unequal eco- nomic efficiency; differently affect changes in the production apparatus and the materials used, and the organization of production. Stimulating the use of innovations in production is associated with the problem of their correct choice. The variety of possible impact of each of the innovative solutions offered by science and technology creates the complexity of their selection for manufacturing applica- tion. 3 Results Innovations are an essential integral part of construction as a branch of business. This situa- tion exists because of the relative duration of the construction of buildings and structures, as well as due to the fact that construction projects, as a rule, are being built and rebuilt with the prospect of operating them for decades (and sometimes centuries). Recently, the primary erection of buildings and structures in urban areas (when con- struction is carried out on previously unbuilt territories) has become rare: the price for land, newly erected or reconstructed utilities and transport routes and roads has risen sharply. More often, they carry out construction of buildings and their reorganization on the already built-up territories (redevelopment, often with the demolition of existing buildings) [4, 5]. 3 3 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2161 https://doi.org/10.1051/e3sconf /201911002161 This allows us to consider the redevelopment, in relation to the previously built-up are- as, as an innovation (urban planning, technical, technological, economic, etc.). Therefore, it is reasonable to consider the innovative investment and construction life cycle of an object (Pic. 3.) as a combination of a series of successive stages (cycles) and their subcycles during the period from the inception of the idea of the initial construction of a building or structure until its demolition, complete disassembly, disposal of construction and demolition waste (CDW) and non-recyclable part of them. Fig. 3. Infographic model of the innovation life cycle (ILC) of the construction project. Fig. 3. Infographic model of the innovation life cycle (ILC) of the construction project. Erection of the construction object (and the cycle of erection of the object, realized at this stage); the content and sequence of implementation of enlarged sub-stages of stage I (from I.1 to I.10 inclusive) are shown in Figure 4; I.1 I.2 I.3 I.4 I.5 I.6 I.7 I.8 I.9 I.10 I.1 I.2 I.3 I.4 I.5 I.6 I.7 I.8 I.9 I.10 I.1 I.2 I.3 I.4 I.5 I.6 I.7 I.8 I.9 I.10 Fig. 4. Notation conventions: Fig. 4. 3 Results Notation conventions: I.1 - development of technical specifications (TS) for the erection of the object (building or structure); I.2 - development of a business plan; I.1 - development of technical specifications (TS) for the erection of the object (building or structur I.2 - development of a business plan; I.1 - development of technical specifications (TS) for the erection of the object (building or structure); I.2 - development of a business plan; I.2 - development of a business plan; I.3 - development of a constructive project for the future facility; I.4 - development of the project of the organization of construction (POC); I.5 - tendering and auctions (tender), objectification of the general contractor (developer); I.6 - development of working documentation for the construction object; I.7 - development of the project of production of works (PPW); I.8 - development of a work organization project (WOP); I.9 - construction and installation works (the actual construction of the object); I.10 - acceptance of the object and (if necessary) auction for the sale of the object. - Stage II: Reorganization of the construction object (and the cycle of reorganization of the object, implemented at this stage Creorganization); - Stage III: Demolition and disposal of the construction object (and the cycle of demolition of the object, implemented at this stage Cdemolition). of the object, implemented at this stage Cdemolition). 4 4 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2161 https://doi.org/10.1051/e3sconf /201911002161 The basis of the innovation life cycle (ILC) is a calendar organizational and technological design and planning (COTDP) of construction and assembly processes, supported by timely investment. The basis of the innovation life cycle (ILC) is a calendar organizational and technological design and planning (COTDP) of construction and assembly processes, supported by timely investment. COTDP is hierarchical functional database accompanying the construction object throughout its innovation life cycle, which reflects all changes in the construction object without any exception, i.e. local adjustments to the constructive project of the construction object of organizational and technological projects: project of the organization of construc- tion (POC), project of production of works (PPW), and work organization project (WOP). 4 Discussion and conclusions During the life cycle, the state of the object is modified. The flexibility of modern building production is a natural result of the variety of types of construction products, natural, social and technical factors influencing the choice of constructive and technological solutions. g g The efficiency of the construction industry is due to the desire to obtain high-quality construction products with the lowest consumption of material, financial, labor, time re- sources. Hierarchical functional database COTDP should be a market product inalienable from the construction project (subject to continuous organizational and technological, software and computer maintenance and support), with market value and being transferred from owner to owner of the construction object as the object is being sold and purchased up to the stage of its demolition and recycling. Only with this approach, each new owner (or operator) of a construction object must treat it consciously and make decisions, possessing all the necessary information about its history and current state of engineering systems of a building or structure, as well as its premises and redevelopment. And only after the completion of the stage of demolition and disposal of the construc- tion object, the hierarchical functional database COTDP can be officially discarded. Alt- hough in this case, it is of archival and historical town-planning value. References 1. A. Komar, A. Kalgin, M. Fakhratov, O. Kikawa, V. Baev, V. Tsyro, Design and reconstruction of enterprises of precast concrete, (Triada Publishing House LLC, Tver, 2012) 2. A. Kalgin, M. Fakhratov, O. Kikawa, Advanced technologies in the production of building materials. (Publishing House International Concrete Manufacturing LLC, Tver, 2009) 3. A. Kalgin, M. Fakhratov, V. Chulkov, Production and use of building materials, products and systems: Volume 1. Finishing materials, products and systems. 2nd edn. SVR-ARGUS LLC, (Moscow, 2009) 4. V. Chulkov, Reconstruction: Organizational and anthropotechnical reliability of construction. A series of “Infographic basis of functional systems”. SVR-ARGUS LLC, (Moscow, 2012) 5. A. Kalgin, V. Sohryakov, M. Fakhratov, V. Chulkov, Production and use of building materials, products and systems: Volume 4. Production of building structures of precast concrete. Design and reconstruction of enterprises. 2nd edn. SVR-ARGUS LLC, (Moscow, 2012). 5
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Dental Post National Cancer Institute National Cancer Institute Qeios ID: F765NH · https://doi.org/10.32388/F765NH Source National Cancer Institute. Dental Post. NCI Thesaurus. Code C66863. A rod that a dentist has positioned and then cemented in the canal space in a tooth's root. Qeios ID: F765NH · https://doi.org/10.32388/F765NH 1/1
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Three-dimensional label-free observation of individual bacteria upon antibiotic treatment using optical diffraction tomography
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1. Introduction Understanding how antibiotics induce bacterial cell death is essential in the study of microbiology and also to general healthcare in order to help develop, improve, and apply treatment to patients [1]. Over the past years, the mechanism of antibiotics has been studied intensively along with the use of new multiple classes of antibiotics that have succeeded in treating infectious diseases [2]. However, the widespread use of antibiotics has resulted in emergence of drug-resistant bacteria, evoking a significant challenge to the use of antibiotics [3, 4]. Since the mainstream approach to overcome this crisis primarily relies on the discovery and development of newer, more efficient antibiotics, understanding how bacteria respond to antibiotics has become more critical. A basic approach to understanding the mechanism of antibiotics is to observe the phenotypic or physical response of bacteria to antibiotics. Phenotypic imaging has been one of the most popular diagnostic methods to achieve rapid antimicrobial susceptibility testing [5-10], as it provides an easy, low cost, real-time single-cell level examination. Furthermore, phenotypic analysis of bacteria has been exploited to extract useful information in various fields such as epidemiology and systems biology [11]. Although the phenotypic response of bacteria to β-lactam antibiotics has been studied [12, 13], detailed phenotypic aspects have been less explored with little available research on the changes in physical quantities such as cell mass, density, and volume. These parameters are direct indicators of cell survival and growth [14, 15], which can provide fundamental insights into the physical and chemical mechanism of bacterial response to antibiotics. p y p To revisit these unattended but physiologically relevant quantities of bacteria, we need an imaging modality that quantifies the parameters mentioned above. However, conventional two-dimensional (2D) microscopic techniques, such as phase contrast microscopy or differential interference contrast (DIC) microscopy, are not suitable for this purpose, because they are unable to measure three-dimensional (3D) properties. Fluorescence microscopy, such as scanning confocal fluorescence microscopy, allows 3D molecular imaging of cells [16], but the use of fluorescent dyes or proteins have limitations for studying the dynamic alterations in bacteria over a long time due to issues of photo-bleaching and photo-toxicity [17]. Scanning electron microscopy or atomic force microscopy is also not suitable for assessing the dynamics of live bacteria with high throughput [18, 19]. JEONGHUN OH,1,2 JEA SUNG RYU,3 MOOSUNG LEE,1,2 JAEHWANG JUNG, 1,2,† SEUNGYUN HAN, 1,2 HYUN JUNG CHUNG,3,4 AND YONGKEUN PARK1,2,5,* JEONGHUN OH,1,2 JEA SUNG RYU,3 MOOSUNG LEE,1,2 JAEHWANG JUNG, 1,2,† SEUNGYUN HAN, 1,2 HYUN JUNG CHUNG,3,4 AND YONGKEUN PARK1,2,5,* 1Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea 2KAIST Institute for Health Science and Technology, KAIST, Daejeon 34141, South Korea 3Graduate School of Nanoscience and Technology, KAIST, Daejeon 34141, South Korea 4Department of Biological Sciences, KAIST, Daejeon 34141, South Korea 5Tomocube Inc., Daejeon 34051, South Korea †Current Affiliation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea *yk.park@kaist.ac.kr 1Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea 2KAIST Institute for Health Science and Technology, KAIST, Daejeon 34141, South Korea 3Graduate School of Nanoscience and Technology, KAIST, Daejeon 34141, South Korea 4Department of Biological Sciences, KAIST, Daejeon 34141, South Korea 5Tomocube Inc., Daejeon 34051, South Korea †Current Affiliation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea *yk.park@kaist.ac.kr j liation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea i k Current Affiliation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea yk park@kaist ac kr Abstract: Measuring alterations in bacteria upon antibiotic application is important for basic studies in microbiology, drug discovery, and clinical diagnosis, and disease treatment. However, imaging and 3D time-lapse response analysis of individual bacteria upon antibiotic application remain largely unexplored mainly due to limitations in imaging techniques. Here, we present a method to systematically investigate the alterations in individual bacteria in 3D and quantitatively analyze the effects of antibiotics. Using optical diffraction tomography, in-situ responses of Escherichia coli and Bacillus subtilis to various concentrations of ampicillin were investigated in a label-free and quantitative manner. The presented method reconstructs the dynamic changes in the 3D refractive-index distributions of living bacteria in response to antibiotics at sub-micrometer spatial resolution. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint Three-dimensional label-free observation of individual bacteria upon antibiotic treatment using optical diffraction tomography JEONGHUN OH,1,2 JEA SUNG RYU,3 MOOSUNG LEE,1,2 JAEHWANG JUNG, 1,2,† S HYUN JUNG CHUNG,3,4 AND YONGKEUN PARK1,2,5,* 2.1 Optical diffraction tomography To measure the 3D RI tomogram of individual bacteria, we employed optical diffraction tomography based on a Mach-Zehnder nterferometer equipped with a digital micromirror device (DMD) [Fig. 1(a)]. To measure the 3D RI tomogram of individual bacteria, we employed optical diffraction tomography based on a Mach-Zehnder interferometer equipped with a digital micromirror device (DMD) [Fig. 1(a)]. Fig. 1. Measurements of the 3D RI distribution. (a) Experimental setup. M1−M4, mirror; L1−L5, lens; FC, fiber coupler; DMD, digital micromirror device. (b) Sequential illumination scanning for bacteria at 71 illumination angles and the corresponding 71 holograms. (c) Images of the retrieved amplitudes and phases corresponding to the holograms. (d) Three-section view of the reconstructed RI distribution. (e) 3D rendered image of bacteria. The color map shows various colors depending on the value of RI and its gradient value. Fig. 1. Measurements of the 3D RI distribution. (a) Experimental setup. M1−M4, mirror; L1−L5, lens; FC, fiber coupler; DMD, digital micromirror device. (b) Sequential illumination scanning for bacteria at 71 illumination angles and the corresponding 71 holograms. (c) Images of the retrieved amplitudes and phases corresponding to the holograms. (d) Three-section view of the reconstructed RI distribution. (e) 3D rendered image of bacteria. The color map shows various colors depending on the value of RI and its gradient value. A coherent laser ( = 532 nm in a vacuum; SambaTM, Cobolt Inc., Sweden) beam was split into a reference beam and sample beam using a 1 × 2 fiber coupler. The sample beam passed through 4-f relay lenses (L1−3), and was diffracted by a DMD (DLPLCR6500EVM, Texas Instruments Inc., USA). A DMD controlled the angle of the sample beam impinging onto a sample, by displaying digital grating patterns [32]. The time-multiplexing illumination technique was utilized to eliminate the unwanted diffractions of a DMD [33]. The first-order diffraction beam illuminated a sample with a specific incident angle controlled by the pattern projected onto the DMD. The beam diffracted from the DMD passes through a lens (L4) and a condenser lens (NA = 1.2, water immersion, UPLSAPO 60XW, Olympus Inc.) in order to magnify the incident angle. The scattered light from the sample was collected by an objective lens (NA = 1.2, water immersion, UPLSAPO 60XW, Olympus Inc.), and projected to the image plane. 2.1 Optical diffraction tomography At the image plane, the sample beam and reference beam interfere and generate a spatially modulated hologram pattern, which is recorded using a camera (FL3-U3–13Y3M-C, Point Grey Research Inc.). To combine two beams and generate the interference pattern with the maximum contrast, a beam splitter and a linear polarizer were used, respectively. The beam impinging onto the sample was controlled to have the maximum elevation angle limited by the NA of the condenser lens and was scanned along a full azimuthal angle with 71 steps [Fig. 1(b)]. The total acquisition time for 71 holograms was approximately 1 s (15 ms per hologram). 1. Introduction py g y g g p [ ] To investigate the aforementioned parameters, optical diffraction tomography (ODT), one of the 3D quantitative phase imaging (QPI) techniques, has several potentials [20]. ODT provides a non-invasive and non-contact 3D optical imaging [21]. ODT reconstructs the 3D refractive index (RI) maps of optically transparent samples based on interferometric imaging at multiple illumination angles. Because RI distributions serve as both intrinsic cell markers and indicators of protein densities, ODT meets the demands of quantitative phenotypic imaging of unlabeled live cells. Thus, its applications have been rapidly expanded for investigations on hematology [22], microalgae [23], immunology [24], infectious diseases [25, 26], plant biology . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: oRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint [27], cell biology [27, 28], neuroscience [29], and drug discovery [30]. Some bacterial studies have also used ODT [31], but its application for bacterial microbiology is in a nascent stage. pp gy g In this study, we investigated the 3D bacterial response to antibiotics by exploiting ODT. Our method provides a label-free, real-time analysis of the phenotypic changes in bacterial cells at given antibiotic conditions. The quantitative analysis of 3D RI maps enables examination of the physical changes in cells induced by antibiotic agents. We demonstrate the capability of ODT for imaging the antibiotic response of bacteria in 3D by investigating the response of Escherichia coli and Bacillus subtilis to a β-lactam antibiotic agent, ampicillin. The dynamics of bacteria were observed by mapping the 3D RI distribution in real time, and the changes in morphological and biochemical parameters were analyzed. With its non-invasive, label-free, and quantitative analysis, our method provides a useful tool for studying and developing antibiotic agents against clinical pathogens that threaten public health. 2.3 Analysis of cell parameters and visualization methods To measure cell volumes, the voxels were selected which have an RI higher than a specific value determined by masking the sample region at the initial time for each cell and averaging the RI values therein. Because the RI value of cell cytoplasm is linearly related to its protein concentration, local protein concentration of a cell can be retrieved from the measured 3D RI tomogram [46, 47]. The concentration at each point of the bacteria was determined from RI increment (RII), an increment of an RI in a solution per an increment of concentration in a solute. We assumed that the value of RII is constant inside bacteria, i.e. n(r) - nmedium = (RII) × C(r), where n is an RI inside the bacteria, nmedium is the RI of surrounding medium, and C is the protein concentration. Therefore, the concentration distribution inside bacteria can be acquired using an RI distribution. In addition, the RII of 0.185 mL/g is known as a typical value for proteins [42, 46]. Because many bacteria have proteinaceous organelles [48], it is reasonable that the typical value of RII for proteins could be used in our analysis. The temporal changes in the cellular dry mass are calculated as the total sum of the concentration at each voxel multiplied by its volume. Cytoplasm concentration was calculated by averaging the concentrations at the internal voxels of bacteria. y g g The 3D rendered image was obtained using commercial software (TomoStudioTM, Tomocube Inc., Republic of Korea), which delineates the overall RI distributions of bacteria more clearly [Fig. 1(e)]. The different colors indicate the presence of subcellular organelles in bacteria with different RI values, suggesting that our technique provides sub-micrometer spatial resolution to investigate individual bacterial cells. 2.2 3D RI tomogram reconstruction The 3D RI distribution of a sample is reconstructed from the measured multiple 2D holograms using the principle of optical diffraction tomography [34, 35]. For each hologram pattern, amplitude and phase delay maps were retrieved using the field retrieval algorithm [Fig. 1(c)] [36, 37]. Then, from these retrieved optical field maps with various angles, the 3D RI distribution was determined [Fig. 1(d)]. ODT solves an inverse scattering problem for monochromatic light propagating in a linear, isotropic, and nonmagnetic medium, which satisfies the 3D inhomogeneous Helmholtz equation [34]. For a slowly varying scattered complex phase, the Rytov approximation was applied, which has shown better reconstruction performances than the Born . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint approximation [38, 39]. According to the Fourier diffraction theorem, each optical field is mapped onto a sphere in the 3D Fourier space [40]. Then, the inverse Fourier transform of the 3D Fourier space provides the RI distribution of the sample [41]. The theoretically calculated lateral and axial resolution of the used optical imaging system is 124 nm and 397 nm, respectively [32, 42]. Due to the limited NAs of both the condenser and the objective lens, side scattering signals are not collected, which is known as the missing cone problem. To resolve this missing cone problem, the regularization algorithm based on non- negativity criteria was used [43-45]. approximation [38, 39]. According to the Fourier diffraction theorem, each optical field is mapped onto a sphere in the 3D Fourier space [40]. Then, the inverse Fourier transform of the 3D Fourier space provides the RI distribution of the sample [41]. The theoretically calculated lateral and axial resolution of the used optical imaging system is 124 nm and 397 nm, respectively [32, 42]. 2.4 Sample preparation Bacterial strains Escherichia coli (E. coli; #1682) and Bacillus subtilis (B. subtilis; #1021) were purchased from Korean Collection for Type Cultures. E. coli cells were cultured in Tryptic soy broth (TSB) and B. subtilis were cultured in Nutrient broth (NB). Both bacteria were cultured to the concentration of 1.0−5.0 × 108 cells/mL to obtain activated bacteria using a nanophotometer (Implen). Centrifugation to wash the cells was repeated twice under the general condition of 8,000 × g for 5 minutes at 4℃ in 200 μL of PBS solution. Cells were then resuspended in TSB and NB to conduct imaging for the bacteria. Ten microliters of the resuspended solution were placed between two coverslips and then measured using the optical imaging system. All the measurements were performed at room temperature (25℃). 2.2 3D RI tomogram reconstruction Due to the limited NAs of both the condenser and the objective lens, side scattering signals are not collected, which is known as the missing cone problem. To resolve this missing cone problem, the regularization algorithm based on non- negativity criteria was used [43-45]. 3. Results In order to systematically investigate the alterations in individual bacteria upon treatment with antibiotics, we measured the 3D RI tomograms of E. coli and B. subtilis using ODT, and analyzed the measured images. 3.1 Changes in a bacterial cell revealed by time-lapse 3D imaging We first validated the 3D dynamic imaging capability of ODT by visualizing bacterial growth in the absence of antibiotics. We imaged the dynamics of E. coli in growth medium with a time interval of 30 minutes up to 6 hours. Both the cross-sections and rendered images of the measured 3D RI tomograms clarify the growth and division of individual E. coli [Figs. 2(a) and 2(b)]. Prominent cell division was observed and the increase in the number of E. coli over time is presented in Fig. 2(c). The number of E. coli doubled constantly at intervals of 56 min, which is consistent with the generation time of E. coli at room temperature [49]. These results clearly validate the label-free imaging capability of ODT for studying the dynamics of individual bacteria. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: oRxiv preprint Fig. 2. (a) Three-section views of the reconstructed RI distributions of E. coli without antibiotics over time (at 0, 120, and 240 minutes). (b) 3D rendered images of bacteria corresponding to (a). The color map shows various colors depending on the value of the RI. (c) The number of E. coli for the control group over time and the fitted curve. R-square = 0.9741. Fig. 2. (a) Three-section views of the reconstructed RI distributions of E. coli without antibiotics over time (at 0, 120, and 240 minutes). (b) 3D rendered images of bacteria corresponding to (a). The color map shows various colors depending on the value of the RI. (c) The number of E. coli for the control group over time and the fitted curve. R-square = 0.9741. 3.2 Antibiotic responses of Escherichia coli and Bacillus subtilis 3.2 Antibiotic responses of Escherichia coli and Bacillus subtilis Next, we investigated the growth dynamics of individual bacteria under antibiotic treatments. As Gram-positive and Gram- negative models, E. coli and B. subtilis were selected, respectively. The bacteria were treated with ampicillin at various concentrations of 20, 100, and 300 g/mL and imaged at time intervals of 30 minutes after drug treatment. As a normal control group, untreated bacterial samples were also measured at the same time intervals. Next, we investigated the growth dynamics of individual bacteria under antibiotic treatments. As Gram-positive and Gram- negative models, E. coli and B. subtilis were selected, respectively. The bacteria were treated with ampicillin at various concentrations of 20, 100, and 300 g/mL and imaged at time intervals of 30 minutes after drug treatment. As a normal control group, untreated bacterial samples were also measured at the same time intervals. The results are shown in Fig. 3. In the absence of antibiotic treatment, both E. coli and B. subtilis exhibited active growth and division. When the bacteria were treated with antibiotics, each cell showed significant morphological alterations, and also presented a decrease in RI values, suggesting cell lysis. To effectively visualize these morphological alterations, the maximum RI projection of each 3D RI tomograms is presented for each group at various time points in Fig. 3. Fig. 3. Maximum RI projection images with ampicillin over time. The red arrows indicate a bulge formed in E. coli. The entry with a red border is presumed to be near the point at which the bacteria die. (a) and (b) are E. coli and B. subtilis, respectively. Fig. 3. Maximum RI projection images with ampicillin over time. The red arrows indicate a bulge formed in E. coli. The entry with a red border is presumed to be near the point at which the bacteria die. (a) and (b) are E. coli and B. subtilis, respectively. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint Figure 3(a) shows the morphological changes in E. coli, due to the ampicillin treatment. As the concentration of ampicillin increased, the bacteria underwent earlier cell lysis. 3.2 Antibiotic responses of Escherichia coli and Bacillus subtilis From the reconstructed 3D RI tomograms, the point where the RI value drops suddenly could serve as the time point when the bacterial lysis initiated, because the decrease in the RI values of bacteria indicates the efflux of cell cytoplasm. When the concentration of the applied ampicillin was 20, 100, and 300 g/mL, the bacterial lysis occurred right after 180, 150, and 30 minutes, respectively. In the control group, E. coli showed stable growth over time, whereas the bacteria treated with ampicillin did not divide. p In addition, the ampicillin-treated bacteria exhibited distinct elongation of cell shape; the cell length increased without cell division until the point when the RI value increased. Interestingly, the cells exposed to the ampicillin concentration of 20 g/mL appeared like filaments, exhibiting a fiber-like cell shape. Notably, formation of bulges could be clearly detected in the measured tomograms [the red arrows in Fig. 3(a)]. Bulge formation was observed in groups treated with ampicillin concentrations of 20 or 100 g/mL. However, a bulge was not formed at 300 g/mL, which is a significantly high concentration. Furthermore, the rate of cell lysis increased noticeably as the concentration of ampicillin increased to 300 g/mL. Compared to the results with E. coli, B. subtilis exhibited significantly different responses to ampicillin [Fig. 3(b)]. First, the bacterial cell lysis time was earlier than that of E. coli treated with low ampicillin concentration. When the ampicillin concentration was 20, 100, and 300 g/mL, the cell lysis points were conjectured to be right after 90, 90, and 40 minutes, respectively. At an even lower concentration, B. subtilis showed faster cell lysis than E. coli. In addition, B. subtilis exposed to the low concentration of ampicillin (20 g/mL) did not exhibit morphological alteration into filament-like shapes as observed with E. coli. Notably, bulge formation was not observed in B. subtilis. 3.3 Quantitative analysis results for morphological and biochemical parameters To investigate the morphological and biochemical characteristics of bacterial growth under antibiotic treatment, the following cellular parameters were retrieved and analyzed from the measured 3D RI tomograms: cell volume, cellular dry mass, cytoplasm concentration (See Methods). These cellular morphological and biochemical parameters were systematically addressed as a function of time and at various antibiotic concentrations [Fig. 4]. Fig. 4. Cell volume, cellular dry mass, and cytoplasm concentration depending on the concentrations of ampicillin (0, 20, 100, and 300 g/mL) and time at the maximum volume. (a−d) and (e−h) are E. coli and B. subtilis, respectively. Fig. 4. Cell volume, cellular dry mass, and cytoplasm concentration depending on the concentrations of ampicillin (0, 20, 100, and 300 g/mL) and time at the maximum volume. (a−d) and (e−h) are E. coli and B. subtilis, respectively. Figure 4(a) shows the cellular volume of E. coli over time for various concentrations of ampicillin. Note that this analysi was conducted at the single cell level, and each line in Fig. 4 indicates individual cells. For untreated E. coli, cells continued growth and division, and thus the volumes of each cell remained within a certain range (0.5−1.4 fL). Furthermore, the volume of untreated E. coli oscillated over time, implying continuous growth and division. Figure 4(a) shows the cellular volume of E. coli over time for various concentrations of ampicillin. Note that this analysis was conducted at the single cell level, and each line in Fig. 4 indicates individual cells. For untreated E. coli, cells continued growth and division, and thus the volumes of each cell remained within a certain range (0.5−1.4 fL). Furthermore, the volumes of untreated E. coli oscillated over time, implying continuous growth and division. Figure 4(a) shows the cellular volume of E. coli over time for various concentrations of ampicillin. Note that this analysis was conducted at the single cell level, and each line in Fig. 4 indicates individual cells. For untreated E. coli, cells continued growth and division, and thus the volumes of each cell remained within a certain range (0.5−1.4 fL). Furthermore, the volumes of untreated E. coli oscillated over time, implying continuous growth and division. Upon ampicillin treatment, E. coli did not exhibit cell division, as shown in Fig. 3. The volumes of treated E. coli showed monotonic increases, followed by sudden plummets [Fig. 3(a)]. These time points are consistent with the cell lysis observed in the 3D RI tomograms. 3.3 Quantitative analysis results for morphological and biochemical parameters agree with our observation that when ampicillin is applied, cell division stops and abnormal elongation and bulge formation occurs. This abnormal growth occurred for a shorter time at high ampicillin concentration, indicating that E. coli burst to death faster at higher antibiotic concentrations [Fig. 4(b)]. The cellular dry mass of E. coli is shown in Fig. 4(c) (See Methods). The graphs for the dry mass are similar to those of cell volume [Fig. 4(a)], indicating that the cytoplasmic concentrations did not significantly change over time. This result can also be confirmed from the measured 3D RI tomograms [Fig. 4(d)]. The cytoplasm concentration increased slightly as the bacteria grew. After bacterial lysis, the internal contents escaped beyond the cell wall, and so, the cytoplasm concentration dropped suddenly. Comparison between the control and ampicillin-treated groups clearly indicates that the sudden drop in cytoplasm concentration was only notable in the latter case. Compared to E. coli, B. subtilis did not show noticeable increases in either cell volume or dry mass upon ampicillin treatment [Figs. 4(e)-4(g)]. Rather, the bacteria rapidly reached a much smaller cell volume than the initial volume. The most discernible change was observed for the temporal graphs of cytoplasm concentration; a decrease in cytoplasm concentration caused by ampicillin was relatively well observed [Fig. 4(h)]. To clarify the different lysis behaviors between E. coli and B. subtilis, we reorganized and compared the species-dependent changes in quantitative parameters. Among the various parameters, we chose to compare cell volumes because they showed the most notable difference [Fig. 5]. At ampicillin concentrations of 20 and 100 g/mL, the difference in cell volume between E. coli and B. subtilis was remarkable [Figs. 5(a) and 5(b)]. This is because abnormal growth was observed in E. coli treated with ampicillin, but no such growth was observed in B. subtilis. Moreover, at ampicillin concentrations of 20 and 100 g/mL, the time to reach a minimal cell volume was shorter for B. subtilis than for E. coli. In other words, B. subtilis appeared to undergo lysis faster than E. coli. However, at the concentration of 300 g/mL [Fig. 5(c)], this tendency was lost. These observations can be seen more clearly in Fig. 5(d), which represents the maximum volume of the bacteria at each concentration as a relative value. These data sufficiently represent how ODT can quantitatively address the dynamics of the antibiotic response of bacteria of bacteria. Fig. 5. 3.3 Quantitative analysis results for morphological and biochemical parameters Comparing the graphs for ampicillin concentrations of 20, 100, and 300 g/mL, as the concentration of ampicillin increased, the bacteria showed earlier lysis, and the maximum volume of bacteria decreased. In addition, the maximum volume of E. coli with ampicillin treatments was significantly larger than that of the control group. These results Upon ampicillin treatment, E. coli did not exhibit cell division, as shown in Fig. 3. The volumes of treated E. coli showed monotonic increases, followed by sudden plummets [Fig. 3(a)]. These time points are consistent with the cell lysis observed in the 3D RI tomograms. Comparing the graphs for ampicillin concentrations of 20, 100, and 300 g/mL, as the concentration of ampicillin increased, the bacteria showed earlier lysis, and the maximum volume of bacteria decreased. In addition, the maximum volume of E. coli with ampicillin treatments was significantly larger than that of the control group. These results Upon ampicillin treatment, E. coli did not exhibit cell division, as shown in Fig. 3. The volumes of treated E. coli showed monotonic increases, followed by sudden plummets [Fig. 3(a)]. These time points are consistent with the cell lysis observed in the 3D RI tomograms. Comparing the graphs for ampicillin concentrations of 20, 100, and 300 g/mL, as the concentration of ampicillin increased, the bacteria showed earlier lysis, and the maximum volume of bacteria decreased. In addition, the maximum volume of E. coli with ampicillin treatments was significantly larger than that of the control group. These results . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint agree with our observation that when ampicillin is applied, cell division stops and abnormal elongation and bulge formation occurs. This abnormal growth occurred for a shorter time at high ampicillin concentration, indicating that E. coli burst to death faster at higher antibiotic concentrations [Fig. 4(b)]. 3.3 Quantitative analysis results for morphological and biochemical parameters (a−c) The relative value of cell volume for E. coli and B. subtilis with the ampicillin concentration of (a) 20 g/mL (b) 100 g/mL (c) 300 g/mL. (d) The relative value of the maximum volume for E. coli and B. subtilis. Fig. 5. (a−c) The relative value of cell volume for E. coli and B. subtilis with the ampicillin concentration of (a) 20 g/mL (b) 100 g/mL (c) 300 g/mL. (d) The relative value of the maximum volume for E. coli and B. subtilis. 4. Discussions and Summary In summary, we present a method to systematically investigate the alterations in individual bacteria upon antibiotic treatment. Employing ODT, the 3D RI of antibiotic-treated bacteria was measured over time in a label-free and non-invasive manner. The 3D RI reconstruction of E. coli and B. subtilis bacteria provides the morphological and biochemical parameters related to their real-time alterations depending on the concentration of ampicillin, which were characterized by the inhibition of cell division, bulge formation, abnormal elongation, and cell lysis. Because the RI inside the bacteria is directly related to the cytoplasm concentration, dynamics of cytoplasmic concentrations and cellular dry mass were also analyzed quantitatively. Moreover, we quantitatively deduced the bacterial lysis time through a sudden change in cell volume or cellular dry mass. This can also be used effectively to study the cell cycle of bacteria [50]. Our measurements and observations with bacteria are consistent with those in the existing literature. Ampicillin from the β-lactam class inhibits cell wall synthesis, leading to various changes in bacterial cell shape and size, and finally resulting in bacterial cell death [51]. It is also intriguing to observe the changes in bacterial appearance depending on the ampicillin concentration. Spratt explains that cell division of E. coli is inhibited even at low concentrations of ampicillin, resulting in filamentation of E. coli; further, at higher concentrations, a bulge is formed in the middle of E. coli, and cell lysis occurs at significantly high ampicillin concentrations [13]. In our experiments, the corresponding concentrations can be considered as 20, 100, and 300 g/mL, respectively. Comparing the cells with 20 g/mL of ampicillin to the control group, cell division was inhibited, and abnormal growth, i.e. filamentation, was observed. As the concentration increased to 100 g/mL, the filamentation phenomenon decreased, and the bulge became more pronounced. This is also consistent with the statement that only lysis was observed at the concentration of 300 g/mL. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 19, 2019. ; https://doi.org/10.1101/775346 doi: bioRxiv preprint The different responses of E. coli and B. subtilis to ampicillin are also noteworthy for discussion. Funding This work was supported by KAIST, BK21+ program, Tomocube, and National Research Foundation of Korea (2017M3C1A3013923, 2015R1A3A2066550, 2018K000396). Disclosures Prof. Park and Mr. Moosung Lee have financial interests in Tomocube Inc., a company that commercializes optical diffraction tomography and quantitative phase imaging instruments and is one of the sponsors of the work. 4. Discussions and Summary Ampicillin hinders transpeptidation by combining with penicillin-binding proteins on maturing peptidoglycan strands, and the resulting reduction in peptidoglycan synthesis causes cell lysis [13, 52]. In the case of Gram-positive bacteria including B. subtilis, the majority of the cell wall is composed of peptidoglycan; thus, the effect on ampicillin is more pronounced [52], resulting in faster cell lysis. This explains the contrast in the E. coli and B. subtilis at ampicillin concentrations of 20 and 100 g/mL. In addition, there is a difference in the reactivity of Gram-positive and Gram-negative bacteria to ampicillin, but ampicillin eventually leads to cell death in both types of bacteria [53]. Our experiments confirmed that both E. coli and B. subtilis reached cell lysis in the presence of ampicillin. Indeed, our results were based on small datasets but were sufficient to epitomize the antibiotic response in a label-free, quantitative, and three-dimensional manner. Previously, 2D QPI techniques have been applied for the study of individual bacteria [54], [55, 56]. However, 2D QPI only measures the optical phase delay maps, an integration of RI values along an optical path, and does not provide tomographic measurements of individual bacteria. As demonstrated in this work, ODT enables the systematic investigation of various volumetric information of individual bacteria. We emphasize that the 3D imaging technique exploited here is not limited to the objects targeted in this paper. The species of bacteria and the type of antibiotics can be expanded as needed. In addition, using this method, it is possible to conduct statistical analysis by increasing the number of microbes regardless of single cells or multiple cells. It is also possible to infer the physical functions and biological characteristics of bacteria from their interior structural features [57, 58]. Moreover, quantitative analysis for other morphological and biochemical parameters can be attempted. For instance, when a bulge is formed in E. coli, each volume or cytoplasm concentration can be quantified by separating the bulge and non-bulge portions. The lifetime of the bulge can also be measured. In this case, combination with the segmentation method will serve as the key to analysis [24]. We believe that the methods presented in this paper will provide microbiologists with broader insights into the various responses of bacteria to antibiotics. Acknowledgments The authors thank Dr. Weisun Park for helpful discussions. References Kohanski, M.A., D.J. Dwyer, and J.J. Collins, How antibiotics kill bacteria: from targets to network Microbiology, 2010. 8(6): p. 423. 2. Pambos, O.J. and A.N. Kapanidis, Tracking antibiotic mechanisms. 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Marine deforestation leads to widespread loss of ecosystem function
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PLOS ONE RESEARCH ARTICLE Marine deforestation leads to widespread loss of ecosystem function Matthew EdwardsID1*, Brenda Konar2, Ju-Hyoung KimID3, Scott Gabara1,4, Genoa Sullaway1, Tristin McHugh1, Michael SpectorID1, Sadie SmallID1 1 Department of Biology, San Diego State University, San Diego, CA, United States of America, 2 College of Fisheries and Ocean Sciences, University of Alaska Fairbanks, Fairbanks, Alaska, United States of America, 3 Marine Applied Biosciences, Kunsan National University, Gunsan, South Korea, 4 Department of Environmental Science and Policy, University of California, Davis, California, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * medwards@sdsu.edu Editor: Maura (Gee) Geraldine Chapman, University of Sydney, AUSTRALIA Editor: Maura (Gee) Geraldine Chapman, University of Sydney, AUSTRALIA Editor: Maura (Gee) Geraldine Chapman, University of Sydney, AUSTRALIA Received: November 20, 2019 Accepted: February 13, 2020 Published: March 4, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0226173 Copyright: © 2020 Edwards 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. Abstract Trophic interactions can result in changes to the abundance and distribution of habitat-form- ing species that dramatically reduce ecosystem functioning. In the coastal zone of the Aleu- tian Archipelago, overgrazing by herbivorous sea urchins that began in the 1990s resulted in widespread deforestation of the region’s kelp forests, which led to lower macroalgal abun- dances and higher benthic irradiances. We examined how this deforestation impacted eco- system function by comparing patterns of net ecosystem production (NEP), gross primary production (GPP), ecosystem respiration (Re), and the range between GPP and Re in rem- nant kelp forests, urchin barrens, and habitats that were in transition between the two habitat types at nine islands that spanned more than 1000 kilometers of the archipelago. Our results show that deforestation, on average, resulted in a 24% reduction in GPP, a 26% reduction in Re, and a 24% reduction in the range between GPP and Re. Further, the transition habitats were intermediate to the kelp forests and urchin barrens for these metrics. These opposing metabolic processes remained in balance; however, which resulted in little-to-no changes to NEP. These effects of deforestation on ecosystem productivity, however, were highly vari- able between years and among the study islands. In light of the worldwide declines in kelp forests observed in recent decades, our findings suggest that marine deforestation pro- foundly affects how coastal ecosystems function. OPEN ACCESS Citation: Edwards M, Konar B, Kim J-H, Gabara S, Sullaway G, McHugh T, et al. (2020) Marine deforestation leads to widespread loss of ecosystem function. PLoS ONE 15(3): e0226173. https://doi.org/10.1371/journal.pone.0226173 * medwards@sdsu.edu PLOS ONE PLOS ONE Introduction Currently, sea otters are largely absent from or are in very low abundances on many of the islands and most of the kelp forests have either disappeared from the archipelago or are in the process of disappearing, although some small forests remain in their ‘historical state’ at scattered locations on most of the islands [14,15] (Fig 1). These remnant forests provide a valuable benchmark against which we evalu- ated the effects of widespread deforestation on an important metric of ecosystem function, namely primary productivity. ME and (OCE1435205) to BK, and the National Research Foundation (NRF-2018R1C1B6008523 and NRF-2015R1C1A1A01054831) to JHK. ME and (OCE1435205) to BK, and the National Research Foundation (NRF-2018R1C1B6008523 and NRF-2015R1C1A1A01054831) to JHK. Competing interests: NO - Authors have no competing interests. Characterizing patterns of biodiversity and primary productivity is essential to fully under- standing ecosystem function [16,17]. The latter includes three basic metrics: gross primary production (GPP), which describes all the CO2 fixed by the autotrophs during photosynthesis, total ecosystem respiration (Re), which describes the release of CO2 during the production of energy by autotrophs, heterotrophs, decomposers and microbes, and net ecosystem produc- tion (NEP), which is the difference between GPP and Re and describes net changes in the total amount of organic carbon in an ecosystem available for consumption, storage and export to adjacent ecosystems, or nonbiological oxidation to carbon dioxide [18–21]. In general, ecosys- tems with high rates of GPP also exhibit high rates of Re, with the central tendency being that GPP and Re are in balance (i.e., similar in magnitude) and therefore have median GPP / Re ratios close to 1.0, and NEP values near zero [21,22]. Indeed, a review of five decades (1950 to 1990) of studies in aquatic ecosystems demonstrated that these two opposing processes are generally in balance, although unproductive ecosystems tend towards net heterotrophy with GPP / Re < 1.0 and NEP < 0, while productive ecosystems tend towards net autotrophy with GPP / Re > 1.0 and NEP > 0 [21,22]. Further, the amount of Re associated with any given GPP in shallow coastal ecosystems tends to be greater when the complete benthic communities are considered [22]. This may be especially true if microbial metabolism, which is an important component of Re, is large compared to GPP [20–22]. This is important for coastal kelp forests, which host a higher diversity of microbes relative to the adjacent ocean waters [23–27]. Introduction Consumers fundamentally affect ecosystems through trophic interactions [1]. These interac- tions are especially important if they result in changes to the abundance or distribution of eco- system engineers, such as forest-forming trees, which can lead to changes in microclimates, biodiversity, primary production, nutrient cycling, and energy flow [2]. For example, the rein- troduction of gray wolves (Canis lupus) into Yellowstone National Park, USA in the 1990s resulted in increased predation on elk (Cervus elaphus) and subsequently reduced herbivory on canopy-forming trees such as aspens (Populus tremuloides), willows (Salix spp.), and cot- tonwoods (Populus spp.) [3]. This ultimately led to changes in the morphology and hydrology Data Availability Statement: Data are available on our NSF bco-dmo data page at https://www.bco- dmo.org/dataset/755658 Funding: This research was funded by grants from the National Science Foundation (OCE1435194) to PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 1 / 21 PLOS ONE Deforestation and loss of ecosystem function of the region’s river systems and its riparian plant communities [4,5]. Similarly, large marine algae, such as kelps, can form subtidal forests whose biogenic structures alter hydrodynamic, nutrient and light conditions, modify patterns of biodiversity, enhance primary production and carbon sequestration, and provide food and habitat for numerous other species [6–9]. Consequently, the loss of these forest-forming kelps and the benthic communities they support can have dramatic impacts to how nearshore ecosystems function, especially if they occur over large geographic areas. Indeed, kelp deforestation has occurred in numerous areas worldwide in recent decades due to a variety of forcing factors [10,11], and the subtidal rocky reefs of the Aleutian Archipelago serve as a model system to investigate the broader impacts of such defor- estation. These forests have historically been dominated by dense populations of the surface canopy-forming kelp Eualaria fistulosa, several species of understory kelps such as Laminaria spp. and Agarum spp., the brown alga Desmarestia spp., and numerous species of fleshy read algae. However, the collapse of sea otter (Enhydra lutris) populations led to large increases in their primary prey, herbivorous sea urchins (Strongylocentrotus polyacanthus), which subse- quently resulted in overgrazing and widespread losses of the region’s kelp forests [12]. This collapse began in the late 1990s, likely in response to a dietary shift by killer whales toward sea otters, and by 2000 sea otter densities had declined throughout the archipelago to around 5–10% of their estimated equilibrium density [13]. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 Introduction Conse- quently, loss of these forests may lead to complex patterns of GPP, Re, and NEP within coastal ecosystems. On one hand, reductions in primary producer biomass should result in lowered GPP and thus reduced NEP. Alternately, deforestation may result in lowered abundances of invertebrates, fishes and microbes, which may lead to reduced Re and thus enhanced NEP. At the same time, loss of the habitat-forming kelps also results in elevated benthic irradiances (measured as photosynthetically active radiation (PAR) [18] and thus potentially to enhanced PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 2 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 1. Three habitat types. Photographs of each habitat type showing (A) high abundance of benthic macroalgae and canopy-forming kelps in the kelp forests, (B) lack of benthic macroalgae but remaining canopy-forming kelps and high abundances of sea urchins in the transition habitats, and (C) lack of benthic macroalgae and canopy-forming kelps, but high abundances of sea urchins in the urchin barrens. Fig 1. Three habitat types. Photographs of each habitat type showing (A) high abundance of benthic macroalgae and canopy-forming kelps in the kelp forests, (B) lack of benthic macroalgae but remaining canopy-forming kelps and high abundances of sea urchins in the transition habitats, and (C) lack of benthic macroalgae and canopy-forming kelps, but high abundances of sea urchins in the urchin barrens. Fig 1. Three habitat types. Photographs of each habitat type showing (A) high abundance of benthic macroalgae and canopy-forming kelps in the kelp forests, (B) lack of benthic macroalgae but remaining canopy-forming kelps and high abundances of sea urchins in the transition habitats, and (C) lack of benthic macroalgae and canopy-forming kelps, but high abundances of sea urchins in the urchin barrens. https://doi.org/10.1371/journal.pone.0226173.g001 https://doi.org/10.1371/journal.pone.0226173.g001 https://doi.org/10.1371/journal.pone.0226173.g001 compensatory production by any remaining fleshy macroalgae, encrusting coralline algae, and microalgae [28–30], which can result in greater NEP. Thus, understanding how GPP, Re, and NEP change with kelp forest change can be instrumental in discerning the broader impacts of deforestation on ecosystem productivity. This may be especially relevant for the Aleutian Archipelago where widespread kelp deforestation has resulted in significant reductions in fishes, invertebrates and fleshy macroalgae, increases in the exposure of encrusting coralline algae [12,31], and elevated benthic irradiances [14]. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 Results We used benthic chambers to study patterns of GPP, Re, and NEP within remnant kelp forests, urchin barrens, and habitats that were in transition to becoming urchin barrens (i.e., they had lost all benthic fleshy macroalgae but still had abundant stands of the canopy-forming Eualaria fistulosa; Fig 1) at nine islands spanning more than 1000 kilometers of the Aleutian Archipel- ago (Fig 2, Table 1). Kelp forests and urchin barrens occur as alternate stable states of one another, often with sharply delineated boundaries between them, and exhibit little-to-no over- lap in community assemblages [15,33] (Fig 1). Indeed, the benthic communities within our chambers reflected these assemblages, with the chambers deployed in the kelp forests having more than a 10-fold greater biomass of fleshy macroalgae, which were predominantly stipitate kelps, than those deployed in the urchin barrens, and the chambers deployed in the urchin bar- rens having a nearly 3-fold greater biomass of urchins than those deployed in the kelp forests (Figs 3 and 4). The chambers deployed within transition habitats contained high abundances of urchins and little-to-no fleshy macroalgae, except for the canopy-forming E. fistulosa. The chambers within all three habitats had high bottom covers of encrusting coralline algae below the fleshy macroalgae, which became exposed following deforestation. Benthic irradiances, measured as photosynthetically active radiation (PAR), varied among the three habitat types (ANOVA: F2,14 = 4.826, p = 0.025), but this was variable among the nine islands and two study years (HabitatIsland(Year) interaction: F14,33, = 4.426, p <0.001; Table 2). Generally, PAR was greatest in the urchin barrens, lowest in the kelp forests, and intermediate in the transition habitats (Fig 5). We examined how GPP, Re and NEP, and the balance between GPP and Re differed among the habitat types by measuring changes in seawater oxygen concentrations within replicate (n = 3) chambers (collapsible benthic incubation tents; hereafter cBITs) that were placed on the benthos over representative assemblages within each habitat type at each island. We pre- dicted that NEP at the benthos would be reduced in the urchin barrens due to the loss of pho- tosynthetic macroalgae. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 Results Instead, we found that NEP did not differ between the habitat types ANOVA: F2,14 = 0.530, p = 0.600), nor did it differ from zero (i.e., GPP = Re) in any of the PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 3 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity (NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity (NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity (NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. https://doi.org/10.1371/journal.pone.0226173.g002 Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity (NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. https://doi.org/10.1371/journal.pone.0226173.g002 https://doi.org/10.1371/journal.pone.0226173.g002 habitat types (Table 2, Figs 6 and 7). The effects of habitat type on NEP, however, varied among the nine islands visited during the two study years (HabitatIsland(Year) interaction: F14,41, = 4.541, p < 0.001; Table 2). Specifically, NEP was greater in the urchin barrens than in the kelp forests on five of the islands and lower in the urchin barrens on three of the islands, with the average difference being 25.58 ± 373.26 mg O2 m-2 day-1 lower in the urchin barrens (i.e., the deforested habitats) (Tables 3 and 4). The change on one island (Attu) was not deter- mined due to lost replication (Tables 1 and 4). Results However, when averaged across all nine islands, NEP was generally lowest (-239.73 ± 425.16 mg O2 m-2 day-1, mean ± SE) in the kelp forests, highest (-59.60 ± 145.32 mg O2 m-2 day-1) in the transition habitats, and intermediate (-120.08 ± 338.07 mg O2 m-2 day-1) in the urchin barrens (Table 3, Fig 4). Benthic GPP also did not vary among the habitat types (ANOVA: F2, 14 = 0.234, p = 0.794), but when averaged across islands, GPP was highest in the kelp forests (1,806.14 ± 521.75 mg O2 m-2 day-1; mean ± SE), lowest in the urchin barrens (1,367.77 ± 483.99 mg O2 m-2 day-1), and intermedi- ate in the transition habitats (1,494.22 ± 452.41 mg O2 m-2 day-1) (Fig 4; Table 3). Like NEP, the effects of habitat type varied among the nine islands visited in the two study years (Habi- tatIsland(Year) interactions: F14,41 = 2.166, p = 0.028; Table 2). Specifically, GPP was lower in habitat types (Table 2, Figs 6 and 7). The effects of habitat type on NEP, however, varied among the nine islands visited during the two study years (HabitatIsland(Year) interaction: F14,41, = 4.541, p < 0.001; Table 2). Specifically, NEP was greater in the urchin barrens than in the kelp forests on five of the islands and lower in the urchin barrens on three of the islands, with the average difference being 25.58 ± 373.26 mg O2 m-2 day-1 lower in the urchin barrens (i.e., the deforested habitats) (Tables 3 and 4). The change on one island (Attu) was not deter- mined due to lost replication (Tables 1 and 4). However, when averaged across all nine islands, NEP was generally lowest (-239.73 ± 425.16 mg O2 m-2 day-1, mean ± SE) in the kelp forests, highest (-59.60 ± 145.32 mg O2 m-2 day-1) in the transition habitats, and intermediate (-120.08 ± 338.07 mg O2 m-2 day-1) in the urchin barrens (Table 3, Fig 4). Benthic GPP also did not vary among the habitat types (ANOVA: F2, 14 = 0.234, p = 0.794), but when averaged across islands, GPP was highest in the kelp forests (1,806.14 ± 521.75 mg O2 m-2 day-1; mean ± SE), lowest in the urchin barrens (1,367.77 ± 483.99 mg O2 m-2 day-1), and intermedi- ate in the transition habitats (1,494.22 ± 452.41 mg O2 m-2 day-1) (Fig 4; Table 3). Results Like NEP, the effects of habitat type varied among the nine islands visited in the two study years (Habi- tatIsland(Year) interactions: F14,41 = 2.166, p = 0.028; Table 2). Specifically, GPP was lower in habitat types (Table 2, Figs 6 and 7). The effects of habitat type on NEP, however, varied among the nine islands visited during the two study years (HabitatIsland(Year) interaction: F14,41, = 4.541, p < 0.001; Table 2). Specifically, NEP was greater in the urchin barrens than in the kelp forests on five of the islands and lower in the urchin barrens on three of the islands, with the average difference being 25.58 ± 373.26 mg O2 m-2 day-1 lower in the urchin barrens (i.e., the deforested habitats) (Tables 3 and 4). The change on one island (Attu) was not deter- mined due to lost replication (Tables 1 and 4). However, when averaged across all nine islands, NEP was generally lowest (-239.73 ± 425.16 mg O2 m-2 day-1, mean ± SE) in the kelp forests, highest (-59.60 ± 145.32 mg O2 m-2 day-1) in the transition habitats, and intermediate (-120.08 ± 338.07 mg O2 m-2 day-1) in the urchin barrens (Table 3, Fig 4). Benthic GPP also did not vary among the habitat types (ANOVA: F2, 14 = 0.234, p = 0.794), but when averaged across islands, GPP was highest in the kelp forests (1,806.14 ± 521.75 mg O2 m-2 day-1; mean ± SE), lowest in the urchin barrens (1,367.77 ± 483.99 mg O2 m-2 day-1), and intermedi- ate in the transition habitats (1,494.22 ± 452.41 mg O2 m-2 day-1) (Fig 4; Table 3). Like NEP, the effects of habitat type varied among the nine islands visited in the two study years (Habi- tatIsland(Year) interactions: F14,41 = 2.166, p = 0.028; Table 2). Specifically, GPP was lower in PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 4 / 21 PLOS ONE Deforestation and loss of ecosystem function the urchin barrens than in the kelp forests on all but two of the islands, by an average of 461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Results Re also did not vary among the habitat types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ± 546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2 Table 1. List of the nine islands in the Aleutian Archipelago where cBITs were deployed to measure NEP, GPP and Re during 2016 and 2017, and the six islands where all macroalgae and invertebrates were collected from within the cBITs to estimate their biomass during 2016. The number cBITs deployed, the deployment year, and whether macroalgae and invertebrates were collected from within the cBITs at each island are noted. Island Year No. cBITs deployed Collections made? Kelp Transition Urchin forests habitats barrens Adak 2016 3 2 3 No Amchitka 2017 3 2 3 Yes Atka 2017 3 3 2 Yes Attu 2017 3 3 1 Yes Chuginadak 2016 2 2 3 Yes Kiska 2017 3 3 2 Yes Nizki 2017 3 3 2 Yes Tanaga 2016 2 1 2 No Yunaska 2017 3 3 3 No Totals 25 22 21 6 https://doi.org/10.1371/journal.pone.0226173.t001 Table 1. List of the nine islands in the Aleutian Archipelago where cBITs were deployed to measure NEP, GPP and Re during 2016 and 2017, and the six islands where all macroalgae and invertebrates were collected from within the cBITs to estimate their biomass during 2016. The number cBITs deployed, the deployment year, and whether macroalgae and invertebrates were collected from within the cBITs at each island are noted. the urchin barrens than in the kelp forests on all but two of the islands, by an average of 461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Re also did not vary among the habitat types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ± 546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2 Table 1. Results List of the nine islands in the Aleutian Archipelago where cBITs were deployed to measure NEP, GPP and Re during 2016 and 2017, and the six islands where all macroalgae and invertebrates were collected from within the cBITs to estimate their biomass during 2016. The number cBITs deployed, the deployment year, and whether macroalgae and invertebrates were collected from within the cBITs at each island are noted. Island Year No. cBITs deployed Collections made? Kelp Transition Urchin forests habitats barrens Adak 2016 3 2 3 No Amchitka 2017 3 2 3 Yes Atka 2017 3 3 2 Yes Attu 2017 3 3 1 Yes Chuginadak 2016 2 2 3 Yes Kiska 2017 3 3 2 Yes Nizki 2017 3 3 2 Yes Tanaga 2016 2 1 2 No Yunaska 2017 3 3 3 No Totals 25 22 21 6 https://doi.org/10.1371/journal.pone.0226173.t001 Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during 2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi.org/10.1371/journal.pone.0226173.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 5 / 21 the urchin barrens than in the kelp forests on all but two of the islands, by an average of 461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Re also did not vary among the habitat types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ± 546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2 the urchin barrens than in the kelp forests on all but two of the islands, by an average of 461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Re also did not vary among the habitat types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ± 546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2 Fig 3. Algae and invertebrate biomass. https://doi.org/10.1371/journal.pone.0226173.t001 Deforestation and loss of ecosystem function Fig 4. Algae and invertebrate biomass. Mean biomass (± SE) of (A) all kelps, and red, brown and green macroalgae, and (B) the most abundant taxonomic groups of invertebrates collected from within the cBITs in each habitat type at six of the islands where the cBITs were deployed in 2017 (Table 1). Fig 5B is divided into two panels, with abundant taxa on the left panel, and rarer taxa on the right panel. https://doi org/10 1371/journal pone 0226173 g004 Fig 4. Algae and invertebrate biomass. Mean biomass (± SE) of (A) all kelps, and red, brown and green macroalgae, and (B) the most abundant taxonomic groups of invertebrates collected from within the cBITs in each habitat type at six of the islands where the cBITs were deployed in 2017 (Table 1). Fig 5B is divided into two panels, with abundant taxa on the left panel, and rarer taxa on the right panel. Table 2. Results of separate three-way nested analyses of variance testing for differences in A) net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem respiration (Re), D) the range between GPP and Re, and E) irradiance (PAR) among the two sample years, nine islands, and three habitat types (kelp forests, transition habitats, and urchin barrens). For each analysis, year and habitat type were fixed factors, and island nested within year was a random factor. The model r2 is given for each analysis. ( ) ) y p ( ) ) g ) ( ) g p y yp (kelp forests, transition habitats, and urchin barrens). For each analysis, year and habitat type were fixed factors, and island nested within year was a random factor. The model r2 is given for each analysis. https://doi.org/10.1371/journal.pone.0226173.t002 Results Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during 2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi org/10 1371/journal pone 0226173 g003 Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during 2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi.org/10.1371/journal.pone.0226173.g003 Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during 2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during 2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi.org/10.1371/journal.pone.0226173.g003 https://doi.org/10.1371/journal.pone.0226173.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 5 / 21 PLOS ONE Deforestation and loss of ecosystem function 6 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 PLOS ONE Deforestation and loss of ecosystem function Deforestation and loss of ecosystem function PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 Deforestation and loss of ecosystem function A) NEP (r2 = 0.74) Source Type III SS df Mean Squares F-ratio p-value Year 146.911 1 146.911 8.319 0.006 Habitat 85.076 2 42.538 0.53 0.6 HabitatYear 365.04 2 182.52 10.336 <0.001 Island(Year) 610.045 7 87.149 4.935 <0.001 HabitatIsland(Year) 1122.729 14 80.195 4.541 <0.001 Error 724.009 41 17.659 B) GPP (r2 = 0.72) Source Type III SS df Mean Squares F-ratio p-value Year 1060.514 1 1060.514 15 <0.001 Habitat 71.658 2 35.829 0.234 0.794 HabitatYear 416.847 2 208.424 2.948 0.064 Island(Year) 3497.967 7 499.71 7.068 <0.001 HabitatIsland(Year) 2144.29 14 153.164 2.166 0.028 Error 2898.811 41 70.703 C) Re (r2 = 0.78) Source Type III SS df Mean Squares F-ratio p-value Year 1456.821 1 1456.821 24.825 <0.001 Habitat 125.51 2 62.755 0.39 0.684 HabitatYear 946.319 2 473.16 8.063 0.001 Island(Year) 3081.525 7 440.218 7.501 <0.001 HabitatIsland(Year) 2254.327 14 161.023 2.744 0.006 Error 2406.048 41 58.684 D) Range (r2 = 0.75) Source Type III SS df Mean Squares F-ratio p-value Year 2503.758 1 2503.758 20.237 <0.001 Habitat 184.788 2 92.394 0.318 0.733 HabitatYear 1267.372 2 633.686 5.122 0.01 Island(Year) 6204.846 7 886.407 7.164 <0.001 HabitatIsland(Year) 4064.628 14 290.331 2.347 0.017 Error 5072.698 41 123.724 E) PAR (r2 = 0.83) Source Type III SS df Mean Squares F-ratio p-value Year 0.898 1 0.898 8.503 0.006 Habitat 4.507 2 2.254 4.826 0.025 HabitatYear 1.231 2 0.616 5.832 0.007 Island(Year) 2.066 7 0.295 2.795 0.021 HabitatIsland(Year) 6.542 14 0.467 4.426 <0.001 https://doi.org/10.1371/journal.pone.0226173.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 7 / 21 PLOS ONE Deforestation and loss of ecosystem function Deforestation and loss of ecosystem PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 8 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 5. Production metrics. Box plots showing (A) Net Ecosystem Production (NEP), (B) Gross Primary Production (GPP), (C) Ecosystem Respiration (Re), (D) the range between GPP and Re (Range), and (E) Irradiance (PAR), as measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at nine islands during 2016 and 2017 (Fig 2, Table 1). Red diamonds represent mean values, and horizontal lines represent median values. Boxes within each graph that do not share letters represent significant differences between habitat pairs. https://doi.org/10.1371/journal.pone.0226173.g005 day-1) (Fig 4; Table 3). Deforestation and loss of ecosystem function As with NEP and GPP, the effects of habitat type varied among the nine islands visited in the two study years (HabitatIsland(Year) interactions: F2,14 = 2.744, p = 0.006; Table 2). Specifically, Re was lower in the urchin barrens than in the kelp forests on four of the islands and greater in the urchin barrens on four of the islands, with the average difference being 472.09 ± 734.70 mg O2 m-2 day-1 lower in the urchin barrens (Table 4). Lastly, the range between GPP and Re, which we believe to be a better measure of ecosystem function regarding productivity than NEP alone, did not differ among the habitat types (ANOVA: F2,14 = 0.318, p = 0.733), but was again greatest in the kelp forests (3,750 ± 1,069.01 mg O2 m-2 day-1), lowest in the urchin barrens (2,860.94 ± 994.44 mg O2 m-2 day-1), and intermediate in the transition habitats (3,047.98 ± 910.36 mg O2 m-2 day-1) (Table 3, Fig 4). This again varied among the study islands visited in the two study years (HabitatIsland(Year) interactions: F14,41 = 2.347, p = 0.017; Table 2). Specifically, the range between GPP and Re was lower in the urchin barrens than in the kelp forests on five of the islands and greater in the urchin barrens on two of the islands, with the average difference being 933.69 ± 1,262.65 mg O2 m-2 day-1 lower in the urchin barrens (Table 4). Although the effects of deforestation on all three metrics of productivity varied among the islands visited in the two study years, some general patterns were evident. When considered across all nine islands, GPP, Re and the range between GPP and Re were each greatest in the kelp forests, intermediate in the transition habitats, and lowest in the urchin barrens. Specifi- cally, GPP was 24% higher, on average, in the kelp forests than in the urchin barrens, and 17% higher, on average, in the kelp forests than in the transition habitats, but it differed by only 7% between the transition habitats and urchin barrens (Table 3, Fig 4). Benthic Re was 26% higher, on average, in the kelp forests than in the urchin barrens, and 22% higher in the kelp forest than the transition habitats, but it differed by less than 1% between the transition habitats and Fig 6. GPP versus Re. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 Deforestation and loss of ecosystem function Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. https://doi.org/10.1371/journal.pone.0226173.g007 https://doi.org/10.1371/journal.pone.0226173.g007 https://doi.org/10.1371/journal.pone.0226173.g007 the urchin barrens. The range was between GPP and Re was 24% greater, on average, in the kelp forests than in the urchin barrens, and 19% greater in the kelp forests than in the transi- tion habitats, but it varied by less than 6% between the transition habitats and the urchin bar- rens. In contrast, GPP was, on average, greatest in the urchin barrens, intermediate in the transition habitats, and lowest in the kelp forests in contrast, but it did not differ from zero (i.e. GPP = Re) in any of the habitats. These patterns, however, were highly variable among the dif- ferent islands visited in the two study years for each of the production metrics. Altogether, this indicated deforestation resulted in widespread but geographically variable losses to primary production and respiration by the ecosystem. As with previous studies in aquatic ecosystems, we found that GPP and Re are generally in balance, resulting in exhibit GPP / Re ratios near 1.0, and NEP values near zero [21,22]. When examined within each cBIT separately, GPP and Re were consistently similar in magnitude with no differences in GPP / Re ratios among habitat types (ANCOVA: F2,62 = 0.16, p = 0.852) (Table 5, Fig 6). Further, the distribution of these ratios was symmetrical around 1.0 in each habitat (Fig 7). Interestingly, the highest individual values of NEP were not observed in the kelp forests but rather in the urchin barrens, which we believe was due to higher irradiances in the urchin barrens than the other two habitats (Fig 5) combined with compensatory produc- tion by the encrusting coralline algae and benthic diatoms [30]. However, those few observa- tions aside, it is clear that all three benthic habitats remain in balance following deforestation, with GPP  Re, GPP / Re ratios  1, and median NEP values  0. Thus, although NEP may help differentiate between productive and unproductive ecosystems [22], it poorly describes changes in primary productivity following large-scale habitat change in the Aleutian Archipel- ago. Deforestation and loss of ecosystem function Instead, it is clear that deforestation results in significant changes to the region’s benthic communities, and these led to geographically variable reductions in GPP, Re and the range between them, which better reflect a reduction in ecosystem functioning. Further, it appears that even partial deforestation, where the benthic macroalgae and invertebrates have been lost but the canopy-forming kelps remain, results in lower GPP and Re at the benthos that is simi- lar to trends found in urchin barrens. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 Deforestation and loss of ecosystem function Relationship between gross primary production (GPP) and ecosystem respiration (Re) for each habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each point represents measurements from a single cBIT. Gray shading denoted 95% confidence intervals. https://doi.org/10.1371/journal.pone.0226173.g006 Fig 6. GPP versus Re. Relationship between gross primary production (GPP) and ecosystem respiration (Re) for each habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each point represents measurements from a single cBIT. Gray shading denoted 95% confidence intervals. https://doi.org/10.1371/journal.pone.0226173.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 9 / 21 PLOS ONE Deforestation and loss of ecosystem function ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 10 / 21 Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. https://doi.org/10.1371/journal.pone.0226173.g007 Deforestation and loss of ecosystem function PLOS ONE Deforestation and loss of ecosystem function Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Discussion Trophic interactions can lead to changes in the distribution and abundance of habitat-forming species, which can have profound impacts on ecosystem function [2,9]. Deforestation, in par- ticular, can result in changes to biodiversity and energy flow [2], altered regional and global cli- mates [34], and even lead to species extinctions [35]. Coastal kelps are a pertinent example of such ecosystem engineers in nearshore habitats that have suffered declines in some locations over the past few decades due to both biological and physical stressors [10,11,36–39]. Conse- quently, while kelp populations remain stable in many of the world’s ecoregions [10,40,41], or may even be expanding in some high latitude regions in response to ocean warming [39,42], our study is relevant to many areas of the world where kelp forests have exhibited local to broad scale declines [10,43–47]. Indeed, recent estimates suggest that global declines in kelp abundances may be as high as 2% per year [11], which can negatively affect numerous other species that depend on them for food and habitat. Certainly, the kelp forests of the Aleutian Archipelago are in critical condition in the face of widespread overgrazing by urchins, and this PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 11 / 21 PLOS ONE Deforestation and loss of ecosystem function Table 3. Community productivity values (measured as mg O2 m-2 day-1) for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem respiration (Re), and D) the Range between GPP and Re (Range) estimated for each habitat on each island. Data reflect the means (SD) of the replicate chambers in each habitat (kelp Forests, Urchin Barrens and Transition Habitats). Positive values for NEP reflect net oxygen production and negative values reflect oxygen consumption (net respiration). Negative values for Re reflect oxygen consumption (i.e. more negative values reflect greater respiration by the ecosystem). “NA” denotes not available due to lack of replication (i.e. data are based on only one chamber at that island; see Table 1). Table 3. Community productivity values (measured as mg O2 m-2 day-1) for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem respiration (Re), and D) the Range between GPP and Re (Range) estimated for each habitat on each island. Data reflect the means (SD) of the replicate chambers in each habitat (kelp Forests, Urchin Barrens and Transition Habitats). PLOS ONE (Continued) Chuginadak 1797.40 (188.72) 3576.43 (462.80) 2057.03 (15.29) Kiska 2606.00 (1597.22) 1589.48 (613.66) 1665.17 (653.88) Nizki 6135.68 (1014.08) 2281.17 (1026.88) 4293.65 (1143.33) Tanaga 5710.56 (1538.90) 5682.77 (515.01) 5201.03 (NA) Yunkasa 890.72 (121.60) 1115.60 (424.16) 3042.63 (1262.13) Average 3750.03 2860.94 3047.98 SE 1069.01 994.44 910.94 https://doi.org/10.1371/journal.pone.0226173.t003 has had profound effects on the region’s benthic communities and on patterns of gross pri- mary production and ecosystem respiration. Whether these forests will recover and return to prior ecosystem functioning regarding these metrics is unknown, but observations of kelp for- ests from other areas of the world suggest it is possible. For example, Laminaria longicruris for- ests recovered from overgrazing following localized disease outbreaks that decimated sea urchin populations in Nova Scotia [48], while L. hyperborea forests recovered in mid-Norway due to low sea urchin recruitment [49]. Ecklonia maxima expanded its range eastward in South Africa, coincident with cooling of the local ocean waters [50]. Likewise, Macrocystis pyri- fera recovered along a ~100 km stretch of the Pacific coast of Baja California, Mexico following nearly two decades of absence after the strong 1997–98 El Niño Southern Oscillation [51]. Recovery of the Eualaria fistulosa forests throughout the Aleutian Archipelago, however, would likely require widespread mortality in the urchin populations, which today seems unlikely. One potential contributing factor for this may lie in the low abundance of other urchin predators, such as the urchin eating sea star, Pycnopodia helianthoides [38,52,53], which historically has not been found in high abundances in the central or western Aleutians. Therefore, until predation on the urchins recovers or the urchin populations suffer widespread disease that reduces their numbers, benthic algal abundances, GPP and Re will likely remain generally lower in areas of kelp forest loss because the high abundance of urchins limits regrowth of macroalgae and maintains the urchin barrens [15]. Thus, we present a benchmark against which we can evaluate this recovery if it occurs, and understand the effects of further deforestation in this ecosystem. Although we have learned much about the effects of the otter-urchin-kelp trophic cascade in the Aleutian Archipelago, this study offers new insights into the consequences of such wide- spread deforestation on the region’s benthic primary productivity. Certainly, benthic GPP, Re and the range between them are generally greatest in the kelp forests where macroalgae, fish, invertebrates, and microbial communities are all most abundant [15,23–26,33], while they are lowest in the urchin barrens. PLOS ONE PLOS ONE Deforestation and loss of ecosystem function has had profound effects on the region’s benthic communities and on patterns of gross pri- mary production and ecosystem respiration. Whether these forests will recover and return to prior ecosystem functioning regarding these metrics is unknown, but observations of kelp for- ests from other areas of the world suggest it is possible. For example, Laminaria longicruris for- ests recovered from overgrazing following localized disease outbreaks that decimated sea urchin populations in Nova Scotia [48], while L. hyperborea forests recovered in mid-Norway due to low sea urchin recruitment [49]. Ecklonia maxima expanded its range eastward in South Africa, coincident with cooling of the local ocean waters [50]. Likewise, Macrocystis pyri- fera recovered along a ~100 km stretch of the Pacific coast of Baja California, Mexico following nearly two decades of absence after the strong 1997–98 El Niño Southern Oscillation [51]. Recovery of the Eualaria fistulosa forests throughout the Aleutian Archipelago, however, would likely require widespread mortality in the urchin populations, which today seems unlikely. One potential contributing factor for this may lie in the low abundance of other urchin predators, such as the urchin eating sea star, Pycnopodia helianthoides [38,52,53], which historically has not been found in high abundances in the central or western Aleutians. Therefore, until predation on the urchins recovers or the urchin populations suffer widespread disease that reduces their numbers, benthic algal abundances, GPP and Re will likely remain generally lower in areas of kelp forest loss because the high abundance of urchins limits regrowth of macroalgae and maintains the urchin barrens [15]. Thus, we present a benchmark against which we can evaluate this recovery if it occurs, and understand the effects of further deforestation in this ecosystem. Al h h h l d h b h ff f h hi k l hi d Table 3. (Continued) Chuginadak 1797.40 (188.72) 3576.43 (462.80) 2057.03 (15.29) Kiska 2606.00 (1597.22) 1589.48 (613.66) 1665.17 (653.88) Nizki 6135.68 (1014.08) 2281.17 (1026.88) 4293.65 (1143.33) Tanaga 5710.56 (1538.90) 5682.77 (515.01) 5201.03 (NA) Yunkasa 890.72 (121.60) 1115.60 (424.16) 3042.63 (1262.13) Average 3750.03 2860.94 3047.98 SE 1069.01 994.44 910.94 https://doi.org/10.1371/journal.pone.0226173.t003 Table 3. Discussion Positive values for NEP reflect net oxygen production and negative values reflect oxygen consumption (net respiration). Negative values for Re reflect oxygen consumption (i.e. more negative values reflect greater respiration by the ecosystem). “NA” denotes not available due to lack of replication (i.e. data are based on only one chamber at that island; see Table 1). (net respiration). Negative values for Re reflect oxygen consumption (i.e. more negative values reflect greater respiration by the ecosystem). “NA” denotes not available due to lack of replication (i.e. data are based on only one chamber at that island; see Table 1). NEP Island Kelp Barren Transition Adak -851.56 (409.34) -431.10 (184.56) -580.85 (89.91) Amchitka -694.81 (217.86) -166.91 (141.38) -151.16 (86.17) Atka -434.43 (72.69) -231.05 (137.95) -208.92 (143.52) Attu -176.96 (167.98) 1104.59 (NA) 98.52 (151.30) Chuginadak 37.74 (15.07) -537.29 (80.57) 105.85 (9.02) Kiska 193.70 (93.03) 418.16 (58.16) 160.13 (63.01) Nizki -608.20 (177.58) -20.29 (102.59) -84.82 (78.95) Tanaga 355.08 (361.67) -909.49 (854.06) 254 (NA) Yunkasa 21.82 (21.50) -307.36 (96.49) -129.13 (132.22) Average -239.74 -120.08 -59.6 SE 245.47 338.07 145.32 GPP Island Kelp Barren Transition Adak 2018.08 (934.63) 2296.04 (1164.46) 2246.55 (184.47) Amchitka 977.21 (552.29) 807.74 (304.87) 416.49 (188.14) Atka 2450.84 (1340.11) 315.90 (60.74) 675.59 (266.57) Attu 2238.97 (479.65) 2445.60(NA) 1826.29 (1482.52) Chuginadak 917.57 (86.83) 1519.57 (199.70) 1081.44 (3.13) Kiska 1399.85 (823.96) 1003.82 (335.91) 912.65 (347.63) Nizki 2763.69 (420.47) 1130.44 (564.73) 2104.32 (610.25) Tanaga 3032.82 (588.61) 2386.64 (684.53) 2727.91 (NA) Yunkasa 456.27 (71.52) 404.17 (169.92) 1456.75 (627.00) Average 1806.14 1367.77 1494.22 SE 521.75 483.99 452.41 Re Island Kelp Barren Transition Adak -2410.85 (763.16) -2439.14(1273.10) -2827.40 (94.57) Amchitka -1672.02 (334.43) -974.65 (184.45) -567.65 (101.96) Atka -2885.26 (1268.85) -546.95 (77.21) -884.52 (406.-6) Attu -2415.93 (564.65) -1509.01 (NA) -1727.77 (1313.82) Chuginadak -879.83 (101.90) -2056.86 (265.44) -975.60 (12.15) Kiska -1206.15 (775.20) -585.66 (277.75) -753.52 (308.09) Nizki -3371.99 (594.26) -1150.73 (462.14) -2189.14 (533.21) Tanaga -2677.74 (950.28) -3296.13 (169.53) -2473.12 (NA) Yunkasa -434.45 (50.10) -711.43 (256.39) -1585.88 (641.96) Average 1994.91 1474.51 1553.84 SE 574.11 546.83 469.81 Range Island Kelp Barren Transition Adak 3969.73 (1770.87) 4735.18 (2432.99) 5073.94 (279.04) Amchitka 2649.23 (886.72) 1782.39 (483.68) 984.15 (290.10) Atka 5336.10 (2608.91) 862.85 (16.48) 1560.11 (671.78) Attu 4654.89 (1033.53) 4122.62 (NA) 3554.07 (2864.05) (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 12 / 21 PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 PLOS ONE Deforestation thus resulted in overall reductions in each of these metrics, identifying a general loss of ecosystem function. This, however, was geographically variable, with some islands showing elevated primary production following deforestation, which we believe is due to higher irradiances combined with compensatory production by microalgae (e.g. diatoms) and the coralline algal crusts. Indeed, we observed some of the high- est production values in a few of the barrens cBITs where diatom mats formed within the chambers during the deployments. These cBITs also tended to have low numbers of urchins within them, and the chambers therefore appeared to exclude urchins from grazing the micro- algae. In contrast, benthic primary productivity and respiration by the ecosystem are all similar in the urchin barrens and transition habitats, which have similarly high abundances of urchins and low biomasses of macroalgae [15,33], suggesting that the transition habitats have already suffered reduced ecosystem functioning. This, of course, reflects patterns at the benthos and PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 13 / 21 PLOS ONE Deforestation and loss of ecosystem function Table 4. The effects of habitat change on patterns of productivity for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem respi- ration (Re), and D the Range between GPP and Re (i.e. Range) estimated for reach island. “Change” reflects absolute differences in each metric (measured as mg O2 m- 2 day-1) as the habitat transitions from Kelp forests to Transition Habitats, Transition Habitats to Urchin Barrens, Kelp Forests to Urchin Barrens (i.e. the total change due to deforestation). Positive values denote greater values for that metric and negative values denote lower values for that metric. NA denotes comparison “not available” due to loss of replicates in one habitat that precluded reliable estimates of the change (see Table 1). At the bottom of each table are the average values and standard errors. Table 4. The effects of habitat change on patterns of productivity for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem respi- ration (Re), and D the Range between GPP and Re (i.e. Range) estimated for reach island. “Change” reflects absolute differences in each metric (measured as mg O2 m- 2 day-1) as the habitat transitions from Kelp forests to Transition Habitats, Transition Habitats to Urchin Barrens, Kelp Forests to Urchin Barrens (i.e. the total change due to deforestation). PLOS ONE Combining these with estimates of macroalgal and invertebrate diversity and abundance revealed that the Aleutian Archipelago suffered geographically variable losses to ecosystem function following widespread deforestation. PLOS ONE PLOS ONE Deforestation and loss of ecosystem function Table 4. (Continued) Chuginadak 259.63 1519.4 1779.03 Kiska -940.83 -75.69 -1016.52 Nizki -1842.03 -2012.48 -3854.51 Tanaga NA NA -27.79 Yunkasa 2151.91 -1927.03 224.88 Average -726.13 -390.51 -933.69 SE 1073.93 754.56 1262.65 htt //d i /10 1371/j l 0226173 t004 not in the mid-water or at the surface where the canopy-forming Eualaria fistulosa remains abundant in the transition habitats. It is likely that these canopy-forming macroalgae would enhance GPP and perhaps result in positive values of NEP in the mid-water and at the surface in both the kelp forests and transition habitats. However, at the benthos, GPP and Re remain in balance following deforestation, leading to similar, near-zero NEP in all three habitats. We believe this reflects balance between the autotrophic and heterotrophic components of the eco- system. Specifically, the macroalgae exhibit positive GPP as they photosynthesize, grow and increase in abundance, but this results in a concomitant increase in heterotrophic metabolism, which enhances Re. In the face of deforestation, both GPP and Re are reduced, resulting in little to no changes in NEP. Thus, we propose that, GPP, Re and the range between them are better measures of changes to primary productivity than NEP. Combining these with estimates of macroalgal and invertebrate diversity and abundance revealed that the Aleutian Archipelago suffered geographically variable losses to ecosystem function following widespread deforestation. not in the mid-water or at the surface where the canopy-forming Eualaria fistulosa remains abundant in the transition habitats. It is likely that these canopy-forming macroalgae would enhance GPP and perhaps result in positive values of NEP in the mid-water and at the surface in both the kelp forests and transition habitats. However, at the benthos, GPP and Re remain in balance following deforestation, leading to similar, near-zero NEP in all three habitats. We believe this reflects balance between the autotrophic and heterotrophic components of the eco- system. Specifically, the macroalgae exhibit positive GPP as they photosynthesize, grow and increase in abundance, but this results in a concomitant increase in heterotrophic metabolism, which enhances Re. In the face of deforestation, both GPP and Re are reduced, resulting in little to no changes in NEP. Thus, we propose that, GPP, Re and the range between them are better measures of changes to primary productivity than NEP. PLOS ONE Positive values denote greater values for that metric and negative values denote lower values for that metric. NA denotes comparison “not available” due to loss of replicates in one habitat that precluded reliable estimates of the change (see Table 1). At the bottom of each table are the average values and standard errors. to loss of replicates in one habitat that precluded reliable estimates of the change (see Table 1). At the bottom of each table are the average values and standard errors. A) NEP Kelp to Transition Transition to Barren Kelp to Barren Island Change Change Change Adak 270.71 808.46 420.46 Amchitka 845.97 -318.07 527.9 Atka 225.51 -22.13 203.38 Attu 275.48 NA NA Chuginadak 68.11 -643.14 -575.03 Kiska -33.57 258.03 224.46 Nizki 523.38 64.53 587.91 Tanaga NA NA -1264.57 Yunkasa -150.95 -178.23 -329.18 Average 253.08 -4.36 -25.58 SE 183.33 265.76 373.26 B) GPP Kelp to Transition Transition to Barren Kelp to Barren Island Change Change Change Adak 687.67 49.49 737.16 Amchitka -560.72 391.25 -169.47 Atka -1775.25 -359.69 -2134.94 Attu -412.68 NA NA Chuginadak 163.87 438.13 602 Kiska -487.2 91.17 -396.03 Nizki -659.37 -973.88 -1633.25 Tanaga NA NA -646.18 Yunkasa 1000.48 -1052.58 -52.1 Average -255.4 -202.3 -461.6 SE 501.77 353.95 578.69 C) Re Kelp to Transition Transition to Barren Kelp to Barren Island Change Change Change Adak 416.55 -388.26 28.29 Amchitka -1104.37 407 -697.37 Atka -2000.74 -337.57 -2338.31 Attu -688.16 NA NA Chuginadak 95.77 1081.26 1177.03 Kiska -452.63 -167.86 -620.49 Nizki -1182.85 -1038.41 -2221.26 Tanaga NA NA 618.39 Yunkasa 1151.43 -874.45 276.98 Average -470.63 -188.33 -472.09 SE 578.86 423.11 734.7 D) Range Kelp to Transition Transition to Barren Kelp to Barren Island Change Change Change Adak 1104.21 -338.76 765.45 Amchitka -1665.08 798.24 -866.84 Atka -3775.99 -697.26 -4473.25 Attu -1100.82 NA NA (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 14 / 21 PLOS ONE Experimental design Our cBITs were made from 0.106 cm polycarbonate plastic triangle sheets glued to fiberglass- reinforced vinyl panels (Fig 8). The frames were reinforced using stainless steel tubes with stainless steel cable to facilitate handling and to ensure they held their pyramidal shape with an internal volume of 192 L and a basal area of (0.64 m2). The cBITs each had 26” skirts around the perimeter, upon which chain was laid to hold them to the benthos and prevent water exchange with the surrounding environment. This was verified by injecting fluorescein dye into the chambers and examining the perimeters for leaks. The polycarbonate walls were thin and flexible to allow hydrodynamic energy transfer into the cBITs, thereby reducing boundary layer formation around the macroalgal thalli. We verified this energy transfer using dissolving plaster blocks placed within cBITs, and by using video analysis of internal seaweed and fluores- cein dye movements within the chambers relative to seaweeds outside them [60]. Sensor arrays that included a Photosynthetic Active Radiation (PAR) sensor (Odyssey Dataflow Systems Ltd), and a Dissolved Oxygen (DO mg/L) and Temperature (˚C) sensor (MiniDOT Logger, PME) were placed at the center of each cBIT (Fig 8). During two cruises aboard the R/V Oceanus in 2016 and 2017, we deployed cBITs in each of the three habitats (kelp forest, urchin barrens, transition habitats) on each of nine islands (Table 1; Figs 1,2 and 8) for 36-hour periods to measure both day and night patterns of NEP and Re, and to ensure we captured a complete diurnal cycle. These islands span more than 1000 km and therefore experience differences in temperature, salinity, wave exposure and other biotic factors [62]. Consequently, all cBITs deployments were done in the summer (i.e. July) of each year, in similar depths (i.e. 6–8 m), and under similar wave exposures (i.e. pro- tected from ocean swells) in order to standardize factors that could affect productivity mea- surements. The three habitat types were selected based on non-overlapping community assemblages (i.e., kelp forests were chosen based on abundant E. fistulosa and dense assem- blages of understory macroalgae; transition habitats were chosen based on abundant E. fistu- losa, little-to-no understory macroalgae, and high abundances of urchins; urchin barrens were chosen based on no E. fistulosa, little-to-no understory macroalgae and abundant urchins). These were then grouped in each island to reduce the effects within-island spatial heterogene- ity in other environmental factors. Materials and methods While many past experiments examining primary production by autotrophic communities have relied on laboratory experiments that do not incorporate natural fluctuations in abiotic conditions, recent studies have identified techniques that measure primary production in situ, thereby increasing the ecological realism of their experiments [54–57]. For example, in situ chamber designs have been developed for estimating primary production by individual species [55,56] and whole benthic communities [29,56,57]. In general, estimates of net ecosystem pro- duction (NEP) on the benthos can be made by measuring changes in dissolved oxygen within chambers that are placed in situ over macroalgae and invertebrate communities. In this study, we deployed collapsible benthic isolation tents (cBITs) modelled after those described by Haas et al. [58] and Calhoun et al. [59] that directly measured in situ benthic oxygen production and allowed us to estimate gross primary production (GPP), ecosystem respiration (Re) and net ecosystem production NEP by the benthic communities [28,29,55]. These cBITs were the same ones used by Sullaway and Edwards [60] to measure loss of primary productivity following the Table 5. Analysis of covariance testing the effect of GPP and habitat on Re. Note the non-significant HabitatGPP interaction hat shows no differences in the slopes (i.e. relationships) between GPP and Re among the three habitat types. See Fig 5 for graphical representation. Source Type III SS df MS F-ratio p-value GPP 8.460E+03 1 8.50E+03 3.20E+02 0.001 HABITAT 20.791443 2 1.00E+01 3.90E-01 0.68 HABITATGPP 8.6140845 2 4.30E+00 1.60E-01 0.852 Error 1.66E+03 62 2.70E+01 https://doi.org/10.1371/journal.pone.0226173.t005 Table 5. Analysis of covariance testing the effect of GPP and habitat on Re. Note the non-significant HabitatGPP interac (i.e. relationships) between GPP and Re among the three habitat types. See Fig 5 for graphical representation. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 15 / 21 PLOS ONE Deforestation and loss of ecosystem function displacement of native giant kelp, Macrocystis pyrifera, by the invasive Sargassum horneri on Catalina Island, CA. Further, because our cBITs encompassed whole benthic communities, species interactions (e.g., shading), and invertebrate and microbial respiration were incorpo- rated into production measurements. These interactions are often not captured in laboratory experiments but are pertinent to understanding GPP, Re, and NEP [61]. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 Experimental design For each deployment, three replicate cBITs were placed on the benthos over targeted assemblages within each habitat type. However, occasionally, repli- cates were lost due to logistical difficulties associated with the chamber-benthos seals (Table 1). The water within each cBIT was replaced once per day by opening the side of the chamber and completely replacing the water with new ambient seawater to reduce “chamber effects” (i.e., the build-up of oxygen and depletion of inorganic carbon and nutrients). After each deployment, the chambers and sensors were retrieved. At six of the islands (Table 1), all organisms within each of the chambers’ benthic footprints were collected, brought back to the ship, enumerated and weighed during our 2017 cruise. We measured NEP over the whole diurnal cycle, Re during the nighttime hours, and calculated GPP during the day for each cBIT during each incubation period separately according to Olive´ et al. [57]. Specifically, measure- ments made during the night (the dark) were used to infer rates of Re, which were then com- bined with measurements of NEP to estimate GPP by the autotrophs [18–20]. Ethical PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 16 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 8. Photograph of cBIT. Photograph of (A) cBIT before deployment showing 26” skirt around perimeter, flexible polycarbonate walls, steel framing, anchor chain used to hold skirt and cBIT to the benthos, and (B) cBIT deployed in kelp forest showing PAR and oxygen sensors placed both inside and outside the chamber. https://doi.org/10.1371/journal.pone.0226173.g008 Fig 8. Photograph of cBIT. Photograph of (A) cBIT before deployment showing 26” skirt around perimeter, flexible polycarbonate walls, steel framing, anchor chain used to hold skirt and cBIT to the benthos, and (B) cBIT deployed in kelp forest showing PAR and oxygen sensors placed both inside and outside the chamber. https://doi.org/10.1371/journal.pone.0226173.g008 https://doi.org/10.1371/journal.pone.0226173.g008 Approval: All procedures performed in studies involving fishes were in accordance with the ethical standards of the institution or practice at which the studies were conducted (University of Alaska Fairbanks Institutional Animal Care and Use Committee; Permit Number: 899401– 4). Acknowledgments We thank S. Lamerdin, and the captain and crew of the R/V Oceanus for excellent ship sup- port. We thank J. Estes for offering historical perspectives on the Aleutian kelp ecosystem, and M. Hatay for designing the cBITs. We are grateful to M. Good, S. Traiger, J. Metzger, A. Bland, A. Ravelo, and B. Weitzman for assistance with field operations. We also thank the Alaska Maritime National Wildlife Refuge for logistical support. Author Contributions Conceptualization: Matthew Edwards, Brenda Konar. Conceptualization: Matthew Edwards, Brenda Konar. Data curation: Matthew Edwards, Michael Spector. Data curation: Matthew Edwards, Michael Spector. Formal analysis: Matthew Edwards, Scott Gabara. Formal analysis: Matthew Edwards, Scott Gabara. Formal analysis: Matthew Edwards, Scott Gabara. Funding acquisition: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim. Investigation: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim, Scott Gabara, Genoa Sull- away, Tristin McHugh, Michael Spector, Sadie Small. Methodology: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim. Project administration: Matthew Edwards, Brenda Konar. Supervision: Matthew Edwards. Supervision: Matthew Edwards. Validation: Ju-Hyoung Kim. Validation: Ju-Hyoung Kim. Visualization: Scott Gabara. Visualization: Scott Gabara. Writing – original draft: Matthew Edwards. Writing – original draft: Matthew Edwards. Writing – review & editing: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim, Scott Gabara, Genoa Sullaway, Tristin McHugh, Michael Spector, Sadie Small. Statistical analyses All analyses were done in either Systat ver. 12 or Primer ver 6. Prior to analyses, all data were evaluated for normality by graphical examination of the residuals, which suggested they were slightly non-normal. Data for NPP, GPP, Re and the Range between GPP and Re were then square-root transformed and re-graphed, which suggested the problems were corrected. Data for PAR were log transformed, which corrected the problem. The transformed data were then PLOS ONE | https://doi.org/10.1371/journal.pone.0226173 March 4, 2020 17 / 21 PLOS ONE Deforestation and loss of ecosystem function examined for equality of variances using Bartlette’s tests, which indicated they were homosce- dastic. We then evaluated if urchin biomass, PAR, GPP, Re, NEP and the range between GPP and Re varied among the three habitats (kelp forests, urchin barrens, and transition habitats), the nine islands, and between the two study years using separate three-way Model III Nested ANOVAs, with year and habitat type as fixed factors, island nested within year as a random factor. We evaluated if the relationship between GPP and Re varied among habitats using ANCOVA, with Re as the response variable, GPP as the covariate, and habitat type as the cate- gorical independent variable. We evaluated if the ratios in any of the habitats differed from 1.0 (i.e. GPP = Re) by assessing if the value 1.0 occurred within the 95% confidence intervals around their average values. References 1. Terborgh J., Estes J. A., Trophic Cascades: Predators, prey and the Changing Dynamics of Nature. Island Press; 2010. 2. Ellison AM, Bank MS, Barton DC, Coulburn E. A., Elliott K, et al. 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Getting clean and harm reduction: adversarial or complementary issues for injection drug users
Cadernos de Saúde Pública
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1 Bloomberg School of Public Health, Johns Hopkins University, Baltimore, U.S.A. 2 Friends Social Research Center, Baltimore, U.S.A. 3 Ethknoworks, Santa Fe, U.S.A. 733 ARTIGO ARTICLE 733 ARTIGO ARTICLE 733 ARTIGO ARTICLE J. Peterson Department of Health Policy and Management, Bloomberg School of Public Health, Johns Hopkins University. Hampton House, 624 North Broadway, Baltimore MD, 21205, Suite 492, U.S.A. jpeterso@jhsph.edu Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Getting clean and harm reduction: adversarial or complementary issues for injection drug users Abstinência versus redução de danos: questões conflitantes ou complementares entre usuários de drogas injetáveis James Peterson 1 Shannon Gwin Mitchell 1 Yan Hong 1 Michael Agar 2,3 Carl Latkin 1 Abstract Traditionally, in the United States, the predom- inant approaches to drug policy and drug treat- ment have been characterized by such phrases as “zero tolerance” and the “war on drugs”. This abstinence-based political and ideological view emerges from the substance use treatment ap- proach embedded within the principals of the moral model 1. This approach supports the be- lief that abstinence is a prerequisite for sub- stance users to modify their behavior. Many contemporary HIV prevention interven- tions targeting injection drug users (IDUs) have been implemented using Harm Reduction as a theoretical framework. Among drug-using indi- viduals, however, the abstinence-based “getting clean” models espoused by Narcotics Anony- mous and other widely adopted approaches to drug treatment are often more readily accepted. This paper describes an ethnographic examina- tion of the ideological dichotomy between Harm Reduction and abstinence-based “getting clean” treatment model which emerged during the pi- loting phase of an HIV prevention intervention in Baltimore City, Maryland, USA. This paper describes how the conflict was identified and what changes were made to the intervention to help resolve the participants’ dichotomous think- ing concerning their substance abuse issues. Street Drugs; Drug Abuse; Harm Reduction Piloting “STEP”: an HIV prevention intervention The “STEP” study, the Harm Reduction pro- gram in question, is a network and peer-orient- ed HIV prevention intervention seeking to train individuals engaged in the injection-drug-us- ing community in Baltimore City. The study’s short-term goal is the promotion of HIV pre- vention behaviors among the program’s partici- pants and their drug and sexual network mem- bers. Based on our previous intervention effi- cacy research 4,5,6,7, STEP utilizes social influ- ence theory to promote the philosophy of Harm Reduction among social network members. The study design is a controlled phase II effica- cy trial with an equal attention control condi- tion. The STEP program consists of four group sessions (peer mentor training sessions), one individual session, one drug (or sex) dyad ses- sion, and periodic booster sessions up to six months after completion of the intervention. The STEP intervention was tested and revised through five waves of piloting, which occurred over the eight-month period proceeding formal program implementation. The process evalua- tion (upon which this paper was based) utilized ethnographic methods and was implemented during the eight-month piloting phase. For many substance users, however, an un- derstanding of recovery from substance use is derived from the philosophy of self-help orga- nizations such as Narcotics Anonymous (NA) and Alcoholics Anonymous (AA) which pro- mote an all-or-nothing belief concerning drug use. The NA philosophy posits that complete abstinence from all mood-changing chemicals is the mark of success and anything less than that is tantamount to failure; in a way, it can be thought of as an individual level of the “zero tolerance” policy. Method The STEP HIV prevention intervention pilot sessions were conducted from July 11, 2003 through March 10, 2004. The STEP HIV prevention intervention pilot sessions were conducted from July 11, 2003 through March 10, 2004. In this article we present salient lessons learned while conducting an ethnographically informed process evaluation during the pilot phase of an HIV prevention intervention trial aimed at injection drug users (IDUs) in Balti- more City, Maryland. We will discuss three key points which emerged during the course of our process evaluation: (1) the program partici- pants’ abstinence orientation, which dominat- ed their views of drug use and initially created tension with the program’s Harm Reduction orientation; (2) how the contradictions between these two orientations manifested themselves in group interactions; and (3) how the facilita- tors and program planners, along with the par- ticipants, augmented the intervention’s lan- guage, highlighting the similarities between the two approaches and reducing the tensions within group sessions. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Correspondence J. Peterson On the other hand, Harm Reduction, and its application within the HIV prevention and sub- stance-use treatment arena, has met with a broad range of acceptance throughout the world 2, particularly among contemporary researchers. Harm Reduction is defined as a set of practical strategies that are used to reduce the negative consequences of substance use. Harm Reduc- tion involves incorporating a continuous spec- trum of strategies, ranging from safer use, to managed use, to complete abstinence from drugs. Harm Reduction tailors behavior change to the unique conditions of substance use along with the substance users, themselves (Ptah A. Speech presented to the Community Planning Leadership Summit by Director of Policy Harm Reduction Coalition. March 2003). Street Drugs; Drug Abuse; Harm Reduction Street Drugs; Drug Abuse; Harm Reduction Proponents of Harm Reduction have been accused of supporting continued drug use, sabotaging efforts toward achieving abstinence, undermining recovery, contradicting current social policy, and selling out or giving in to the Peterson J et al. 734 Piloting “STEP”: an HIV prevention intervention presence of illicit drug use in the society. How- ever, those who adopt the principles of Harm Reduction do so with the understanding that drug use is a complex problem related to other quality of life issues such as poverty, class, racism, social isolation, and discrimination. They espouse that an approach to drug use that attempts to mitigate its negative effects on the drug-using individuals, their families, and the communities where they live, should in- clude non-coercive intervention strategies 3 and should not paint an overly simplistic pic- ture of the problem. In addition, Harm Reduc- tion allows for the creation of a non-adversari- al and supportive milieu that engages drug-us- ing individuals in a process where rewards and continued supports are not just the consequence of fundamental change, but are the incentives for incremental gains. Based on a vast body of empirical research, harm reduction is widely accepted among public health professionals as the cornerstone of HIV prevention among sub- stance abusers. Ethnography The use of qualitative methods in conducting process evaluations has demonstrated utility and success in the evaluation realm. Patton 8 elucidates four characteristics that make quali- tative inquiry “highly appropriate” for process evaluation: (1) depicting process requires de- tailed descriptions of how people engage with each other; (2) the experience of process typi- cally varies for different people, so their experi- ence needs to be captured in their own words; (3) process is fluid and dynamic so it can not be fairly summarized on a single rating scale at one point in time; and (4) participants’ percep- tions are a key process which should be taken into account. During the field observations, ethnographers also identified information-rich opinion lead- ers and key informants. In some cases, further interviews were scheduled to elicit more feed- back concerning the participants’ perspectives on relevant social and cultural factors. These interviews were also recorded. Specific to this study, LeCompte & Schensul 9 provide a detailed dialogue describing the use of ethnography in the process of developing in- tervention programs. Consistent with their un- derstanding, ethnographic methods were used in the STEP HIV prevention intervention devel- opment process to (1) examine the acceptabili- ty of the intervention to the staff as well as the intervention participants; (2) assess the pro- grammatic integrity of the intervention; (3) monitor the usefulness of the intervention; and (4) inform the design and development of the intervention outcome evaluation. Participants Eligibility criteria for STEP participants includ- ed: (1) at least 18 years of age, (2) willing to in- vite at least one sex or drug network member, and (3) self-reported unprotected sex or injection drug use within previous 3 months. Pilot par- ticipants were recruited using word-of-mouth, advertising in community newspaper, flyers posted throughout the neighborhoods, and street outreach. The participants were primari- ly African Americans from low-income, high- crime, urban neighborhoods and all resided within Baltimore City. A total of 29 individuals were recruited and participated in the piloting phase of the intervention. Among these partic- ipants 21 were male, 8 were female. Twenty- four of the participants were African American and 5 were White. The intervention utilized a team of two co-fa- cilitators (one Caucasian male and one African- ADVERSARIAL OR COMPLEMENTARY ISSUES FOR INJECTION DRUG USERS 735 American female). The research team consist- ed of 4 ethnographers, the primary investigator of the intervention, the project’s director, the two facilitators, and several staff members. community. The procedure was thoroughly ex- plained to each participant, and additional in- formed consent was received prior to any field observation work. Field work took place, by definition, out in the community. The purpose of the communi- ty-based field work was for the ethnographer to observe the STEP participant actually imple- menting the risk reduction skills and dissemi- nating HIV prevention information learned in the intervention sessions. The participants were asked to conduct the sessions with either their sex partner or a drug-using network member. Detailed field notes were taken by the ethnog- rapher and recorded immediately after leaving the field and entered into an ethnographic database. Relevant activities and conversations were audio-taped, when appropriate. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Data analysis Several types of ethnographic data were in- cluded in the analysis: (1) recordings and de- tailed notes from training sessions; (2) written notes of the session observations; (3) record- ings and written field notes from the fieldwork; and (4) recordings and written notes from in- terviews with key informants. The group notes, along with the field obser- vation notes, were uploaded to an ethnograph- ic database so that the ethnography team and the STEP intervention project planners could have easy access to the notes, track the progress of the evaluation, and make adjustments to the pilot program as necessary. Ethnographers coded the field notes using themes that paral- leled the domains and/or content areas identi- fied as “session objectives” as well as identify- ing new and emerging themes 9. Detailed sum- maries were made with substantial retention of original quotes and notes so as minimize the “summary” bias and reproduce the true situa- tions. Ethnography was utilized to inform the process evaluation during the project’s piloting phase, from August 2003 to March 2004. Data were collected through a variety of methods, including group observations, field observa- tions, and the content analysis of session tapes and interviews with STEP participants. In each of the piloting waves, at least one ethnograph- er was present during all of the classroom ses- sions. An ethnographer conducted observa- tions concerning the pilot sessions’ content and the interactions among the facilitators and the participants. Detailed notes of the observa- tion were recorded. During session breaks an ethnographer con- ducted informal interviews and participated in casual conversations with the participants in order to elicit feedback about the sessions, as well as to establish and build rapport. After earning the trust of the participants, an ethno- grapher would individually query participants as to whether they were willing to demon- strate skills during observation activities in the Regular meetings were scheduled with ethnographic team members in order to dis- cuss and cross-validate preliminary insights and observations. These insights were then shared with the larger project research team and the facilitators. These discussions provid- ed opportunities to examine preliminary find- ings from the perspectives of both the ethnog- Peterson J et al. 736 the idea of getting clean than on the Harm Re- duction information presented during the pilot sessions. Longer drug use history among par- ticipants also created barriers to conducting outreach. The use of AA/NA language by program participants The participants’ conversations during the ses- sions invariably focused on their drug use and their desire to get clean or help their network member get clean. For instance, in the early pi- loting sessions, participants were asked about their individual goals through their participa- tion in the STEP project. Many of the partici- pants responded by making “not using” their primary goal rather than simply reducing HIV risks associated with their drug use. In the conversations during group sessions, as well as in their outreach activities, the language of the participants was liberally peppered with clichés and language often heard in AA and NA meetings. Comments such as “I’m gonna keep coming back”, “sick and tired of being sick and tired”, and “I have to change people, places, and things”, among others. Conversations with par- ticipants and their network members suggested that AA/NA significantly influenced their think- ing and dialogue about their substance use and concomitant issues. In many cases, the partici- pants incorporated AA/NA language into their risk reduction conversations with their network members and it seemed as if participants had internalized the “all-or-nothing” attitude un- derlying the AA/NA model of drug dependence. The following field note includes the response from one ethnographer’s observation of an out- reach episode that a participant had coordinat- ed in which several of his drug-using associates were participants: “Well, I thought I was at an NA meeting and I also thought the participant planned it that way. The order that it [the con- versation] fell in and the way everyone shared behind each other. Then, when they started cry- ing right behind each other. It was strange, but then I thought maybe these people really needed to talk to someone, and they just got emotional”. Th “ ll hi ” i d h b There are some plausible explanations for this finding. The ethnographic team hypothe- sized that many of the participants may have talked about getting clean because they thought it was socially acceptable and what they need- ed to say in order to obtain personal influence within the program structure. Also, it is likely that some participants actually wanted to get clean, but may have had little or no other re- sources to achieve this goal. In response, they decided to utilize the Harm Reduction-based research project as if it were a treatment pro- gram. The project’s focus on Harm Reduction The STEP philosophy can be summed up in the following sentence from session one, “we believe that any positive change you are willing to make is an important step towards Harm Reduction”. Session content centered upon various methods and ways to reduce the participants’ potential exposure to HIV by engaging in safer injection practices and using condoms. All of the sessions (group, individual, and dyad) were focused on reducing injection and sex risks for contracting HIV. This emphasis was clearly identified in the facilitators’ statements and the discussions they led during the sessions. Data analysis For instance, a 50-year-old African American male participant who reported a 20- year drug-use history discussed, in detail, the resistance to his attempt at outreach that he had to deal with as he initiated a conversation with his drug-using partner, who he also iden- tified as his sex partner. The conversation, as he reported, was about him articulating the in- formation he learned while participating in the sessions. The participant was emphatic when he mentioned the argumentative and con- frontational behavior he met with from his partner. The resistance the participant was ex- periencing may have been the result of his ap- proach being flavored with his subjective and unrelenting understanding of getting clean, an idea that had the potential to greatly overshad- ow any “incremental change” notions garnered from STEP’s Harm Reduction-based sessions. raphy team members and the project planning staff and to make recommendations regarding the intervention session content. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Blending divergent viewpoints At first glance the views of many of the partici- pants appeared to be diametrically opposed to the views espoused by the STEP project. How- ever, once the “conflict” was identified, an ef- fort was made to emphasize during the pro- gram’s sessions that getting clean was just one step along the path to a Harm Reduction plan. Facilitators began to emphasize that complete abstinence was at the far end of the Harm Re- duction continuum and that each incremental step towards reducing exposure to HIV was al- so a step towards their ultimate goal of getting clean. Other key factors added to the sessions which assisted the participants in the integra- tion of the two perspectives were: The intervention sessions were conceived with a flexible design that allowed for tailoring the prevention intervention to the needs and satisfaction of the participants. Thus, one of the sessions in the STEP program was designed to convey that to “talk the talk” you need to “walk the walk” or you may be seen as hypo- critical and less effective in your outreach ac- tivities. Such an accomplishment required the intervention planning group to reach a delicate balance between the HIV prevention practices espoused by the Harm Reduction arena and the goals of the targeted group. Thus, the STEP program emphasized that walking the walk could be about harm reduction, that is, prac- tice and preach safer injection practices. • They were taught that the Harm Reduction information and incremental changes they made could help them get clean; The group facilitators mentioned that they initially experienced some tension introducing the Harm Reduction concept to the partici- pants. The participants, while not being able to clearly articulate their lack of understanding of the concept of Harm Reduction, demonstrated this fact in a variety of ways. During the group sessions several of the participants articulated a sense of pride when a participant announced that he or she had accumulated any amount of “clean-time” or amassed one day or more with- out using drugs. However, while accruing “clean- time” was a worthwhile goal, many of the par- ticipants found that maintaining these efforts at staying clean seemed insurmountable for any length of time. The use of AA/NA language by program participants It is also likely that as drug-dependent individuals tended to focus their behavior on acquiring and using drugs, they also viewed drug use as the cause of the major problems in their lives. From this standpoint, stopping drug use would alleviate all or most of their other problems, such as poor family relationships, unemployment, and depression. Results from observations of the group ses- sions suggest that many participants with a long history of drug use were more focused on The “all-or-nothing” attitude may have been a consequence of the AA/NA mindset that one ADVERSARIAL OR COMPLEMENTARY ISSUES FOR INJECTION DRUG USERS 737 has to “hit a bottom” before asking for and, in many cases, receiving help. Several of the par- ticipants articulated discussions and responses throughout the piloting of the intervention ses- sions which echoed the NA/AA language and seemed to further convey the getting clean phi- losophy rather than the program’s main focus of Harm Reduction. with the concept of getting clean in the AA/NA abstinence model. An example of this type of behavior is inferred from the following note, which describes the participants’ focus on their own behavior during conversation in the group sessions. An ethnographer wrote: “In this session, the most prominent problem is the tension between ‘partner’ and ‘myself’. Partici- pants could not get out of the notion of ‘getting clean’. Many times the discussion moved from ‘helping others’ to ‘helping myself’”. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Blending divergent viewpoints • That helping others was a way to help them- selves; • That there were other goals in their lives be- sides getting clean and that reaching those goals might assist them in getting clean; and • That getting clean is a process, not just an end result, and that it requires positive steps. The group facilitators took special care to celebrate any small gains toward risk and harm reduction reported by participants. Discussion cated, interconnected components of the in- tervention, the relationship of those compo- nents to the program as a whole, and the im- mediate reactions of the participants. Using ethnographic methods to collect data, which informed a process evaluation of the imple- mentation of an intervention program, can fos- ter a reflexive exercise, which enables immedi- ate and responsive feedback for program de- velopment. In the current study an ethnograph- ically informed process evaluation was used to obtain information during the pilot sessions and assist with program development and de- cision-making 12. The design and implementation of an HIV pre- vention intervention is an arduous and com- plex undertaking. Weeks et al. 10 suggest that in communities such as the locales from where the participants of this study were recruited, is- sues of poverty, gender, ethnic identity, and lev- el of acculturation come together to create a unique set of high-risk conditions for the trans- mission of drug-related epidemics. Strategies to reduce the high risk of disease transmission among the drug-using population have to be culturally relevant to the participants and im- plemented within multiple contexts 11. The par- ticipants engaged in the pilot sessions of the STEP HIV prevention intervention show that “cultural relevance” means taking seriously the issue of getting clean and the influence of tra- ditional abstinence models like AA/NA. Traditionally, evaluation strategies have pri- marily been used to determine cost effective- ness and the impact of the prevention efforts on the targeted group. Thus, outcome evalua- tions provide a systematic method to deter- mine (1) whether the program achieved the stat- ed objectives and (2) whether the intervention was a cost-effective activity. Obtaining an un- derstanding of the aforementioned aspects and characteristics of an intervention may have some usefulness for evaluating whether a pro- gram has achieved the intended objectives. However, describing key aspects of the pro- gram’s dynamics has significant utility for the design, development, proper implementation, and facilitation of interventions that are tai- lored to meet the specific needs of its targeted group. Within the context of the public health are- na, the relationship between the philosophical principles underpinning the STEP approach to Harm Reduction can be seen as challenging the traditional abstinence-only models 2. Discussion Re- searchers attempting to design and implement a Harm Reduction-based intervention for IDUs must ask the question, “How many drug users ‘just say no’ to Harm Reduction and why do they do so?”. Injection drug users who have the tradi- tional “getting clean” ideology may initially struggle with the messages of an HIV preven- tion intervention that has Harm Reduction as its philosophical base. This preliminary and ex- ploratory study shows that many of the IDUs who participated in this pilot study focused on abstinence as their only goal and not abstinence as the end result of a process, as it is viewed from the Harm Reduction perspective. Designing and implementing culturally rel- evant HIV prevention interventions is a com- plex and arduous task. Contemporary preven- tion interventions designed to address the transmission of HIV and other diseases among specific populations are intricate and typically are implemented within multiple contexts. Consequentially, evaluating HIV prevention in- terventions is an indispensable component of the overall effort. The IDU participants recruited to the STEP program were at various stages in their drug- using careers and brought a variety of needs and experiences to the intervention that im- pacted their receptivity to the project’s Harm Reduction message. Through the use of ethnog- raphy in the process evaluation phase of the HIV prevention intervention, these needs and prior experiences became clear to the research team and provided both a need and an oppor- tunity to include strategies merging the absti- nence and Harm Reduction models. It is important for researchers and program planners to know the extent to which all com- ponents of the intervention are actually imple- mented as planned. The use of ethnographic methods within the process evaluation offered a strategy, which allowed for systematically documenting the step-by-step procedures in- volved in the implementation of the interven- tion, disentangling the factors that may con- tribute to successful outcomes, and explicating the components of the intervention that are in- herent to the effectiveness and success of the intervention effort. Reduction setting The AA/NA abstinence model advocates that if a person does not remain clean or drug free, then a negative label is attached to their efforts. Thus the participants’ perception was that they must “walk the walk” in order to “talk the talk”. The participants consistently articulated their discomforts with disseminating the recently learned HIV prevention information if they were not “clean” or they continued to use drugs while participating with the outreach compo- nent of the intervention. The idea, from their perspective, was that they thought of them- selves as being hypocritical if they conducted outreach work while still using drugs. This no- tion of hypocrisy appeared to be directly linked As the intervention developed, the facilita- tors became more and more adept at eliciting Harm Reduction-based “positive steps” while acknowledging the heroic efforts that the par- ticipants were making to reduce their risks of contracting HIV and other infectious diseases and, ultimately, get clean. The conflict resolved with the concept of any positive change, which was flexible and did not focus on either the me- chanics of risk reduction or the ultimate goal of getting clean. Peterson J et al. 738 Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Contributors Muitas intervenções para prevenir a infecção pelo HIV entre usuários de drogas injetáveis adotam a técnica de redução de danos como referencial teórico. Entre- tanto, os próprios usuários tendem a preferir modelos baseados na abstinência, defendidos pelos Narcóticos Anônimos, além de outras abordagens adotadas am- plamente para o tratamento da dependência química. O artigo descreve uma avaliação etnográfica da dico- tomia ideológica entre redução de danos e o modelo terapêutico baseado na abstinência (getting clean) du- rante a fase piloto de uma intervenção para a pre- venção de HIV na Cidade de Baltimore, Maryland, Es- tados Unidos. O artigo descreve como o conflito foi identificado e que mudanças foram introduzidas na intervenção para ajudar a resolver as idéias dicotômi- cas dos participantes em relação às questões de de- pendência química. J. Peterson is the primary author of the manuscript. S. G. Mitchell assisted with manuscript development and Y. Hong with consultation, proofreading, and writing. M. Agar provided conceptualization, consul- tation, proofreading, and scientific editing. C. Latkin performed proofreading and scientific editing. Implications for research and program development Delineating the particular group norms, such as language use and the means by which specific group participants get their needs met, Process evaluations that include ethnographic methods are useful for explicating the compli- Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 ADVERSARIAL OR COMPLEMENTARY ISSUES FOR INJECTION DRUG USERS 739 is vital to determining the intervention strate- gies and structure. The course of integrated ac- tions that are included in achieving a specific planned or stated outcome has value in dupli- cating those program activities that have proven to be effective, determining the integrity of the program, assessing the acceptability of the program by the targeted group, and identifying key factors of the intervention that are being carried out as planned. volved with deciphering the interactions be- tween the intervention components, the par- ticipants, and the facilitators, however ethno- graphic methods were indispensable in obtain- ing information needed to mitigate these chal- lenges and were critical in furthering our un- derstanding of the change process within the HIV prevention intervention. Ethnographic methods not only assisted with addressing the specific questions related to obtaining an un- derstanding of the overall project, but this ap- proach facilitated efforts to bring those ques- tions to light, one step at a time. Gaining an understanding of the process involved in carrying out change efforts, such as the STEP HIV prevention intervention, can prove challenging. There are complexities in- Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 References 7. Dickson-Gomez J, Knowlton A, Latkin C. Hoppers and oldheads: qualitative evaluation of a volun- teer AIDS outreach intervention. AIDS Behav 2003; 7:303-15. 1. Brickman P, Rabinowitz VC, Karuza K, Coates D, Cohn E, Kidder L. Models of helping and coping. Am Psychol 1982; 37:368-84. 2. Heller D, McCoy C, Cunningham C. An invisible barrier to integrating HIV primary care with Harm Reduction services: philosophical clashes between the Harm Reduction and medical models. Public Health Rep 2004; 119:32-9. 2. Heller D, McCoy C, Cunningham C. An invisible barrier to integrating HIV primary care with Harm Reduction services: philosophical clashes between the Harm Reduction and medical models. Public Health Rep 2004; 119:32-9. 8. Patton MQ. Qualitative research & evaluation methods. Thousand Oaks: Sage Publications; 2002. 9. LeCompte M, Schensul J. Analyzing and inter- preting ethnographic data in ethnographer’s tool- kit. v. 5. Walnut Creek: AltaMira Press; 1999. 3. Harm Reduction Coalition. Principles of harm re- duction. Newsline People AIDS Coalition N Y 1998 Jun; 7-8. 3. Harm Reduction Coalition. Principles of harm re- duction. Newsline People AIDS Coalition N Y 1998 Jun; 7-8. 10. Weeks MR, Singer M, Himmelgreen DA, Rich- mond P, Grier M, Radda K. Drug use patterns of substance abusing women: gender and ethnic dif- ferences in an AIDS prevention program. Drugs and Society 1998; 13:35-61. 4. Latkin C, Sherman S, Knowlton A. HIV prevention among drug users: outcome of a network-orient- ed peer outreach intervention. Health Psychology 2003; 22:332-9. 4. Latkin C, Sherman S, Knowlton A. HIV prevention among drug users: outcome of a network-orient- ed peer outreach intervention. Health Psychology 2003; 22:332-9. 11. Steckler AB, Linnan L. Process evaluation for public health interventions and research. San Francisco: John Wiley and Sons; 2002. 5. Hopson RK, Peterson JA, Lucas KJ. Tales from the ‘hood’: framing HIV/AIDS prevention through in- tervention ethnography in the inner city. Addic- tion Research & Theory 2001; 9:339-63. 12. Hong Y, Mitchell S, Peterson J, Latkin CA, Tobin K, Gann D. Ethnographic process evaluation: pilot- ing an HIV prevention intervention program among injection drug users. International Jour- nal of Qualitative Methods 2005; 4(1). http:// www.ualberta.ca/~iiqm/backissues/4_1/html/ho ng.htm. 6. Hopson RK, Lucas KJ, Peterson JA. HIV/AIDS talk: implications for prevention intervention and eval- uation. In: Hopson RK, editor. How and why lan- guage matters in evaluation: new directions for evaluation. San Francisco: Jossey-Bass Publish- ers; 2000. p. 29-42. 6. Hopson RK, Lucas KJ, Peterson JA. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Submitted on 23/May/2005 Approved on 30/Jun/2005 Acknowledgements This work was funded by the National Institute on Drug Abuse, grant R01DA016555. Drogas Ilícitas; Abuso de Drogas; Redução de Danos Drogas Ilícitas; Abuso de Drogas; Redução de Danos Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Peterson J et al. 740 References References HIV/AIDS talk: implications for prevention intervention and eval- uation. In: Hopson RK, editor. How and why lan- guage matters in evaluation: new directions for evaluation. San Francisco: Jossey-Bass Publish- ers; 2000. p. 29-42. Submitted on 23/May/2005 Approved on 30/Jun/2005
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Decolonisation and the “Federal Moment”
Diplomacy and statecraft
2,013
cc-by
11,674
© Michael Collins. 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 use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. © Michael Collins. Diplomacy & Statecraft, 24:21–40, 2013 Published with license by Taylor & Francis ISSN: 0959-2296 print/1557-301X online DOI: 10.1080/09592296.2013.762881 Decolonisation and the “Federal Moment” Federations involved alternative ways of thinking about sovereignty, territoriality and political economy: a re-examination of the rationale for their creation and the reasons for their failure may be of some significance for historians looking back at the successes and failures of the post-colonial nation-state. This article is an early attempt to sketch some of the possible contours of what is a wide area of historical research. The article proceeds on the basis that the intersection between international and imperial history constitutes a key pathway for making sense of some of the most important trans-national and world historical developments in twentieth century history.3 Downloaded by [University College London] at 08:18 08 September 2014 Specifically, the paper looks at the British Labour governments of 1945–51, and some of the imperial challenges faced by Britain in south- ern and central Africa immediately following the South African election in 1948 and up to the creation of the Central African Federation (CAF) under the Conservatives in 1953. These two events were interconnected, with fears of Afrikaner territorial, economic and political expansion influenc- ing British policy making in the region.4 The CAF—a federal realm of the British crown formed in 1953 by combining northern and southern Rhodesia with Nyasaland—involved a denial of the principle of nationality. It thus ran counter to a broad, if loosely defined acceptance of the emergence of African nationalism by senior Labour figures. The abstract idea of national self- determination gave way to the practical developmentalist imperialism that came naturally to many senior Labour politicians and policy-makers. In addi- tion, acute and multiple problems of race and identity arose in the southern and central African region in the late 1940s, which impacted upon both policy-making and wider, less tangible perceptions of Britain’s imperial and national self. British policy makers redefined the terms of collaboration with both white colonists and black nationalists, allowing them to secure on-going control of political power and resources. Federation was thus ambivalent, a move towards decolonisation as well as a means of securing imperial con- trol. But insofar as federation can be read as a form of the “imperialism of decolonisation,”5 the politics of Britain’s imperial identity were causal as the British re-articulated justifications for the control of African peoples and resources. Decolonisation and the “Federal Moment” MICHAEL COLLINS Post-1945 decolonisation involved the universal acceptance of nation-statehood as the alternative to imperialism. Nationalism vanquished its transnational competitors, notably imperialism and Marxism. Alternatives to imperial rule that avoided sovereign states on national lines, such as federations in the later 1940s and 1950s, have received less attention from historians. Federations involved alternative ways of thinking about sovereignty, territorial- ity, and political economy. British interest in creating federations, for example the Central African Federation (CAF) in 1953, offers some new perspectives on the strength of imperial ideology and the determination to continue a missionary imperialism after the Second World War. Federal thinking and practice was prominent at this time in other European empires too, notably the French and Dutch ones. The federal idea was also an aspect of the emerg- ing European community. This is suggestive of a wider “federal moment” that points to the importance of linking international, trans-national, imperial, and world historical approaches. Downloaded by [University College London] at 08:18 08 September 2014 Downloaded by [University College Londo Post-1945 decolonisation involved the universal acceptance of nation- statehood as the alternative to imperialism.1 Nationalism vanquished its transnational competitors, notably imperialism and Marxism. Alternatives to imperial rule that resisted the logic of sovereign states on national lines, such as the federations created by the British in the later 1940s and 1950s, as well as post-imperial federations created by decolonising political elites, have arguably received less attention from historians than they might.2 From a British imperial perspective, the interest in creating federations offers an opportunity to re-examine—within the context of the “decline, revival and fall” of the British Empire—the role of imperial ideology and Britain’s 21 22 M. Collins determination to continue a missionary imperialism after World War II. But federal thinking and practice was also prominent at this time in other European empires too, notably the French and Dutch ones. Equally, the federal idea was an aspect of the emerging European community. This is suggestive of a wider post-1945 “federal moment” that is linked, in con- trast to the longer history of thinking about federations or forms of “closer union” in British imperial circles, to the failures of the inter-war nation-state and the specific challenges posed by decolonisation and the creation of national states after 1945. Decolonisation and the “Federal Moment” It is significant—especially for those who are interested in lega- cies of empire and decolonisation in the metrople—that this took place at a relatively late stage, within the era of decolonisation itself. The events and Decolonisation and the “Federal Moment” 23 debates leading up to the creation of the CAF in 1953 reveal a conjuncture of problems relating to imperial identity, imperial politics and international pressures. Underlying these is a wider historical space in which the very idea of sovereignty and nation-statehood was being contested. Variations of the federal idea have a long history in British imperial thinking. The motivations for federal plans were most often a mixture of practical, administrative, and cost-saving schemes, and sometimes sugared with romantic notions of imperial unity. As early as the seventeenth century, a federal model was floated as a solution to reducing the financial burden of defending the West Indian plantations.6 In the late eighteenth century, figures including Adam Smith and Lord Shelburne met the crisis in the American colonies with calls for federation.7 But it was the end of the nineteenth and beginning of the twentieth century that saw the emergence of a more concerted and widespread movement for imperial federation and greater imperial unity. Downloaded by [University College London] at 08:18 08 September 2014 The “colonial question” had come to the fore increasingly from the 1860s with “empire unity” occasionally demanded, and numerous schemes “for federation, confederation, union, a colonial council, council represen- tatives in Parliament, or occasional colonial conferences” advanced. “Little emerged from the talk,” and yet “many of these ideas were to crop up again and again for nearly a century.”8 One notable though short-lived venture was the Imperial Federation League in 1884. Looking to Canada by way of example, the League advocated an imperial parliament for Britain and the self-governing territories gaining support from familiar figures in the “Greater Britain” debate such as J. R. Seeley and J. A. Froude.9 The British branch of the League was formally dissolved in 1894, undermined by divisions over the necessity of putting forward formal proposals for federation versus the development of a forum for an ongoing conversation about imperial ties and closer association. The increasingly fraught debate over free trade and tariff protection also split enthusiasts for federation at this time. Decolonisation and the “Federal Moment” p p Even so, the theme was given renewed impetus by the creation of the Round Table Movement in 1909, with the journal of the same name founded in 1910. The Movement initially pressed for organic union, but ultimately for a Commonwealth of independent nation states, ironically the antithesis of such a union. The Round Table Movement consisted largely of acolytes of Lord Milner, his so-called “kindergarten” men such as Lionel Curtis. The union in South Africa prompted thinking about a wider conception of union. In his more lofty and idealistic moments Curtis envisaged “an organic union to be brought about by some establishment of an Imperial government, constitutionally responsible to all the electors of the Empire, and with power to act directly on individual citizens.”10 More concretely, even in light of the recent Entente with France and Agreement with Russia, Curtis argued that a “business arrangement for the support and control of Imperial defence and foreign policy of the Empire” was necessary, or the empire “must break up.”11 24 M. Collins The impact of World War I on this kind of thinking was ambiguous. On the one hand, there was a well-documented increase in Dominion assertiveness, nationalism even. On the other, it also led to the development of greater imperial participation in decision making at the centre. When, in December 1916, Lloyd George became Prime Minister, with the Milnerite and founding member of the Round Table Philip Kerr as his Private Secretary, his call for a special imperial war conference in 1917 and the creation of an Imperial War C bi “ l b i h fi i f h h l i ”12 p p p Cabinet “came close to becoming the first executive for the whole empire.”1 What is noticeable is that calls for federation or closer union between Britain and its empire, by which its advocates almost always meant the colonies of white settlement, tended to be more vigorous and achieve greater resonance at times of perceived weakness or vulnerability. If the Balfour Declaration of 1926 and the Statute of Westminster constituted shifts towards greater independence for the Dominions, the Great Depression forced a shift in the opposition direction, towards greater economic inter-dependence. Here practical necessity tended to crowd out the loftier rhetoric of an earlier period. Decolonisation and the “Federal Moment” This is interesting14 and per- tinent to the federal moment of the 1950s in which will, desire and idea are indeed stressed over and above means. The post war “federal moment” is thus explicitly associated with decolonisation and the challenges of colonial nationalism, and yet the desire and imagination to maintain Britain’s world role were arguably of an earlier vintage. In his work on Sir Andrew Cohen, Ronald Robinson attributed a degree of progressive foresight to the British Colonial Office in terms of its post- 1945 attitude towards decolonisation, specifically in Africa.15 With India liberated, Palestine abandoned, and Malaya under threat, active steps were taken to reorient British imperialism towards the strategically important site of the Middle East, and to re-think the British role in Africa. In fact this pro- cess and the ideas that pushed it forward had been underway before the war, partially embodied in the progressive figure of Malcolm MacDonald, Secretary of State for the Colonies from May 1938 to May 1940, and the personal zeal that he brought to the Colonial Development and Welfare Act of 1940. The shift in colonial policy towards Africa should not be seen as a direct consequence of the war and the loss of imperial assets or prestige that the British subsequently endured. Lord Hailey’s influential Africa Survey, published in 1938, was an important moment in the steady re-evaluation of the Lugardian idea of “trusteeship” that had framed African societies and cul- tures as static, essentially ahistorical entities, with the British as benevolent guardians, and in this regard at least primus inter pares when compared to rival colonial powers.16 Downloaded by [University College London] at 08:18 08 September 2014 The flattering and deeply misleading self-image embodied in famous Together wartime propaganda poster was indicative of British attempts to portray their diverse “multi-racial” empire as the nemesis of Fascism’s attempted racial holocaust. Their efforts were complicated, amongst other things, by the obvious on-going economic exploitation underpinning the empire, the brutal suppression of Gandhi’s 1942 “Quit India” campaign and the awkward colour bar between black and white American G.I.s that drew so much attention in wartime London, and in the British West Indies. Decolonisation and the “Federal Moment” Even so, the shift towards a more clearly defined and articulated national identity on the part of the Dominions pointed toward a future Commonwealth rather than any previously imagined imperial federation. Downloaded by [University College London] at 08:18 08 September 2014 The significance of this for our understanding of post-1945 fed- eral experiments is twofold. The problem of colonial nationalism would inevitably grow beyond the Dominions and India and would thus threaten British control and influence in colonial territories. At the same time, the strength of the association between Britain and its empire clearly remained fundamental to the maintenance of a British world role. Commonwealth would be central to this, and this would shape the British approach to nation- alism. Federation would no longer be conceived as an imperial endeavour formally constituting the relations between metropole and colony, but rather as a way of maintaining British influence in particular parts of the empire, a way of reconfiguring the politics of collaboration so as to defy the logic of nationalism with its fetishisation of sovereign territoriality and hence to maintain key British spheres of influence. What is surprising, perhaps, is the enthusiasm with which some key British officials in the later 1940s pursued this objective, for example Sir Andrew Cohen at the Colonial Office. In this sense, although the formal structure of the relationship may have changed, the central objective of maintaining Britain’s world role and the ideological belief in a liberal civilising mission is suggestive of a longer run connection between the post-war period and the earlier liberal imperialists such as Milner. As Jack Gallagher put it some time ago, “between the wars British statesmen had been fearful for their empire. It had seemed so fragile that another great war might well knock it to pieces. That did not hap- pen. Whatever caused the end of empire, it was not the Second World War, although this conclusion will not please those who think that the world came to an end in 1945, or those who think that the world’s great age began anew Decolonisation and the “Federal Moment” 25 in 1945.”13 Gordon Martel, in his critique of what he saw as Paul Kennedy’s economic determinist view of decline, stressed the importance of will and ideas in this process of imperial reconfiguration. Decolonisation and the “Federal Moment” The Atlantic Charter of 1941, which Churchill had done so much to resist, changed the tone of international public opinion.17 With the 1945 Charter of the United Nations declaring “the respect for the principle of equal rights and self-determination of peoples” as one of its basic purposes, a sec- ond and, from the perspective of nationalists, more promising “Wilsonian moment” was well under-way.18 Amongst its many foreign policy challenges, the Labour government would have to respond to this new environment. Colonial Office policy shifts towards Africa certainly accelerated in the later 1940s. It was not only domestic and international public opin- ion that was changing, but also the politics of collaboration in the colonies 26 M. Collins themselves. In the face of rising African nationalism, which would increase in the foreseeable future, the Colonial Office deemed it necessary that the aforementioned “trusteeship model” of indirect rule—whereby the British struck deals with African chiefs in order to maintain control of territory— needed revision. Increasing urbanisation was underway in Britain’s African colonies, and labour unrest was taking on surprisingly violent forms, notably in Accra in 1948.19 Urgent action was required to improve Britain’s position. 9 g q p p A major component of this was to be the so-called “new approach” to local government in Africa advanced by Arthur Creech Jones, Secretary of State for the Colonies, and Andrew Cohen, Assistant Under-Secretary of State, and Creech Jones’ “alter ego.”20 The purpose was to bring greater African participation into the affairs of government. Robinson, in one of the only significant scholarly works on Cohen, claimed that the May 1947 “Cohen Report” which outlined four stages of constitutional advance to incor- porate Africans into local democracy, foresaw most African dependencies being self-governing within a generation. Cohen’s “revolutionary manifesto” was “inspired by the belief that if the dependencies were to be devel- oped economically, their administration would have to be democratised and nationalised.”21 But the general political radicalism of Labour’s approach and support for nationalism and decolonisation has been grossly overstated.22 If we are to understand why Labour begins to acquiesce in the creation of the CAF by the late 1940s this misperception needs to be cleared up. In fact, in the 1947 report only the Gold Coast was viewed as being capable of self- government “in a generation.” Elsewhere, internal self-government would take substantially longer. Decolonisation and the “Federal Moment” Independence is not even discussed.23 Downloaded by [University College London] at 08:18 08 September 2014 Cohen is a key figure here, whom Robinson presents as embodying the spirit of the new policy, labelling him the “proconsul of African national- ism,” rather misleadingly suggesting that his “constitution mongering” was awakening the “slumbering genius” of national politics.24 Early in his career at the Colonial Office, Cohen had gravitated towards the Fabian wing of the Labour Party. The Fabian Colonial Bureau (FCB), founded in 1940, might be seen as a spiritual home, and along with Creech Jones, Cohen corresponded with, and highly regarded the opinion of, key figures in the FCB such as Rita Hinden and Marjory Perham. In true Fabian spirit, the new approach to Africa was not primarily constitutional but socio-economic and socio-political.25 In some senses, Fabians and democratic socialists from the British Labour Party could have their cake and eat it too. They could make efforts at colonial development which sought to increase dollar earning exports and thus benefit the metropole in a period of desperate dollar shortage, whilst simultaneously (and arguably sincerely) believing that in the longer run this new economic development would create the socio-economic under- pinnings of “de-tribalised” societies with a functioning civil society: trade unions, political parties, news media. Modernised indigenous elites would make excellent future collaborators—not least for Labour governments—in Decolonisation and the “Federal Moment” 27 a post-imperial world where formal constitutional decolonisation gave way to looser but still substantial economic and political cooperation through Commonwealth. Rita Hinden, looking back from the perspective of 1959 on the FCB and its role in post-war imperial policy, put Labour’s predicament thus: To this massive legacy, the socialists were heirs; they had the duty to decide on its use. Enjoy it? No, that would have been a violation of social- ist principle. Reject it outright, and so remain true to the anti-imperialism which socialists had always preached? Or, better still perhaps, accept the heritage, but with the determination to nurse and develop it for the advantage of its rightful owners till they themselves should have come of age?26 Downloaded by [University College London] at 08:18 08 September 2014 Hinden recognised the “poverty and backwardness” of the colonies had been deepened by imperialism, by “extorting the . . . wealth and alienating . . . Decolonisation and the “Federal Moment” land from the people, by taxation and forced labour, by the despoliation of the soil.” But, she believed that “even before the entry of imperialist powers, the colonial territories were poor and economically backward” and that poverty might “be sooner cured by prolonging imperial rule” than by moving too quickly toward independence.27 Getting Labour’s approach to empire straight is thus crucial in understanding not simply the acceptance of federation in central Africa, but ultimately its advocacy. Neither nationalism nor democracy was of paramount importance. What mattered was steady and consistent social and economic development, and inter alia, continued British control. British missionary imperialism was in rude health under Labour. Margaret Joy Tibbets, a sharp-witted political officer at the United States Embassy in London, saw the point early on. In a memorandum on “British Colonial Development and Welfare Programs” she wrote that “it is the opin- ion of the United States Embassy at London, that [Labour’s approach to colonial development] is intended primarily to provide increased foodstuffs and other primary products for the British people.”28 This was undoubtedly true, but the realties of the Cold War, the apparent unpreparedness of African peoples to govern themselves and the predatory threat of Communist infiltra- tion were already softening the United States government’s attitude towards the British Empire.29 The early Cold War made the southern African region a strategic asset first and foremost because of South Africa’s rich source of uranium, and instability in the region caused by political and racial ten- sions offered opportunities to the Soviet Union to foment unrest, threatening this rich and vital source of nuclear material.30 Hence the extent to which “London retained a good deal of its old determination to shape the terms of collaboration and make things happen on the ground”31 became increas- ingly important to the United States, and opened up new opportunities for the Labour government to reinvigorate colonial policy. In fact, Labour’s 28 M. Decolonisation and the “Federal Moment” Collins redeployment of imperial power in Africa after World War II, with hun- dreds of new bureaucrats and the burgeoning of a technocratic, imperialist developmentalism, has been called a “second colonial occupation”.32 The United States would not stand in the way of the British if this meant Britain’s empire would remain a bulwark against the encroachments of communism in Africa, and to this end Marshall Aid was allowed to be spent within the empire.33 p Despite some recent corrections from Cooper and Hyam on the Labour Party and Africa c. 1945–51, Robinson’s triumphalist account of the Colonial Office—particularly his version of Cohen’s role—continues to deflect our attention from an important moment in the decolonisation process in which both trusteeship and a new civilising mission make a return. The creation of the CAF is an important case in point. Some kind of amalgamation or feder- ation of Britain’s central African territories had been mooted for some time, an idea largely pushed forward by the white settler community for its own purposes. Yet in early 1948 Creech Jones had declared to Roy Welensky, Prime Minister of Northern Rhodesia, that the Labour Government, indeed “no government” would “abandon” millions of black Africans in Nyasaland and the Rhodesias to rule by a minority of white settlers.34 But in September of the same year Cohen’s private meetings with United States diplomats in London led them to surmise that the British government believed the interests of Africans in southern and central would suffer “if Whitehall’s pro- tecting hand were withdrawn.”35 Federation was looking increasingly likely. By September 1950 American diplomats produced a secret Memorandum of Conversation for Washington’s consumption in which they directly quote Cohen as saying that “it would obviously be advantageous to have a strong federated Rhodesia as a counterweight to the Union [of South Africa].”36 In the context of this particular federal project, the “race problem” and the South African bogeyman were crucial drivers of British policy, allowing a paternalistic imperialism to side-line black African nationalism (specifically in Nyasaland) in favour of a new British dominion in central Africa. The spectre of a pan-Afrikaner alliance that had prompted Milner and Chamberlain to go to war in 1899 reared its head again.37 More broadly, the denial of national sentiment in favour of non-national geo-political reform begs the question of whether a more pervasive strategy is underway. Decolonisation and the “Federal Moment” Downloaded by [University College London] at 08:18 08 September 2014 The problems Britain faced on its southern African imperial periphery after 1945 were manifold. Sympathy for newly defeated German fascism in some quarters of the Afrikaner community re-opened and accentuated old divisions between the Afrikaner and Anglo worlds. From London’s per- spective, the growing political power of the Nationalist Party under Daniel Malan did not augur well. The South African election of May 1948 brought the (Afrikaner) National Party to power. The Nationalists did not con- ceal their commitment to the most rigid separation of the races. In July 1949 the Prohibition of Mixed Marriages Act became law in South Africa, Decolonisation and the “Federal Moment” 29 making a marriage between a “European” and a “non-European” a criminal offence. Further efforts to increase segregation followed. The long-standing discrimination against the South African Indian community, which had equally been a feature of Smuts’ United Party rule, was now internation- alised via Jawaharal Nehru’s vigorous efforts to censure South Africa through the United Nations and its new status as a Dominion within the British Commonwealth. South Africa’s desire to incorporate the British protectorates of Swaziland, Basutoland and Bechuanaland existed since the Union was created and Dominion status conferred in 1910. Under Nationalist Party government from 1948 incorporation was pursued with renewed vigour. In addition, South African ambitions to annex South West Africa (mod- ern day Namibia) and the underlying possibility that Britain’s dependent white settler colonies in southern and northern Rhodesia may gravitate—by desire, design, or accident—towards South African-style apartheid was an omnipresent concern in Whitehall. South African sensitivities over all these matters caused chronic anxiety in London about the possibility of South Africa declaring itself a republic and leaving the Commonwealth (as it would eventually do in May 1961). Downloaded by [University College London] at 08:18 08 September 2014 y y All these issues, falling as they do under the umbrella of South African expansionism, were further exacerbated by indigenous black political mobil- isation. In Nyasaland, a new sense of black African political resistance gained ground. Northern Rhodesia contained a large indigenous population, which suffered exploitation in the copper mines and other extractive industries that dominated its economy. Decolonisation and the “Federal Moment” Moreover, the way in which the Seretse affair taps into pre-existing beliefs about the superiority of British civilisation helps us to understand the turn towards federation in central Africa. Soon after Malan’s election, Seretse Khama and Ruth Williams were declared prohibited immigrants in the Union of South Africa. This may not have come as a surprise to Labour ministers, nor would it, in and of itself, have been particularly destabilising of British policy in the region. The com- plicating factor was the calls of successive South African governments to have the three High Commission Territories—Seretse’s native Bechuanaland, as well as Swaziland and Basutoland—incorporated into the Union of South Africa.39 Under these circumstances, Labour ministers were advised that to give the title of “Native Authority” to an African who had married a white woman would be regarded as a deliberate provocation in South Africa. Malan would use the opportunity to renew calls for the transfer of the Territories, and to do so with the cross-party backing of the whites in the Union, as a way of setting his own back yard in order, as Pretoria saw it. He would also have the support of Sir Godfrey Huggins, the Prime Minister of Southern Rhodesia, which was made clear in a statement to the all white Legislative Assembly in Salisbury (Harare) on 7 July 1949.40 Downloaded by [University College London] at 08:18 08 September 2014 On the same day, after a meeting with Seretse Khama in Mafeking, Evelyn Baring, High Commissioner in Bechuanaland, had, in his own words, “his first opportunity of seeking help” from Douglas Forsyth, the Secretary of South Africa’s Department of External Affairs. A top secret and per- sonal letter was then sent to the Permanent Under-Secretary of State in the Commonwealth Relations Office (CRO London), Sir Percival Leisching. Baring made it clear to Leisching that Forsyth had been very clear with him. Forsyth, Baring reported, had discussed the whole matter with Malan, who had in turn discussed the question with his ministers and was greatly worried and distressed. Decolonisation and the “Federal Moment” The struggles of indigenous peoples in South West Africa were brought to the attention of international public opinion through the United Nations and the political activism of the English Anglican reverend Michael Scott, considered a “communist” by the United States.38 Amidst these myriad complications, the event that captured the British domestic mood most strongly, and in turn re-shaped the terrain upon which Labour had to make its southern African policy, is the marriage of Seretse Khama to Ruth Williams. It was September 1948 when Seretse Khama, a black African man from the Bechuanaland Protectorate, studying law in England, married a white English girl, Ruth Williams, at the Kensington Registry Office in London. Unfortunately for the newly married couple, Seretse was not just any old law student from Africa. He was the grandson of Khama the Great, destined to become the Kgosi (Chief) of the Bangwato, the largest and politically most significant tribe in the Bechuanaland Protectorate. Seretse’s uncle, Tshekedi, was the current Regent, recognised by the British Administration as the Native Authority pending Seretse’s accession, once he was of the right age. The marriage of Seretse Khama and Ruth Williams caused problems for successive British governments, first under Attlee’s Labour Party and then under the Conservatives. Most importantly for our purposes, in the late 1940s these fears fed into the on-going discussions about the CAF. In short, the 30 M. Collins “white settler problem” and the advent of Pretoria’s Nationalist apartheid policy were key drivers of British policy towards southern Africa and the CAF. The extent to which the Malan government in Pretoria was dictating British policy in southern Africa reveals the link between race politics and Britain’s self-perception as a benign power still capable of a progressive liberal imperialism. Moreover, the way in which the Seretse affair taps into pre-existing beliefs about the superiority of British civilisation helps us to understand the turn towards federation in central Africa. “white settler problem” and the advent of Pretoria’s Nationalist apartheid policy were key drivers of British policy towards southern Africa and the CAF. The extent to which the Malan government in Pretoria was dictating British policy in southern Africa reveals the link between race politics and Britain’s self-perception as a benign power still capable of a progressive liberal imperialism. Decolonisation and the “Federal Moment” Baring told Leisching that two points had emerged, and they warrant quoting in full: First, official recognition of Seretse as chief, so long as it implies the resi- dence in Serowe [Bechuanaland] of his English wife and the performance by her of the duties of the first wife of a chief is what really matters to the members of the government and probably to most South Africans. The mere residence of Seretse and his wife in Serowe without official recognition is objectionable to them, but in Forsyth’s view of subsidiary importance. He hopes that it might be avoided, but it is the recognition of Seretse as chief which will be the match to set off the gunpowder. Huggins has also written to me taking exactly the same point of view.41 Decolonisation and the “Federal Moment” 31 Secondly, Baring added: Secondly, Baring added: [t]he political consequences in the Union of recognition would be far more serious than I had realised. . . . The more extreme Nationalists will use the Seretse incident to add fuel to those flames. They will argue that our action demonstrates the folly of allowing the existence side by side in southern Africa of two systems of native administration diametrically opposed to one another. They will go on to say that South Africa should not and cannot remain associated with a country which recognises offi- cially an African chief married to a white woman, and they will make Seretse’s recognition the occasion of an appeal of the country for the establishment of a republic, and not only of a republic but a republic outside the Commonwealth. Decolonisation and the “Federal Moment” Collins it added, uncomfortably for the Labour government, that Seretse’s “prospects of success as a Chief are as bright as those of any native in Africa. . . . He is admittedly the lawful and legitimate heir and, save for his unfortunate marriage, would be in our opinion, a fit and proper person to assume the chieftainship.”45 In light of this Noel-Baker warned Attlee that the Harragin Report was “an inflammable document.”46 Attlee was suitably alarmed. “The document is most disturbing,” he said. “In effect we are invited to go contrary to the desires of the great majority of the Bangwato tribe, solely because of the attitude of the governments of the Union of South Africa and Southern Rhodesia. It is as if we had been obliged to agree to Edward VIII’s abdication so as not to annoy the Irish Free State and the United States of America.47 Attlee decided that the Report should be referred to the Cabinet but then accepted the advice of Gordon Walker that it should not be published. The Harrigan Report was suppressed for 30 years. Downloaded by [University College London] at 08:18 08 September 2014 Following the February 1950 General Election Gordon Walker was promoted to Secretary of State for Commonwealth Relations, replacing Noel- Baker. With Leisching now his Permanent Under-Secretary, Gordon Walker took the matter to the Cabinet with a new set of proposals. Seretse and Ruth would be banished from the Bechuanaland Protectorate. His proposals were accepted and a statement seeking approval was to be made to Parliament.48 Gordon Walker faced concern about the course of action proposed from all sides of the House of Commons. The integrity of the government was in question. The Seretse affair had become a cause celebre amongst the anti-colonial left in Britain, and a propaganda coup for the Soviet Union.49 Churchill called the whole affair a “very disreputable transaction.”50 Following the February 1950 General Election Gordon Walker was promoted to Secretary of State for Commonwealth Relations, replacing Noel- Baker. With Leisching now his Permanent Under-Secretary, Gordon Walker took the matter to the Cabinet with a new set of proposals. Seretse and Ruth would be banished from the Bechuanaland Protectorate. Decolonisation and the “Federal Moment” Malan is desperately worried and feels he could not successfully oppose an extremist offensive on these lines.42 Downloaded by [University College London] at 08:18 08 September 2014 In a minute to Philip Noel-Baker—the Secretary of State for Commonwealth Relations—Leisching informed him that in addition to the communication from Baring, he had been separately told by General Byers, Chief of the South African General Staff, that the British government’s recog- nition of Seretse as chief of the Bangwato would “light a fire through all the British colonial territories in Africa which would not soon be quenched.” Leisching went on to tell Noel-Baker that “the very existence of white settle- ment in these territories depended, in light of the numerical inferiority and defencelessness of the white population, upon the principle that the native mind regarded the white woman as inviolable.” He himself, he added, had been unable to accept the “ultimate logical consequences of this principle of non-discrimination when it takes practical forms affecting oneself or one’s family in terms of miscegenation.” He did not “believe that many who hold to their antipathy to the colour bar would, if confronted with this matter in personal terms, view with equanimity, or indeed without revulsion, the prospect of their son or daughter marrying a member of the Negro race.”43 The most senior civil servant at the CRO clearly viewed the problem through the prism of both race and gender. Gordon Walker responded with his sup- port for Baring’s proposal: “I would not put out of court the possibility of declaring that a chief cannot have a white wife. There is a lot to be said for this argument, and we should consider facing the uproar that would result. We must all think about this carefully.”44 Baring’s encounter with Forsyth appears to have decisively influenced his thinking, and in turn influenced the position of the British govern- ment. Having previously been minded to recognise Seretse as Chief, Baring changed his position, saying the right course was now to “play for time” via a commission of enquiry, which was subsequently set up under the chair- manship of Lord Harrigan, a course of action that Noel-Baker was willing to follow. For political reasons, the Harrigan Report came to the required con- clusion that Seretse should not be recognised as Kgosi of the Bangwato. But 32 M. Decolonisation and the “Federal Moment” His proposals were accepted and a statement seeking approval was to be made to Parliament.48 G d lk f d b h f i d f In our understanding of the extent to which the race issue shaped British government policy, it is important to note that during the House of Commons debate on Seretse, Reginald Sorenson, a Labour member, asked Gordon Walker whether South Africa had had any influence on the gov- ernment’s decision. Gordon Walker replied in unambiguous language: “we have had no communication from the government of the Union nor have we made any communication to them. There have been no representations and no consultation in this matter.”51 Gordon Walker’s statement to Parliament was followed by a White Paper which said: “His Majesty’s Government were of course aware that a strong body of European opinion in South Africa would be opposed to recognition; but as stated in the House of Commons on the 8 March, no representations on this matter have been received from the government of the Union of South Africa or Southern Rhodesia.” Hyam has referred explicitly to this incident and suggested Gordon Walker was “perilously close” to lying.52 In fact, it is almost certainly clear that this was indeed a lie to the House. On 30 June 1949, prior to Baring being persuaded by Pretoria, Lief Egeland, South African High Commissioner in London, had a meeting with Gordon Walker’s predecessor, Noel Baker, in which he made the very same case about the impact of recognising Seretse’s marriage on Decolonisation and the “Federal Moment” 33 white opinion in the Union and beyond. He had made clear to the Secretary of State that his visit was on the instructions of his Prime Minister, Malan, and Gordon Walker could not reasonably claim he, or his civil servants, were unaware of this.53 Either way, senior Labour ministers were paranoid about the race issue and the impact it could have on Britain’s strategic position in Africa. A paper presented by Gordon Walker to Cabinet in September 1950 claimed, fear- ing South American expansion, that “the policies we detest in the Union,” could be “established far to the North, and in the heart of this part of our Colonial Empire.” He envisaged “terrible wars . . . Decolonisation and the “Federal Moment” Collins Robinson’s so-called proconsul of African nationalism was taking a different stance. Cohen is perhaps better understood as a defender of a particular vision of British liberalism, one with a long pedigree, and for historical pur- poses this may be more interesting than the anodyne picture presented by Robinson. It is important to recognise the ways in which this vision was redeployed after World War II, indicating as it does the on-going vitality of the British imperial mission long after 1945, the scepticism about the nation-state that cut across party lines, and divisions between the civil service and politicians. p In August 1952, Henry Hopkinson, now Minister of State at the Colonial Office under Winston Churchill’s Conservative Government, held sixty-eight meetings in Northern Rhodesia and Nyasaland, coming to the conclusion that any further delay would merely encourage “nascent nationalism.”58 By the time the CAF came into being in 1953 it was accepted that Southern Rhodesia would benefit economically from northern raw materials, and that Nyasaland would provide a large pool of black labour. The issue of the colour bar in the northern copper belt was fudged. With this in mind, the suggestion that it was only after the Conservatives came to power in 1951 that we see the return of “old-fashioned imperialism” is surely misleading.59 There were some cases of consistency. Attlee, for example, stuck to his belief that federation in central Africa ran counter to “the entire premise of Labour’s African policy” because it set back the cause of African nationalism.60 But we have seen how, in this instance, earlier talk in Labour circles about political reform and the advance of black African nationalism came to be superseded by a renewed vision of British imperial mission, a vision that was accompanied by the implementation, eventually under the Conservatives, of a tremendously ambitious, ultimately foolhardy project of imperial state-building, a civilising mission, familiar in content but new in form. Decolonisation and the “Federal Moment” between a white ruled East Africa and a black ruled Western Africa,” and warned his colleagues that “our whole work in Africa would be undone.”54 Fred Cooper has suggested that after World War II, “[t]he old claims to colonial authority based on superior- ity of race and civilisation were thoroughly discredited.”55 This, as Cooper expertly shows, required Labour to justify its developmental imperialism in technocratic terms. And yet arguments about race and civilisation do remain of great importance. Downloaded by [University College London] at 08:18 08 September 2014 The conjuncture of race alongside geo-strategic considerations trumped the idea that amalgamation and federation in Central Africa should be postponed in order to protect the native African from white supremacist ideologies, both from within the Rhodesias and spreading out of the Union. Now, members of the Labour Party took heed of the warnings out of Pretoria, because they fed into pre-existing beliefs about the superiority of British values and the on-going need for Britain to nurture and protect the black African. They therefore acted against Seretse’s interests, persuaded that a new dominion north of the Limpopo (the river separating the Union of South Africa from Southern Rhodesia) was a higher goal. The fact that this British bloc was supposed to stand for British “multi-racial” values against the alien impact of the Afrikaner merely adds to the irony of Seretse’s treat- ment. Cohen’s conversations with American diplomats in September 1950, in which he reveals the radical shift from the position he held in 1949—now believing that “it would obviously be advantageous” to have a federated central Africa as a counterweight to South Africa—was contemporaneous to Gordon Walker’s apocalyptic vision of race war in Africa, the Cabinet dis- cussions about Seretse, the suppression of the Harrigan Report and Gordon Walker’s deceitful statement to the House of Commons. By April 1951, in a confidential minute, Cohen warned of the “Afrikaner danger” and Britain’s “duty to the welfare of Africans.” In the context of Northern Rhodesia and Nyasaland, Cohen stated that the native problem was one of bringing them round to a “true realisation of their own interests.”56 In these matters it is difficult to establish a forensic causal link, but the connection here seems to be irresistible. By the end of 1951 Cohen felt that it was now crucial not to abandon the field to “black nationalists,” which would have “disastrous consequences.”57 34 M. Decolonisation and the “Federal Moment” Downloaded by [University College London] at 08:18 08 September 2014 For the black Africans of Nyasaland and northern Rhodesia, talk of socio-economic advancement and preparation for self-government had been replaced by a new trusteeship and the re-mergence of pre-Word War II approaches to collaboration on the British imperial periphery, embodied in Hopkinson’s Lugardian conclusion that the black Africans of the CAF would accept federation “taking their lead from the chiefs.”61 The history of British imperial policy in southern Africa under Labour suggests that the great changes apparently signalled by the Cohen Report of 1947 stalled in the face of Afrikaner nationalism, white settler politics, and the complexities of race relations. The missionary imperialism of key Fabian influences on the Labour Party had always been pronounced. The turn to federation in central Africa, what Hyam has called “the most controversial large-scale imperial exercise in constructive state-building ever undertaken by the British government,”62 enabled the continuation of British control in central Africa and constituted a late flowering of a deep-rooted, cross-party and inter-governmental belief in Decolonisation and the “Federal Moment” 35 Britain’s liberal imperial mission. It is suggestive that will and ambition could still imagine their triumph over a paucity of resources.63 The CAF is but one instance of federation, and the story has only been told from the perspective of high politics in the metropole. If there are links between the missionary imperialism of an earlier age, which animated ideas about federation, union, and commonwealth prior to 1945, the post-1945 fed- eral moment is different, being explicitly a problem of decolonisation. The nascent Commonwealth would be a commonwealth of independent nation states. The federal option as deployed by the British meant resisting the logic of nationalism as a response to the challenges of decolonisation, and in the case of the CAF the pressures of white settler politics. Having acted as bridge between Labour and Conservative administra- tions in the negotiations over the CAF, Cohen went on to become Governor of Uganda in 1952. His time there is remembered rather well by many Ugandans, especially for his liberal and tolerant views, his focus on social and economic development, and his racial inclusivity. But it was not with- out controversy. Decolonisation and the “Federal Moment” Yet the compression of the historical space between empire and nation-state has arguably prevented us from asking how it was that for the purposes of decolonisation, national states became the only alternative to empires.68 In the post-war period federal questions arose in regard to other European empires.69 The United States was supportive of the idea of European federation.70 Questions of federal and “world government” continued to coalesce around debates about the United Nations.71 These may not be incidental connections, but may point to a historical moment in which decolonisation and the relationships between empire, nation, sovereignty, identity, and political economy were being questioned. This comes at a time when, as Charles Maier has suggested, the territorial basis of nation-statehood was already weakening.72 Downloaded by [University College London] at 08:18 08 September 2014 Raising our sights a little we realise that the CAF was not isolated. There were numerous federations or mooted federations across the British Empire during this period, including the Malay Federation, 1948–1963; the East African High Commission, 1948–1961; the previously discussed Central African Federation, 1953–1963; the West Indies Federation, 1958–1962; and the Federation of South Arabia, 1962–1967. These were not always British ini- tiatives, with nationalists and other agents in the colonies themselves thinking through the possibilities of federation for their own ends. But the sheer breadth of federal experiments is suggestive of a deeper historical connec- tion to ideologies of power and structures of identity, the significance of which may be lost if we understand these manoeuvres strictly in terms of a peripheral theory of collaboration and control. The idea of a federated empire has long and deep roots in British imperial ideology. Clearly the Federation of Rhodesia and Nyasaland is distinct from the dream of a fed- erated white Empire-Commonwealth imagined by the advocates of Greater Britain in the late nineteenth century, or the protagonists of the Round Table Movement which pushed the idea forward throughout the twentieth cen- tury. The British had already imposed a federal constitution on India. But exactly what is the relationship between idea of federation in the late 1940s and 1950s and that previous tradition? How did questions of empire, nation- hood, sovereignty, and territoriality play themselves out across the canvas of a decolonising British Empire? Moreover, that canvas was in fact far bigger than the British Empire. Decolonisation and the “Federal Moment” Cohen exiled the Kabaka of Buganda in 1953, essentially because of the Kabaka’s attempt to steer the course of Ugandan decoloni- sation toward a state based on Buganda, rather than the whole protectorate established by the British in 1894. In fact, Cohen was also very interested in an East African Federation to match the Central African one. The idea of an east African federation prompted vigorous political debate, support and opposition, in London and in east Africa itself. It would be picked up by local political leaders and newly independent states as a possible way to realise greater cooperation and put pan-African ideals into practice. As Julius Nyerere put it in 1964, “there is one way in East Africa that the present unity of opposition should become a unity of construction. The unity and freedom movements should be combined, and the East African territories achieve independence as one unit . . . this means Federation of the Territories now administered separately.”64 The story of the failure of east African federation needs to be told, both from the perspective of colo- nial authorities and, crucially, from the perspective of indigenous political elites.65 Why did federation seem like a good idea? In asking this ques- tion, the possible flaws of the nation-state model itself would be central to the discussion of the failure of the post-colonial state, alongside the neo-colonialism of former imperial powers or the inequities of the world economy.66 Downloaded by [University College London] at 08:18 08 September 2014 Answering these kinds of historical questions would take us into a wider field of inquiry, one in which the historical constitution of the nation-state in the post-war period could be re-examined. Whereas the Atlantic Charter and the Charter of the United Nations had envisaged the triumph of national self- determination, the 1950s was also a moment of deep aporia surrounding the viability of the nation-state. As Ernest Gellner articulated so precisely, nation- alism as an ideology holds that “the political and the national unit should be 36 M. Collins congruent.”67 The application of that principle had been one of the key drivers of two world wars. Paradoxically, empire explicitly resists national- ist logic. So too does federation. Decolonisation and the “Federal Moment” Decolonisation and the “Federal Moment” 37 NOTES 1. Versions of this article were presented at the International History Conference in Honour of Kathy Burk, UCL, 14 June 2012 and the Brady–Johnson Colloquium on Grand Strategy, International Security Studies, Yale University, 4 December 2012. I am grateful for the comments I received at both events. I am also incredibly thankful to all participants and convenors at the July 2012 International Seminar on Decolonization, National History Center, Washington DC, for their careful and sharp reading of an earlier draft. 2. For a near-contemporary assessment, see R. L. Watts, New Federations: Experiments in the Commonwealth (Oxford, 1966). My thanks to Jason C. Parker at Texas A&M for alerting me to this source. 2. For a near-contemporary assessment, see R. L. Watts, New Federations: Experiments in th Commonwealth (Oxford, 1966). My thanks to Jason C. Parker at Texas A&M for alerting me to th source. 2. For a near-contemporary assessment, see R. L. Watts, New Federations: Experiments in the Commonwealth (Oxford, 1966). My thanks to Jason C. Parker at Texas A&M for alerting me to this source. 3. For example, in the context of Anglo–American and imperial histories, see K. Burk, Old World, New World: The Story of Britain and America (New York, 2007); B. J. C. McKercher, Transition of Power: Britain’s Loss of Global Preeminence to the United States, 1930–1945 (Cambridge, 1999); or, more broadly, the Adventures With Britannia series, beginning with W. R. Louis, ed., Adventures With Britannia: Personalities, Politics and Culture in Britain (Austin Texas, 1996). 4. See R. Hyam, “The Political Consequences of Seretse Khama: Britain, the Bangwato and South Africa, 1948–1952,” Historical Journal, 29 (1986), pp. 921–47; and R. Hyam, “The Geopolitical Origins of the Central African Federation: Britain, Rhodesia and South Africa, 1948–1953,” Historical Journal, 30(1987), pp. 145–72. Both are reprinted in R. Hyam and P. Henshaw, The Lion and the Springbok: Britain and South Africa Since the Boer War (Cambridge, 2003). 5. W.R. Louis & R. Robinson, “The Imperialism of Decolonization,” Journal of Imperial an Commonwealth History, 22(1994), pp. 462–511. 6. E. Wallace, The British Caribbean: From the Decline of Colonialism to the End of Federation (Toronto, 1977), p. 86. This reference is attributable to Jason C. Parker’s as-yet-unpublished manuscript on federation in the West Indies. 7. Smith called for a “federal union with America.” See E. C. Mossner, I. Simpson Ross, R.H. Campbell, D. D. Raphael, and A. S. 18. On the first “Wilsonian moment” see E. Manela, The Wilsonian Moment: Self-Determination and the International Origins of Anticolonial Nationalism (New York, 2009). On the second one, see S. 15. R.E. Robinson, “Andrew Cohen and the Transfer of Power in Tropical Africa, 1940–1951,” in W.H. Morris-Jones & G. Fischer, eds, Decolonisation and Africa: The British and French Experience (London, 1980), pp. 50–72. Since Robinson himself was at the Colonial Office in the late 1940s working alongside men such as Andrew Cohen, this raises questions about how the historian is to historicise peo- ple and institutions with which he was so intimately involved. For another positive interpretation of the Labour government’s approach to Africa see R. Pearce, “The Colonial Office and Planned Decolonisation in Africa,” African Affairs, 83 (1984), pp. 77–93. Decolonisation and the “Federal Moment” The decolonising federal moment was a genuinely trans-national, world his- torical one, which deserves deeper, comparative historical analysis.73 This will hopefully reveal the complex ways in which historical actors at both the core and periphery of empires imagined alternative forms of sovereignty and political organisation, forms which it my yet serve us well to re-examine from our postcolonial present. NOTES Collins Moyn, “Imperialism, Self-Determination, and the Rise of Human Rights,” in Akira Iriye et al., eds., Th Human Rights Revolution: An International History (New York, 2012), pp. 159–78 19. For a comparative and comprehensive approach to labour questions in British and French Africa, see F. Cooper, Decolonization and African Society: The Labor Question in French and British Africa (Cambridge, 1996). On British East Africa, see P. Kelemen, “Modernising Colonialism: the British Labour Movement and Africa,” Journal of Imperial and Commonwealth History, 34(2006), pp. 223–244. 20. R.E. Robinson, “Cohen, Sir Andrew Benjamin (1909–1968),” in H.C.G. Matthew and Brian Harrison, eds, Oxford Dictionary of National Biography (Oxford, 2004), online edition, L. Goldman, ed., January 2011, http://www.oxforddnb.com/view/article/32478. 21. Robinson, “Transfer of Power in Tropical Africa,” p. 62. 22. Pearce called the 1947 report “a mature and systematic exposition of Colonial Office thinking, a consistent and conscious strategy of decolonisation.” R. Pearce, “Planned Decolonisation in Africa,” p. 86. Hyam has criticised the “wilder misinterpretations” that have been placed upon the pronouncements of Cohen and others in 1947 but cites D. Williams’ Cambridge History of Africa, Vol. VII: From c. 1940 to c. 1975 (Cambridge, 1984), 341 as an example, not mentioning Robinson. See R. Hyam, “Africa and the Labour Government, 1945–1951,” in R. Hyam, Understanding the British Empire (Cambridge, 2010), p. 242. Downloaded by [University College London] at 08:18 08 September 2014 23. See Cooper, Decolonization and African Society, pp. 202–215; R. Hyam, “Africa and the Labour Government,” p. 242. 24. Robinson, “Transfer of Power in Tropical Africa,” passim. 25. The possible convergence of Fabian thinking about empire and the slightly later American modernisation theory, for example Seymour Martin Lipset’s ideas about the socio-economic underpin- nings of democracy, is perhaps worth further investigation. See S.M. Lipset, “Some Social Requisites of Democracy: Economic Development and Political Legitimacy,” American Political Science Review, 53(1959), pp. 69–105; S.M. Lipset, Political Man: The Social Bases of Politics (New York, 1960). 26. R. Hinden, “Socialism and the Colonial World,” in A.C. Jones, ed., New Fabian Colonial Essays (London, 1959), p. 9. I owe this quote to Charlotte Riley, UCL History Department. 27. Ibid., p. 13. 28. United States Embassy, London, memorandum on “British Colonial Development and Welfare Programs,” p. 37. As Gallagher puts it with regard to the Second Anglo–Boer War, “indeed a good deal of what is commonly described as imperialist aggression during this period should rather be seen as imperialist counter-punching; or, to put it more demurely, as reactions by British policy-making to developments outside Europe.” J. Gallagher, Decline, pp. 81–82. The parallels with southern Africa in 1948–1953 are intriguing. NOTES Skinner, eds, The Correspondence of Adam Smith (Oxford, 1987), p. 383. On Shelburne, see H. M. Scott, British Foreign Policy in the Age of the American Revolution (O f d 1990) 224 I b h f h f S h C UCL (Oxford, 1990), p. 224. I owe both of these references to Stephen Conway, UCL. 8. W.D. McIntyre, The Commonwealth of Nations: Origins and Impact, 1869–1971 (Minneapolis, 1977), p. 30 9. D. Bell, The Idea of Greater Britain: Empire and the Future of World Order, 1860–1900 (Princeton NJ, 2007), pp. 6–62. 10. J. Kendle, Round Table Movement and Imperial Union (Toronto, 1975), pp. 70–71 10. J. Kendle, Round Table Movement an 11. Curtis to Milner, 1908, in Ibid., p. 56. 12. Macintyre, Commonwealth of Nations, p. 172. 13. J. Gallagher, The Decline, Revival and Fall of the British Empire (Cambridge, 1982), pp. 141–142. 13. J. Gallagher, The Decline, Revival and Fall of the B 14. G. Martel, “The Meaning of Power: Rethinking the Decline and Fall of Great Britain,” International History Review, 13(1991), passim; P. Kennedy, The Realties Behind Diplomacy: Background Influences on British External Policy, 1865–1980 (London, 1983); idem., The Rise and Fall of Great Powers (New York, 1987). 15. R.E. Robinson, “Andrew Cohen and the Transfer of Power in Tropical Africa, 1940–1951,” in W.H. Morris-Jones & G. Fischer, eds, Decolonisation and Africa: The British and French Experience (London, 1980), pp. 50–72. Since Robinson himself was at the Colonial Office in the late 1940s working alongside men such as Andrew Cohen, this raises questions about how the historian is to historicise peo- ple and institutions with which he was so intimately involved. For another positive interpretation of the Labour government’s approach to Africa see R. Pearce, “The Colonial Office and Planned Decolonisation in Africa,” African Affairs, 83 (1984), pp. 77–93. 16. J. Flint, “Planned Decolonization and Its Failure in British Africa,” African Affairs, 82(1983), pp. 389–411; R. Pearce, The Turning Point in Africa: British Colonial Policy in Africa, 1938–1948 (London, 1982), p. 43. 17. E. Borgwardt, A New Deal for the World: America’s Vision for Human Rights (Cambridge MA 2007). 18. On the first “Wilsonian moment” see E. Manela, The Wilsonian Moment: Self-Determination and the International Origins of Anticolonial Nationalism (New York, 2009). On the second one, see S. 38 M. 35. United States Embassy, London, “The Proposed Federation of Rhodesia and Nyasaland 28 October 1948, RG84, Classified General Records, Box 158, File 350 Britain-Commonwealth. 36. United States Embassy, London, “British Colonial Policies in Africa,” 20 September 1950, Ibid 33. This money has proved difficult to trace but the figure usually used is £98 million. See R. Schreurs, “A Marshall Plan for Africa? The Overseas Territories Committee and the Origin of European Cooperation in Africa,” in R.T. Griffiths, ed., Explorations in OEEC History (Paris, 1997). 31. J. Darwin, The End of the British Empire, p. 104 32. See F. Cooper, “Modernising Bureaucrats, Backward Africans and the Development Concept in F. Cooper and R.M. Packard, eds, International Development and the Social Sciences: Essays on th History and Politics of Knowledge (Los Angeles, 1997) pp. 64–92. 34. R. Welensky, Welensky’s 4000 Days (London, 1964). 31. J. Darwin, The End of the British Empire, p. 104 34. R. Welensky, Welensky’s 4000 Days (London, 1964). 38. C. Anderson, “International Conscience, the Cold War, and Apartheid: The NAACP’s Alliance with the Reverend Michael Scott for South West Africa’s Liberation, 1946–1952,” Journal of World History, 19 (2008), pp. 297–326; R. Skinner, “Christian Reconstruction, Secular Politics: Michael Scott and the Campaign for Right and Justice, 1943–45” in S. Dubow and A. Jeeves, eds., South Africa’s 1940s: Worlds of Possibilities (Cape Town, 2005). Decolonisation and the “Federal Moment” 39 Decolonisation and the “Federal Moment” 38. C. Anderson, “International Conscience, the Cold War, and Apartheid: The NAACP’s Alliance with the Reverend Michael Scott for South West Africa’s Liberation, 1946–1952,” Journal of World History, 19 (2008), pp. 297–326; R. Skinner, “Christian Reconstruction, Secular Politics: Michael Scott and the Campaign for Right and Justice, 1943–45” in S. Dubow and A. Jeeves, eds., South Africa’s 1940s: Worlds of Possibilities (Cape Town, 2005). 39. These calls found their impetus in the 1909 South Africa Act, which included consideration of the possibility of handing over the three territories to South Africa at some undefined point in the future. 40 “Th i d b ” i ld h “ h h ib ’ d i i [ S 39. These calls found their impetus in the 1909 South Africa Act, which included consideration of the possibility of handing over the three territories to South Africa at some undefined point in the future. 40. “There is no doubt,” Huggins told the House, “that the tribesmen’s decision [to support Seretse as Kgosi] is a disastrous one. First it shows lack of racial pride in Bechuanaland; secondly, it is disastrous from the effect it will have on neighbouring territories.” He told members that he had already written to the High Commissioner, but would write again “informing him of the opinion of this House and how disastrous it would be if this fellow is allowed to become Chief of Khama’s people.” S. Williams, Colour Bar: The Triumph of Sereste Khama and His Nation (London, 2006), pp. 69–70. 40. “There is no doubt,” Huggins told the House, “that the tribesmen’s decision [to support Seretse as Kgosi] is a disastrous one. First it shows lack of racial pride in Bechuanaland; secondly, it is disastrous from the effect it will have on neighbouring territories.” He told members that he had already written to the High Commissioner, but would write again “informing him of the opinion of this House and how disastrous it would be if this fellow is allowed to become Chief of Khama’s people.” S. Williams, Colour Bar: The Triumph of Sereste Khama and His Nation (London, 2006), pp. 69–70. 41. Baring to Leisching, 11 July 1949, DO (Dominions Office Archives, The National Archives, Kew, London) 119/1283. 42. Ibid. 43. Minute by Leisching, 14 July 1949, DO119/1283, Williams, Colour Bar, pp. 72–3. 45. Decolonisation and the “Federal Moment” Harrigan Report, 1 December 1949, PREM (Prime Minister’s Office Archives, Archives, The National Archives, Kew, London) 8/1308, Part 1, Ibid., p. 107. 45. Harrigan Report, 1 December 1949, PREM (Prime Minister’s Office Archives, Archives, The National Archives, Kew, London) 8/1308, Part 1, Ibid., p. 107. 46. Noel Baker to Attlee, 21 December 1949, Ibid.; Williams, Colour Bar, p. 110. 47. Attlee to Gordon Walker, 22 January 1950, PREM 8/1308, Williams, Colour Bar, p. 113 48. Gordon Walker would lose the Smethwick constituency seat at the General Election of 1964 to a Conservative Party candidate who ran an anti-immigration campaign with the slogan: “If you want a nigger neighbour, vote Labour.” 48. Gordon Walker would lose the Smethwick constituency seat at the General Election of 1964 to a Conservative Party candidate who ran an anti-immigration campaign with the slogan: “If you want a nigger neighbour, vote Labour.” 49. R. Skinner, “The Moral Foundations of British Anti-Apartheid Activism 1946–60,” Journal of Southern African Studies, 35 (2009), pp. 399–416. Cf. Hyam, “Political Consequences of Seretse Khama.” . R. Skinner, “The Moral Foundations of British Anti-Apartheid Activism 1946–60,” Journal of n African Studies, 35 (2009), pp. 399–416. Cf. Hyam, “Political Consequences of Seretse Khama.” . J. Redfern, Ruth and Seretse: A Very Disreputable Transaction (London, 1955), p. 7. 50. J. Redfern, Ruth and Seretse: A Very Disreputable Transaction (London, 1955), p. 7. 51. Hansard, 8 March 1950, House of Commons Debates, Series 5, Vol. 472, p. 288 m, “Political Consequences of Seretse Khama,” p. 939 53. Egeland sent a telegram to Malan, copied to the CRO, and then followed up his telegram with a private letter to Malan, telling him that at the very start of his meeting with Noel Baker in June he had made it clear that his visit to the Secretary of State was made “on your [Malan’s] instructions.” See Egeland to Malan, 24 March 1950, PM (National Archives of South Africa) Vol. 1/4/21, 1/15. / / / 54. R. Hyam, ed., British Documents on the End of Empire, Vol. 2: The Labour Government and the End of Empire 1945–1951 (London, 1992), p. 311. 54. R. Hyam, ed., British Documents on the End of Empire, Vol. 2: The Labour Government and the End of Empire 1945–1951 (London, 1992), p. 311. 55. Cooper, Decolonization, p. 173. 56. Hyam, “Geopolitical Origins,” p. 163. 57. Quoted in R. NOTES 9, circulated to Certain American Diplomatic and Consular Officers by the State Department, Washington D.C., 7 July 7 1948, RG 84 (National Archives and Records Administration, College Park, MD) Classified General Records, Box 158, File 350 Britain-Colonies. 29. Cohen did not miss the opportunity to remind the American Embassy in London that whilst in his view (and he was thought by American diplomats to be “probably the best-informed man in London on the subject”) communism had not yet penetrated deeply into Africa, keeping it out would be possible in large part due to the presence of British administration and control. See W. Stratton Anderson, United States Embassy, London, “Communism in Africa: Cohen’s Comments,” 26 October, 1948, Ibid. 30. On United States policy towards South Africa see T. Borstelmann, Apartheid’s Reluctant Uncle: The United States and Southern Africa in the Early Cold War (New York, 1993); and A. Thomson, U.S. Foreign Policy Towards Apartheid South Africa, 1948–1994 (New York, 2008). 30. On United States policy towards South Africa see T. Borstelmann, Apartheid’s Reluctant Uncle: The United States and Southern Africa in the Early Cold War (New York, 1993); and A. Thomson, U.S. Foreign Policy Towards Apartheid South Africa, 1948–1994 (New York, 2008). 31. J. Darwin, The End of the British Empire, p. 104 32. See F. Cooper, “Modernising Bureaucrats, Backward Africans and the Development Concept,” in F. Cooper and R.M. Packard, eds, International Development and the Social Sciences: Essays on the History and Politics of Knowledge (Los Angeles, 1997) pp. 64–92. 33. This money has proved difficult to trace but the figure usually used is £98 million. See R. Schreurs, “A Marshall Plan for Africa? The Overseas Territories Committee and the Origin of European Cooperation in Africa,” in R.T. Griffiths, ed., Explorations in OEEC History (Paris, 1997). 47. Attlee to Gordon Walker, 22 January 1950, PREM 8/1308, Williams, Colour Bar, p. 113. 68. Partha Chatterjee argued persuasively that anti-colonial nationalism was a “derivative discourse.” atterjee, Nationalist Thought and the Colonial World: A Derivative Discourse (London, 1986). Critics 72. C. S. Maier, “Consigning the Twentieth Century to History: Alternative Narratives for the Modern Era,” in American Historical Review, 105 (2000), pp. 807–31. Maier highlights “the spatially anchored structures for politics and economics that were taken for granted from about 1860,” and puts in a “plea for historians to envisage a historical era that took shape in the second half of the nineteenth century and, just as important, effectively unraveled in the two or three decades before the [twentieth] century formally ended.” Maier, “Consigning the Twentieth Century to History,” p. 808. Decolonisation and the “Federal Moment” Hyam, “Containing Afrikanerdom: The Geopolitical Origins of the Central Africa 57. Quoted in R. Hyam, “Containing Afrikanerdom: The Geopolitical Origins of the Central African Federation, 1948–1953,” in Hyam & Henshaw, Lion and the Springbok, p. 219. 58. Ibid., p. 165. Federation, 1948–1953,” in Hyam & Henshaw, Lion and the Springbok, p. 219. 58. Ibid., p. 165. Federation, 1948–1953,” in Hyam & Henshaw, Lion and the Springbok, p. 219. 58. Ibid., p. 165. 59. Pearce, “Planned Decolonization,” p. 88. 60. Hyam, “The Labour Government and Africa,” note 54, pp. 266. Hyam contrasts this with Cohen’s “determination to bring federation into being,” note 54, pp. 266. 61. Hyam, “Geopolitical Origins,” pp. 165. 61. Hyam, “Geopolitical Origins,” pp. 165. 62 Ibid 63. Cf. Martel, ‘The Meaning of Power’, passim. 63. Cf. Martel, ‘The Meaning of Power’, passim. 64 d d 4 ( 64) 4 64. J. Nyerere, “Freedom and Unity,” Transition, 14 (1964), p. 40. 65. There seems to be relatively little work done in this area, although east African federation did attract the attention of some prominent political scientists in the 1960s. See J.S. Nye, Pan–Africanism and East African Integration (Cambridge MA, 1965); C. Leys and P. Robson, eds, Federation in East Africa (Oxford, 1965). 66. For an idiosyncratic and stimulating discussion of this problem see B. Davidson, The Black Man’s Burden: Africa and the Curse of the Nation-State (New York, 1992). 66. For an idiosyncratic and stimulating discussion of this problem see B. Davidson, The Blac Man’s Burden: Africa and the Curse of the Nation-State (New York, 1992). 67. E. Gellner, Nations and Nationalism (Oxford, 1983), p. 1. 68. Partha Chatterjee argued persuasively that anti-colonial nationalism was a “derivative discourse.” P. Chatterjee, Nationalist Thought and the Colonial World: A Derivative Discourse (London, 1986). Critics 68. Partha Chatterjee argued persuasively that anti-colonial nationalism was a “derivative discourse.” atterjee, Nationalist Thought and the Colonial World: A Derivative Discourse (London, 1986). Critics M. Collins 40 of empire who resisted nationalism were few and far between. Rabindranath Tagore had denounced both imperialism and nationalism in the early twentieth century, but his lone voice was drowned out by the rising tide of anti-colonial nationalism, the seductive political power of which not even Gandhi could deny. See M. Collins, Empire, Nationalism and the Postcolonial World: Rabindranath Tagore’s Writings on History, Politics and Society (London, 2012). 69. On the Malay federation see A.J. 73. Cf. F. Cooper and J. Burbank, Empires in Word History: Power and the Politics of Differenc (Princeton, 2010), p. 10. 71. M. Mazower, Governing the World: The History of an Idea (London, 2012). 70. P. Winand, Eisenhower, Kennedy and the United States of Europe (Basingstoke, 1996). Decolonisation and the “Federal Moment” Stockwell, “Malaysia: The Making of a Neo-Colony?,” Journal of Imperial and Commonwealth History, 26 (1998), pp. 138–156; and M. Jones, Conflict and Confrontation in South East Asia, 1961–1965: Britain, the United States and the Creation of Malaysia (Cambridge, 2002). On federal experiments in French West Africa see F. Cooper, “Alternatives to Empire: France and Africa After World War II,” in D. Howland and L. White, eds, The State of Sovereignty: Territories, Laws, Populations (Bloomington, 2009). On the Dutch East Indies see J. Foray, “A Unified Empire of Equal Parts: The Dutch Commonwealth Schemes of the 1920s–1940s,” forthcoming in the Journal of Imperial and Commonwealth History. Downloaded by [University College London] at 08:18 08 September 20
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Seven naphtho-γ-pyrones from the marine-derived fungus Alternaria alternata: structure elucidation and biological properties
Organic and medicinal chemistry letters
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Seven naphtho-g-pyrones from the marine- derived fungus Alternaria alternata: structure elucidation and biological properties Mohamed Shaaban1,2*†, Khaled A Shaaban2† and Mohamed S Abdel-Aziz3 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Open Access © 2012 Shaaban 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. * Correspondence: mshaaba_99@yahoo.com † Contributed equally 1Chemistry of Natural Compounds Department, Pharmaceutical Industries Division, National Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt Full list of author information is available at the end of the article Abstract Eight bioactive pyrone derivatives were identified from the culture of Alternaria alternata strain D2006, isolated from the marine soft coral Denderonephthya hemprichi, which was selected as its profound antimicrobial activities. The compounds were assigned as pyrophen (1), rubrofusarin B (2), fonsecin (3), and fonsecin B (5) beside to the four dimeric naphtho-g-pyrones; aurasperone A (6), aurasperone B (7), aurasperone C (8), and aurasperone F (9). Structures of the isolated compounds were identified on the basis of 1D and 2D NMR spectroscopy and mass (EI, ESI, HRESI) data, and by comparison with the literature. Configuration of the four dimeric naphtho-g-pyrones 6-9 was analyzed by CD spectra, exhibiting an identical stereochemistry. Keywords: pyrone derivatives, Alternaria alternata, marine fungi, biological activity at Safaga coasts, Egypt) was selected due to its distinc- tive features in the chemical and biological assays. We therefore performed a bioassay-guided fractionation. 1. Background g Infectional diseases and drug resistance phenomena are the most effective reasons for the death of ca. 20 mil- lions yearly. For example, tuberculosis (TB) was the leading cause of ca. two million deaths due to a bacterial pathogen, Mycobacterium tuberculosis, among them more than 80% of TB patients living in sub-Africa and Asia [1-4]. Thus, new and more-powerful drugs are necessary to solve these problems. Marine microorgan- isms, especially fungi, are still a less investigated resource of bioactive substances [5,6]; recent investiga- tions indicated their tremendous potential as source of new drugs [7-13]. y g The crude extract possessed in the agar diffusion test potent activity against Pseudomonas aeruginosa, Staphy- lococcus aureus and Candida albicans. For isolation of the bioactive constituents, A. alternata D2006 was up- scaled as a shaker-culture using GYMP medium [15] (100% seawater) for 10 days. Thereafter, the obtained black broth was worked up [16] and separated by a ser- ies of chromatographic steps, yielding colourless semiso- lid of pyrophen (1) and seven naphtho-g-pyrones (2, 3, 5-9) as yellow solids, among them four dimeric analo- gues (6-9). Herein, we describe their separation, struc- ture elucidation (using 1D and 2D NMR and MS (EI, ESI, HRESI) data and antimicrobial properties. In this article, a report on the antimicrobial activity of naphtho-g-pyrones (naphthopyran-4-ones) attracted our interest [14]. During the investigation of fungal strains for the production of structurally novel active com- pounds from marine microorganisms, we found that the EtOAc extract of the marine-derived fungal strain Alter- naria alternata D2006 (isolated from a red soft coral, Denderonephthya hemprichi, collected from the Red Sea * Correspondence: mshaaba_99@yahoo.com † Contributed equally 1Chemistry of Natural Compounds Department, Pharmaceutical Industries Division, National Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt Full list of author information is available at the end of the article © 2012 Shaaban 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. 2. Taxonomy and characterization The fungal isolate was identified as A. alternata (Dema- tiaceae) according to Barnett [17]. Microscopically, the conidiophores were dark, simple, rather short or elon- gate and contained simple or branched chains of coni- dia. Conidia were dark, typically with both cross and longitudinal septa, with various shapes, obclavate to elliptical or ovoid. The fungal spores were multicellular, dark and having thick cell walls. Page 2 of 8 Page 2 of 8 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 for 1 using the 2D NMR experiments for the first time (Figure 1 and Table 1 [see Additional file 1]). for 1 using the 2D NMR experiments for the first time (Figure 1 and Table 1 [see Additional file 1]). Compound 2 showed a molecular weight of m/z 287.09137 (HRESI MS), corresponding to the molecular formula C16H15O5 [M+H]+. The 1H NMR spectra (Table 2) displayed a chelated hydroxyl group (δ 14.96), two m-coupled doublets (δ 6.56, 6.38) and two singlets (δ 6.94 and 5.98), along with two methoxy signals (δ 3.99, 3.91) and an sp2 linked methyl (δ 2.35). The 13C/ HMQC spectra (Table 2) indicated the presence of 16 carbon signals, including 4 sp2 methines (δ 107.3-97.2), 3 sp2-oxy carbons (δ 162.6-160.6), 1 carbonyl of g -lac- tone (δ 184.2) [20], 5 non-oxygenated sp2, 2 aromatic- attached methyl ethers (δ 56.0, 55.4) and 1 sp2-attached methyl (δ 20.6). Full assignment of the 2D NMR experi- ments (Figure 2 and Table 2) established the structure of 2 as rubrofusarin B, and excluded the structure of the isomeric asperxanthon (11) in the same way [21]. Structure of 2 was not fully assigned using 2D NMR before, which we report her to first time (see Additional file 2). The closely related compound 3 afforded a molecular weight of 290 Da (C15H12O5 by HRESI MS); EI MS gave easily an ion peak at m/z 272 by expulsion of water molecule. The 1H NMR spectrum exhibited aromatic m-coupled doublets (δ 6.47, 6.31, J ~ 1.1 Hz) and a methine singlet (δ 6.41), but in contrast to 2, two phe- nolic hydroxy signals (δ 14.19, 10.18), and only one methoxy signal (δ 3.84). In addition, an AB signal of dia- stereotopic methylene protons (δ 3.14, 2.72, J ~ 16.8) and a methyl singlet (δ 1.60) were visible. Based on 13C/ HMQC spectra (Table 2) and HMBC experiment (as it was not fully assigned before using 2D NMR) (Figure 2), 3. Results and discussion The expectation of a dimeric rubrofusarin B ( was confirmed by 1H NMR spectra, where six s Table 1 13C and 1H NMR data of pyrophen (1) in CDCl3 in [Hz]) Number δc δh Number δc δh 2 164.7 - 7-NHCOCH3 170.3 - 3 88.0 5.43 (d, 2.2) 7-NHCOCH3 22.3 1.95 (s 4 171.0 - 8 38.1 3.09 (m 4-OCH3 55.7 3.73 (s) 1’ 136.0 - 5 100.6 5.90 (d, 2.2) 2’,6’ 128.6 7.16 (m 6 161.9 - 3’,5’ 128.2 7.25 (m 7 52.3 4.98 (q, 7.8) 4’ 126.5 7.21 (m 7-NHCOCH3 - 7.79 (d, 8.4) Table 2 13C and 1H NMR data of rubrofusarin B (2) and fonsecin (3) in CDCl3 (J in [Hz]) Number 2 3 δc δH δc δH 2 167.4 - 100.0 - 2-CH3 20.6 2.35 (s) 27.6 1.60 (s) 2-OH - - - 6.95 (brs) 3 107.3 5.98 (s) 47.6 3.14 (d, 16.8), 2.72 (d, 16. 4 184.2 - 197.5 - 4a 104.3 - 102.5 - 5 162.6 - 164.2 - 5-OH - 14.96 (s) - 14.19 (s) 5a 108.4 - 105.2 - 6 160.6 - 161.4 - 6-OCH3 56.0 3.99 (s) 55.6 3.84 (s) 7 97.2 6.38 (d, 2.2) 96.6 6.31 (brd, 1.1) 8 161.5 - 160.7 - 8-OH - - - 10.18 (brs) 8-OCH3 55.4 3.91 (s) - - 9 97.8 6.56 (d, 2.2) 101.5 6.47 (s) 9a 141.0 - 142.9 - 10 101 0 6 94 (s) 101 0 6 41 (s) Table 1 13C and 1H NMR data of pyrophen (1) in CDCl3 (J in [Hz]) Number δc δh Number δc δh 2 164.7 - 7-NHCOCH3 170.3 - 3 88.0 5.43 (d, 2.2) 7-NHCOCH3 22.3 1.95 (s) 4 171.0 - 8 38.1 3.09 (m) 4-OCH3 55.7 3.73 (s) 1’ 136.0 - 5 100.6 5.90 (d, 2.2) 2’,6’ 128.6 7.16 (m) 6 161.9 - 3’,5’ 128.2 7.25 (m) 7 52.3 4.98 (q, 7.8) 4’ 126.5 7.21 (m) 7-NHCOCH3 - 7.79 (d, 8.4) Table 1 13C and 1H NMR data of pyrophen (1) in CDCl3 (J in [Hz]) methine protons were visible, which were classified into two m-coupled protons, two a-methines of the conse- quent g -pyrones (δ 6.15, 6.08) and two singlet methines (δ 7.35 and 7.24), together with six methyls, among them four methoxy signals. 3. Results and discussion Based on these data and search in literature, compound 6 was identified as auras- perone A [22] (see Additional file 5) Compound 7 exhibited a close structural similarity with fonsecin B (5); the molecular weight was determined as 606 Da, corresponding to the molecular formula C32H30O12 (HRESI MS). EI MS of 7 displayed an ion sig- nal at m/z 570 as base peak, resulting from the expulsion of two water molecules, affording the molecular weight of aurasperone A (6). The 1H NMR spectrum established a dimeric pattern of fonsecin B (5), where four sp2 methines protons being of two m-coupled protons and two singlet methines; two methylene signals (δ 3.02 and 2.89) instead of the two a-methines of the consequent-g- pyrones shown in 6, along with six methyls, among them four methoxy signals and two sp3-bounded methyl signals (δ 1.79, 1.46). In accordance, structure of 7 was assigned as aurasperone B (7) [22]. (see Additional file 6) compound 3 was finally established as fonsecin (3) (see Additional file 3). The facile loss of water by EI MS cor- responded to the formation of TMC-256 A1 (4). Compound 5 displayed similar chromatographic prop- erties and the same 1H NMR pattern as 3. The molecu- lar weight of 5 was deduced as 304 Da, which is 14 amu higher than that of 3, attributing to the methylation of the phenolic hydroxyl group at 8-position, hence com- pound 5 was identified as fonsecin B [22] (see Addi- tional file 4) A third dimer 8 had a molecular weight of 592 Da and a corresponding molecular formula C31H28O12. Three consecutive fragment ions (m/z 574, 556 and 525) on EI MS corresponded to the expulsion of one H2O molecule (to afford aurasperone F, 9), two H2O (dianhy- droaurasperone C, 10) and 2H2O + OCH3, respectively. The 1H NMR spectrum displayed the same pattern as in aurasperone B (7), except that the methoxy signal (δ 3.78) of 8-OCH3 in 7 was replaced by a phenolic hydro- xyl group, pointing to aurasperone C (8) [23]. (see Addi- tional file 7) 3. Results and discussion The expectation of a dimeric was confirmed by 1H NMR spec Table 1 13C and 1H NMR data of pyro in [Hz]) Number δc δh Numbe 2 164.7 - 7-NHCO 3 88.0 5.43 (d, 2.2) 7-NHCO 4 171.0 - 8 4-OCH3 55.7 3.73 (s) 1’ 5 100.6 5.90 (d, 2.2) 2’,6’ 6 161.9 - 3’,5’ 7 52.3 4.98 (q, 7.8) 4’ 7-NHCOCH3 - 7.79 (d, 8.4) Table 2 13C and 1H NMR data of rubr fonsecin (3) in CDCl3 (J in [Hz]) Number 2 3 δc δH δc δH 2 167.4 - 100.0 - 2-CH3 20.6 2.35 (s) 27.6 1.6 2-OH - - - 6.9 3 107.3 5.98 (s) 47.6 3.1 4 184.2 - 197.5 - 4a 104.3 - 102.5 - 5 162.6 - 164.2 - 5-OH - 14.96 (s) - 14 5a 108.4 - 105.2 - 6 160.6 - 161.4 - 6-OCH3 56.0 3.99 (s) 55.6 3.8 7 97.2 6.38 (d, 2.2) 96.6 6.3 8 161.5 - 160.7 - 8-OH - - - 10 8-OCH3 55.4 3.91 (s) - - 9 97.8 6.56 (d, 2.2) 101.5 6.4 9a 141.0 - 142.9 - 10 101.0 6.94 (s) 101.0 6.4 10a 153.3 - 153.4 - compound 3 was finally established as fonsecin (3) (s Additional file 3). The facile loss of water by EI MS co responded to the formation of TMC-256 A1 (4). Compound 5 displayed similar chromatographic pro erties and the same 1H NMR pattern as 3. The molec lar weight of 5 was deduced as 304 Da, which is 14 am higher than that of 3, attributing to the methylation the phenolic hydroxyl group at 8-position, hence com pound 5 was identified as fonsecin B [22] (see Add tional file 4) 3.1. Aurasperones A-C and F Compound 6 was obtained from fraction II as midd polar yellow solid, displaying a molecular weight at m 570. 3. Results and discussion The fungal extract showed several UV absorbing (254 nm) yellow bands, exhibiting yellowish-green UV fluor- escence at 366 nm. On spraying with anisaldehyde/sul- phuric acid and heating they turned orange to dark red, but showed no colour change with sodium hydroxide, thus excluding peri-hydroxyquinones. O NH O C H3 H O C H3 O 1 3 5 7 8 1' 4' Figure 1 Selected HMBC (®) and H, H-COSY (bold lines) correlations of pyrophen (1). The molecular formula of compound 1 was deter- mined by HRMS as C16H17NO4; the 1H NMR spectrum revealed signals for a phenyl residue, an amino NH doublet, and two m-coupled methines (δ 5.90, 5.43). Further signals were a methine quartet, a methylene 2H multiplet and two methyl singlets. The 13C NMR/ HMQC spectra indicated the existence of 16 carbons corresponding to a phenyl residue, 2 up-field sp2 methines (δ100.6, 88.0), 4 quaternary sp2 atoms (δ171.0- 161.9), representing carbonyls or phenolic carbons, and 4 sp3 carbon signals (δ55.7-22.3). According to these data, compound 1 was identified as pyrophen (1) [5], which was isolated and reported previously from Asper- gillus niger [18,19] and elucidated by crystal structure analysis. Here, we report the full NMR assignments data Figure 1 Selected HMBC (®) and H, H-COSY (bold lines) correlations of pyrophen (1). Figure 1 Selected HMBC (®) and H, H-COSY (bold lines) correlations of pyrophen (1). Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Page 3 of 8 Page 3 of 8 compound 3 was finally established Additional file 3). The facile loss of w responded to the formation of TMC Compound 5 displayed similar chr erties and the same 1H NMR pattern lar weight of 5 was deduced as 304 D higher than that of 3, attributing to the phenolic hydroxyl group at 8-p pound 5 was identified as fonsecin tional file 4) 3.1. Aurasperones A-C and F Compound 6 was obtained from fr polar yellow solid, displaying a mole 570. 3.1. Aurasperones A-C and F Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Page 4 of 8 O O OH O C H3 O CH3 C H3 1 3 5 7 9 10 O O OH O C H3 O H CH3 OH 1 3 5 7 9 10 Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). O O OH O C H3 O CH3 C H3 1 3 5 7 9 10 Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). O O OH O C H3 O CH3 C H3 1 3 5 7 9 10 Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). The optical rotations of the dimers had the same negative sign and similar values indicating that the opti- cal rotation value was dominated by the chiral axes between the two naphthopyranone moieties (atropi- somerism). The absolute configurations of dimeric naphtho-g-pyrones have been determined by circular- dichroism (CD). According to the literature [25], (S)- configured dimeric naphtho-g-pyrones exhibit a first positive Cotton Effect in the long-wavelength region, a negative Cotton Effect at middle wavelength and then a positive Cotton Effect at shorter one. In our experimen- tal data, the CD spectra for three representative dimeric naphtho-g-pyrones (6-8) showed closely related values with pronounced Cotton Effects, recognizing them to have the same patterns. In accordance, the ellipticity of aurasperones A-C (6-8) showed three Cotton Effects, one peak was shown firstly in the region of [θ]284-285 +359274-22843.4, then one trough between [θ]270-267 -151670-339938 and the last elliptical peak was shown at [θ]227-219 +107899-5629. As the dimer 6 has no further chiral elements, the chiral axis is dominating the absolute configuration. Based on the revealed features from the CD spectroscopic data, the four dimeric com- pounds (6-9) have identical (S)-configurations around their corresponding axis between C-10’ and C-7 (see Additional file 9) studied compounds here showed weak cytotoxicity (approx. 4-11%). 4.1. Sampling and isolation of the fungal strain 4.1. Sampling and isolation of the fungal strain The reddish soft coral D. hemprichi was collected from the Red Sea; approx. 30 km offshore from Safaga (east Egypt) at a depth of approx. 30 m. Pieces of the coral were rinsed three times with sterile seawater and then aseptically cut into smaller pieces and shaken for 2 h. The aqueous supernatant was serially diluted, and each 200 μL were inoculated onto 15-cm Petri dishes, each containing 50 mL of yeast extract/starch agar (yeast extract 0.2 g/L, soluble starch 1.0 g/L, agar 20 g/L, chloramphenicol 50 mg/L natural seawater at pH 6.0) [7]. The black single colonies were picked from the plates after inoculation for 25 days at 30°C and sub- cultured on the same medium without chlorampheni- col. The strain is deposited in the culture collection of the Department of Microbial Chemistry, NRC, Cairo, Egypt. 3.1. Aurasperones A-C and F Compound 6 was obtained from fraction II as middle polar yellow solid, displaying a molecular weight at m/z 570. The expectation of a dimeric rubrofusarin B (2) was confirmed by 1H NMR spectra, where six sp2 Table 2 13C and 1H NMR data of rubrofusarin B (2) and fonsecin (3) in CDCl3 (J in [Hz]) Number 2 3 δc δH δc δH 2 167.4 - 100.0 - 2-CH3 20.6 2.35 (s) 27.6 1.60 (s) 2-OH - - - 6.95 (brs) 3 107.3 5.98 (s) 47.6 3.14 (d, 16.8), 2.72 (d, 16.8) 4 184.2 - 197.5 - 4a 104.3 - 102.5 - 5 162.6 - 164.2 - 5-OH - 14.96 (s) - 14.19 (s) 5a 108.4 - 105.2 - 6 160.6 - 161.4 - 6-OCH3 56.0 3.99 (s) 55.6 3.84 (s) 7 97.2 6.38 (d, 2.2) 96.6 6.31 (brd, 1.1) 8 161.5 - 160.7 - 8-OH - - - 10.18 (brs) 8-OCH3 55.4 3.91 (s) - - 9 97.8 6.56 (d, 2.2) 101.5 6.47 (s) 9a 141.0 - 142.9 - 10 101.0 6.94 (s) 101.0 6.41 (s) 10a 153.3 - 153.4 - Table 2 13C and 1H NMR data of rubrofusarin B (2) and fonsecin (3) in CDCl3 (J in [Hz]) Compound 9 was a fourth dimer with a molecular for- mula C31H26O11; on EI MS, it displayed a fragment ion at m/z 556 corresponding to an aromatized structural analogue (dianhydroaurasperone C, 10), and a further fragment at m/z 286 corresponded to rubrofusarin B (2). The 1H NMR spectra displayed five sp2 methines (δ 6.87-6.08), one less than in 6, replaced by an AB signal of a methylene group (δ 3.35-3.25). Accordingly, one of the b-bounded methyls of the lactones was up-field shifted (δ 1.65), while the other one was retained at δ 2.16 as in 6. In contrast to 6, only three methoxy signals (δ 3.95-3.43) were visible, while the fourth one was replaced by a phenolic OH. Based on these spectro- scopic features, structure 9 was confirmed as aurasper- one F [24] (see Additional file 8) The four dimeric naphtho-g-pyrones (6-9) were pre- sently constructed from two naphtho-g-pyrone units, which are not symmetrically linked; i.e. the first pyrone (above) unit is linked via a middle aromatic moiety (10’- position) to a terminal aromatic residue (7-position) of the second pyrone (down) unit. Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. 4. Experimental The NMR spectra were measured on a Bruker AMX 300 (300.135 MHz), a Varian Unity 300 (300.145 MHz) and Varian Inova 500 (499.876 MHz) spectro- meters. EI mass spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV). ESI MS was recorded on a Finnigan LCQ with quaternary pump Rheos 4000 (Flux Instrument). HRMS were recorded by ESI MS on an Apex IV 7 Tesla Fourier-Transform Ion Cyclotron Resonance Mass Spectrometer (Bruker Daltonics, Bill- erica, MA, USA). Optical rotation was measured on a Perkin-Elmer Polarimeter, model 343. Flash chromato- graphy was carried out on silica gel (230-400 mesh). Rf values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co., Düren, Germany). Size exclu- sion chromatography was done on Sephadex LH-20 (Lipophilic Sephadex, Amersham Biosciences Ltd.; pur- chased from Sigma-Aldrich Chemie, Steinheim, Germany). Pyrophen (1) l l Colourless semisolid, UV-absorbing, no colour reaction on spraying with anisaldehyde/sulphuric acid; Rf = 0.86 (CH2Cl2/5% MeOH); 1H NMR (300 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 1; EI MS m/z (%) = 287.2 ([M]+, 28), 228.1 (8), 196.1 (40), 154.2 (100), 125.1 (16), 111.1 (6), 91.1 (12), 43.1 (11); (+)-ESI MS m/z (%) = 596.9 ([2M+Na]+, 85), 310 [M+Na]+, 36), 288 ([M+H]+, 100); (-)-ESI MS m/z 286 [M+H]-; (+)-HRESI MS m/z 288.12301 ([M+H]+, calcd: 288.12303 for C16H18NO4); 310.10490 ([M+Na]+, calcd: 310.10497 for C16H17NO4Na). Table 4 Antimicrobial activities of the fugal extract (60 μg/disc (5-mm diameter) Test organism Extract activity (mm) P. aeruginosa 17 S. aureus 23 C. albicans 20 A. niger ND ND, not detected. Table 4 Antimicrobial activities of the fugal extract (60 μg/disc (5-mm diameter) Test organism Extract activity (mm) P. aeruginosa 17 S. aureus 23 C. albicans 20 A. niger ND ND, not detected. 3.2. Biological activities The antibiotic activity of compounds 1-8 was exam- ined against 11 microbial test organisms using the agar diffusion method (40 μg/disc) (Table 3). According to the antimicrobial assay, the crude extract of the fungal strain exhibited high activity against bacteria and yeasts (Table 4). Nevertheless, only three of the iso- lated metabolites were found to exhibited activity: pyr- ophen (1) and rubrofusarin B (2) displayed high (28 mm) and moderate (12 mm) activity against C. albi- cans, respectively, while aurosperone A (6) was active (13 mm) against the plant pathogenic fungi, Rhizocto- nia solani. In the brine shrimp assay (10 μg/mL), all Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Page 5 of 8 Page 5 of 8 Table 3 Antimicrobial (40 μg/disc (∅9 mm; [mm]) and cytotoxic (10 μg/mL) activities of compounds 1-8 Compound number BSa SAb SVc ECd CAe MMf CVg CSh SSi PSj PUk Brine shrimp 1 ND ND ND ND 28 ND ND ND ND ND ND 4.2% 2 ND ND ND ND 12 ND ND ND ND ND ND 11% 3 ND ND ND ND ND ND ND ND ND 13 ND Nt 4 ND ND ND ND ND ND ND ND ND ND ND 8.8% 5 ND ND ND ND ND ND ND ND ND ND ND 5.0% 6 ND ND ND ND ND ND ND ND ND ND ND 9.7% 7 ND ND ND ND ND ND ND ND ND ND ND 6.4% 8 ND ND ND ND ND ND ND ND ND ND ND 9.7% aBacillus subtilis, bS. aureus, cStreptomyces viridochromogenes (Tü 57), dEscherichia coli, eC. albicans, fMucor miehi, gChlorella vulgaris, hChlorella sorokiniana, iScenedesmus subspicatus, jR. solani; kPythium ultimum ND, not detected. microbial (40 μg/disc (∅9 mm; [mm]) and cytotoxic (10 μg/mL) activities of compounds 1-8 b d f h i j k /disc (∅9 mm; [mm]) and cytotoxic (10 μg/mL) activities of compounds 1-8 aBacillus subtilis, bS. aureus, cStreptomyces viridochromogenes (Tü 57), dEscherichia coli, eC. albicans, fMucor miehi, gChlorella vulgaris, hChlorella sorokiniana, iScenedesmus subspicatus, jR. solani; kPythium ultimum ND, not detected. and they were combined therefore, and concentrated in vacuo, affording 5.5 g as black crude extract. 4.3. Isolation of the active constituents The obtained extract was applied to column chromato- graphy on silica gel eluted by CH2Cl2-MeOH gradient and monitored by TLC to afford five fractions: I (0.62 g), II (1.21 g), III (0.71 g), IV (1.52 g) and V (0.22 g). Fraction I was re-purified on silica gel column (DCM) followed by Sephadex LH-20 (DCM/40% MeOH) to afford a colour- less semisolid of phyrophen (1) (468.0 mg). Application of Fraction II to PTLC (DCM/3% MeOH) followed by purification on Sephadex LH-20 (DCM/40% MeOH) lead to isolation of two yellow solids of rubrofusarin B (2, 11.0 mg) and aurosperone A (6, 13.0 mg), respectively. Frac- tion III was purified using a silica gel column (DCM- MeOH) followed by Sephadex LH-20 (DCM/40% MeOH) to give a yellow solid of aurasperone F (9, 15.0 mg). Purification of the middle polar fraction IV via PTLC (DCM/5% MeOH) followed by Sephadex LH-20 (MeOH) yielded three yellow solids of aurasperone B (7, 8.0 mg), aurasperone C (8, 14.0 mg) and fonsecin (3, 11.5 mg). As the same for IV, the polar fraction V afforded three yellow solids of fonsecin B (5, 12.0 mg), aurasper- one B (7, 3.4 mg) and aurasperone C (8, 4.1 mg). The well-grown agar slants of the fungal strain D2006 were served to inoculate 60 of 1-L Erlenmeyer flasks, each containing 300-mL of GYMP medium (g/L): malt extract (3), yeast extract (3), peptone (5), glucose (10), agar (20) and 1000 mL of 100% seawater at pH approx. 6.5. The inoculated media was applied to additional cul- tivation using a rotary shaker (150 rpm) for 10 days. After harvesting, the obtained black culture broth was mixed with celite (approx. 1.5 kg) and then filtered in vacuo. The afforded two phases, filtrate and mycelium, were applied to exhaustive extraction by ethyl acetate. TLC of both organic extracts recognized their unique, 4.2. Fermentation and working up The well-grown single colonies of A. alternata were inoculated in subculture agar slants containing malt extract medium: malt extract (30 g/L), peptone 5 g/L), agar (20 g/L), natural sea water (1000 mL); at pH approx. 5.5 for 7 days at 30°C). The obtained grown agar slants were served to inoculate 500-mL Erlenmeyer flasks, each containing 100 mL of GYMP medium (g/L): malt extract (3), yeast extract (3), peptone (5), glucose (10) and 1000 mL natural seawater at pH approx. 6.5 at 30°C. The culture media was in turn applied to cultiva- tion on a rotary shaker (10 days). After harvesting, the afforded black broth was centrifuged (7,000 rpm for 15 min), and the obtained two phases, mycelial cake and supernatant, were individually extracted with ethyl acet- ate. The obtained unique black organic extracts were applied to biological and chemical screenings. Aurasperone A (6) Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; Rf = 0.82 (CH2Cl2/5% MeOH); [a]D 20 = -18.9 (c = 0.19, MeOH); CD (c 1.1929 × 10-5 mol/L [c 6.8 μg/mL], MeOH) [θ]400 0 [θ]284 +22843, [θ]270 -36396, [θ]219 +5629; 1H NMR (300 MHz, CD3OD) δ = 7.35 (s, 1H, H-10), 7.25 (s, 1H, H-9), 6.51 (brd, 1H, J ~ 1.1 Hz, H-7’), 6.23 (brd, 1H, J = 1.1 Hz, H-7), 6.15 (s, 1H, H-3), 6.08 (s, 1H, H-3’), 3.95 (s, 3H, 6’-OCH3), 3.79 (s, 3H, 8-OCH3), 3.59 (s, 3H, 8’- OCH3), 3.46 (s, 3H, 6-OCH3), 2.42 (s, 3H, 2-CH3), 2.13 (s, 3H, 2’-CH3); EI MS m/z (%) = 570.5 ([M]+, 44), 539.5 ([M-OCH3]+, 10), 513.4 (5), 286 (7), 167.2 (7), 145.2 (44), 116.2 (100), 85.1 (39), 55.1 (22), 43.1 (24). Aurasperone B (7) Aurasperone F (9) Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; Rf = 0.55 (CH2Cl2/5% MeOH); 1H NMR (300 MHz, CD3OD) δ = 6.87 (s, 1H, H-10), 6.55 (s, 1H, H-9), 6.51 (d, 1H, J ~ 1.1 Hz, H-9’), 6.36 (brd, 1H, J ~ 1.1 Hz, H-7’), 6.08 (s, 1H, H-3’), 3.95 (s, 3H, 6’-OCH3), 3.63 (s, 3H, 8’-OCH3), 3.43 (s, 3H, 6-OCH3), 3.35-3.25, (m, 2H, 3-H2), 2.16 (s, 3H, 2’-CH3), 1.65 (s, 3H, 2-CH3); EI MS m/z (%) = 556.5 ([M-H2O]+, 5), 286.3 ([rubrofusarin B (2)]+, 8), 84.1 (12), 57.2 (10), 44.1 (100); (+)-ESI MS m/z (%) = 1172 ([2M+Na+H]+, 19), 575 ([M+H]+, 100); (-)-ESI MS m/z (%) = 1721 ([3M-H]-, 31), 1147 ([2M-H]-, 22), 573 ([M- H]-, 100). 16 15 6) Fonsecin B (5) Yellow solid, UV-green fluorescence (365 nm), turned dark red with anisaldehyde/sulphuric acid; Rf = 0.44 (CH2Cl2/5% MeOH); 1H NMR (300 MHz, DMSO-d6) δ = 14.09 (brs, 1H, 5-OH), 7.00 (brs, 1H, 2-OH), 6.68 (brd, 1H, J ~ 1.1 Hz, H-9), 6.55 (s, 1H, H-10), 6.38 (brd, 1H, J ~ 1.1 Hz, H-9), 3.84 (s, 6H, 6,8-OCH3), 3.14 (d, 1H, J ~ 16.8 Hz, H-3a), 2.72 (d, 1H, J ~ 16.8 Hz, H-3b), 1.61 (s, 3H, 2-CH3); EI MS m/z (%) = 304.3 ([M]+, 56), 286.3 ([M-H2O]+, 8), 262.3 (8), 247.2 (28), 246.2 (60), 220.2 (20), 218.2 (10), 149.2 (20), 145.2 (34), 127.2 (12), 116.2 (64), 101.2 (48), 84.1 (36), 66.1 (24), 59.1 (63), 43.1 (100). Fonsecin (3) Yellow solid, UV-green fluorescence (365 nm), turned dark red with anisaldehyde/sulphuric acid; Rf = 0.38 (CH2Cl2/5% MeOH); 1H NMR (300 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 2; EI MS m/z (%) = 290.2 ([M]+, 24), 272.2 ([M-H2O]+, 16), 243.2 (8), 232.1 (21), 189.1 (7), 175.1 (16), 101.1 (15), 85.1 (22), 59.1 (36), 43.1 (100); (+)-ESI MS m/z (%) = 291 ([M+H] +); (-)-ESI MS m/z (%) = 289 ([M-H]-); (+)-HRESI MS m/z 291.08631 ([M+H]+, calcd: 291.08631 for C16H15O6). Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; Rf = 0.26 (CH2Cl2/5% MeOH); [a]D 20 = -33.5 (c = 0.17, MeOH); CD (c 4.29 × 10-5 mol/L [c 24 μg/mL], MeOH) [θ]400 0, [θ]285 +359273, [θ]268 -339938, [θ]226 +107899.87; 1H NMR (300 MHz, CD3OD): δ = 6.84 (s, 1H, H-10), 6.57 (s, 1H, H-9), 6.38 (d, 1H, J ~ 1.2 Hz, H-9’), 6.20 (d, 1H, J ~ 1.2 Hz, H-7’), 3.93 (s, 3H, 6’-OCH3), 3.60 (s, 3H, 8’- OCH3), 3.50 (s, 3H, 6-OCH3), 3.30-3.29 (m, 4H, 3,3’- H2), 1.69 (s, 3H, 2-CH3), 1.49 (s, 3H, 2’-CH3); EI MS m/ z (%) = 574.3 ([M-H2O]+, 6), 556.3 ([M-2H2O]+, 42), 525.3 ([M-(2H2O+OCH3)]+, 32), 264.2 (7) 58.2 (28), 43.1 (100); (+)-HRESI MS m/z 615.14779 ([M+Na]+, calcd: 615.14729 for C31H28O12Na), m/z 593.16570 ([M+H]+, calcd: 593.16534 for C31H29O12). Rubrofusarin B (2) Yellow solid, UV-green fluorescence (365 nm), orange with anisaldehyde/sulphuric acid; Rf = 0.78 (CH2Cl2/5% Page 6 of 8 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Hz, H-9’), 3.99 (s, 3H, 6’-OCH3), 3.78 (s, 3H, 8-OCH3), 3.63 (s, 3H, 8’-OCH3), 3.39 (s, 3H, 6-OCH3), 3.02, (d, 2H, J ~ 16.3 Hz, 3-H2), 2.89 (m, 2H, 3’-H2), 1.79 (s, 3H, 2-CH3), 1.46 (s, 3H, 2’-CH3); EI MS m/z (%) = 570.3 ([M-2H2O]+, 100), 539.4 ([M-(2H2O+OCH3)]+, 74), 524.3 (5), 299.2 (12), 272.2 (13), 269.7 (24), 230.2 (18), 193.1 (12), 154.2 (14), 149.1 (19), 130.1 (48), 91.1 (54), 57.1 (30), 43.1 (57); (+)-HRESI MS m/z 607.18100 ([M +H]+, calcd: 607.18100 for C32H31O12), m/z 629.16294 ([M+Na]+, calcd: 629.16295 for C32H30O12Na). Aurasperone C (8) MeOH); 1H NMR (300 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 2; EI MS m/z (%) = 286.2 ([M]+., 100), 268.1 ([M-H2O]+, 12), 257.2 ([M-CHO]+, 44), 240.2 (8) 213.2 (5), 43.1 (7); (+)-ESI MS m/z (%) = 594.9 ([2M+Na]+, 14), 287 ([M+H]+, 100); (+)-HRESI MS m/z 287.09137 ([M+H]+, calcd: 287.09139 for C16H15O5). References 1. DWHO Report (2006) Global tuberculosis control: surveillance, planning, financing E. World Health Organization, Geneva p 1 1. DWHO Report (2006) Global tuberculosis control: surveillance, planning, financing E. World Health Organization, Geneva p 1 2. Alland D, Kalkut GE, Moss AR, McAdam RA, Hahn JA, Bosworth W, Drucker E, Bloom BR (1994) Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods. N Engl J Med 330(24):1710–1716. doi:10.1056/ NEJM199406163302403. 3. Whalen C, Horsburgh CR, Hom D, Lahart C, Simberkoff M, Ellner J (1995) Accelerated course of human immunodeficiency virus infection after tuberculosis. Am J Respir Crit Care Med 151(1):129–135 Acknowledgements g The authors are deeply thankful to Prof. H. Laatsch for his Lab facilities and unlimited support. We are appreciated greatly R. Machinek for the NMR spectra, Dr. H. Frauendorf for the mass measurements, F. Lissy for biological activity tests and A. Kohl for technical assistance. Dr. Mohamed Shaaban is deeply thankful to the DAAD offices in Cairo and Bonn for kindly financing of the project during the visiting period in Germany. Brine shrimp microwell cytotoxicity assay Supplementary Information accompanying this paper includes MS, NMR and CD spectra. The cytotoxic assay was performed according to Taka- hashi et al. [27] and Sajid et al. [28]. Author details 1 1Chemistry of Natural Compounds Department, Pharmaceutical Industries Division, National Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt 2Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany 3Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt 4.4. Biological activities Antimicrobial activity Compounds 1-8 were dissolved in CH2Cl2/10% MeOH at a concentration of 1 mg/mL. Aliquots of 40 μL were soaked on filter paper discs (9 mm ∅, no. 2668, Schlei- cher & Schüll GmbH, Germany) and dried for 1 h at room temperature under sterilized conditions. The paper discs were placed on inoculated agar plats and incubated for 24 h at 38°C for bacterial and 48 h (30°C) for the fungal isolates, while the algal test strains were incubated at room temperature in day light. Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; Rf = 0.48 (CH2Cl2/5% MeOH); [a]D 20 = -18.3 (c = 0.12, MeOH); CD (c 2.83 × 10-5 mol/L [c 17.2 μg/mL], MeOH) [θ]400 0, [θ]284 +143232, [θ]267 -151670, [θ]227 +46610; 1H NMR (300 MHz, CDCl3) δ = 14.51 (brs, 1H, 5’-OH), 14.08 (brs, 1H, 5-OH), 6.84 (s, 1H, H-9), 6.72 (s, 1H, H- 9), 6.37 (d, 1H, J ~ 1.1 Hz, H-7’), 6.14 (d, 1H, J ~ 1.1 For the fungal extract examination, representative test microbes; P.aeruginosa, S. aureus, C. albicans and A. Page 7 of 8 Page 7 of 8 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 niger were used. Both bacterial and yeast strains were grown on nutrient agar medium (g/L): Beef extract 3; peptone, 10; and agar, 20. The pH was adjusted to 7.2. The fungal strain was grown on Czapek-Dox medium (g/l): Sucrose, 30; NaNO3, 3; MgSO4.7H2O, 0.5l; KCl, 0.5; FeSO4, 0.01; K2HPO4, 1; and agar, 20. The pH was maintained at 6.0. The disc diffusion test has been done according to Collins and Lyne [26]. Filter paper discs (5 mm diameter) were saturated with 200 μg from the cul- ture extract, and located on the surface of the agar plates (150 mm diameter containing 50 mL of solidified media). The paper discs were placed on inoculated agar plats and incubated for 24 h at 38°C (bacteria and yeast) and 48 h at 30°C (fungi). Additional file 7: Spectral data of Aurasperone C (8). Four charts (chart 40-43) containing the mass (ESI, HRESI MS) and NMR (1HNMR) spectral data of Aurasperone C (8) Additional file 7: Spectral data of Aurasperone C (8). Four charts (chart 40-43) containing the mass (ESI, HRESI MS) and NMR (1HNMR) spectral data of Aurasperone C (8) Additional file 8: Spectral data of Aurasperone F (9). 5. Conclusions In this research article, eight bioactive pyrone derivatives were identified from the culture of A. alternata strain D2006, isolated from the marine soft coral D. hemprichi. Selection of the strain was based on its profound anti- biotic and antimicrobial activities. Structures of the iso- lated compounds were identified on the basis of 1D and 2D NMR spectroscopy and mass (EI, ESI, HRESI) data, and by comparison with the literature. Configuration of the four dimeric naphtha-g-pyrones 6-9 was analyzed by CD spectra, exhibiting an identical stereochemistry. The biological activity (antimicrobial and cytotoxicity) of the fungal extract and its corresponding isolated compounds were comparatively studied. This is as a trial to find out new leading drugs to overcome some of the recently dis- covered diseases. 4.4. Biological activities Antimicrobial activity Three charts (chart 44-46) containing the mass (ESI, EI MS) and NMR (1HNMR) spectral data of Aurasperone F (9) Additional file 9: CD Spectra of Aurasperones A-C (6-8). Three charts (chart 47-49) containing the CD spectral data of Aurasperones A-C (6-8). Additional file 9: CD Spectra of Aurasperones A-C (6-8). Three charts (chart 47-49) containing the CD spectral data of Aurasperones A-C (6-8). Additional file 9: CD Spectra of Aurasperones A-C (6-8). Three charts (chart 47-49) containing the CD spectral data of Aurasperones A-C (6-8). Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 21 October 2011 Accepted: 29 February 2012 Published: 29 February 2012 Additional material p 4. Zumla A, Grange J (1998) Tuberculosis. BMJ 316(719):1962–1964 5. Laatsch H AntiBase, a data base for rapid structural determination of microbial natural products, and annual updates, chemical concepts, Weinheim, Germany.http://wwwuser.gwdg.de/~hlaatsc/antibase.htm Additional file 1: Spectral data of Pyrophen (1). Ten charts (chart 1- 10) containing the mass (ESI, HRESI, EI MS) and NMR (1HNMR, 13CNMR, H, H COSY, HMQC, HSQC, HMBC) spectral data of Pyrophen (1). 6. Laatsch H (2006) Marine bacterial metabolites. In: Proksch P, Müller WEG (ed) Frontiers in marine biotechnology. Horizon Bioscience, Norfolk, UK pp 225–288. ISBN 1-904933-18-1 Additional file 2: Spectral data of Rubrofusarin B (2). Thirteen charts (chart 11-23) containing the mass (ESI, EI, HRESI MS) and NMR (1HNMR, 13CNMR, H, H COSY, HMQC, HSQC, HMBC) spectral data of Rubrofusarin B (2). 7. Shiono Y, Tsuchinari M, Shimanuki K, Miyajima T, Murayama T, Koseki T, Laatsch H, Takanami K, Suzuki K (2007) Fusaristatins A and B, two new cyclic lipopeptides from an endophytic Fusarium sp. J Antibiot 60(5):309–316. doi:10.1038/ja.2007.39. Additional file 3: Spectral data of Fonsecin (3). Nine charts (chart 24- 32) containing the mass (ESI, EI MS) and NMR (1HNMR, 13CNMR, H, H COSY, HMQC, HSQC, HMBC) spectral data of Fonsecin (3). 8. Al-Zereini W, Schuhmann I, Laatsch H, Helmke E, Anke H (2007) New aromatic nitro compounds from Salegentibacter sp. T436, an Arctic sea ice bacterium. Taxonomy, fermentation, isolation and biological activities. J Antibiot 60(5):301–308. doi:10.1038/ja.2007.38. 8. Al-Zereini W, Schuhmann I, Laatsch H, Helmke E, Anke H (2007) New aromatic nitro compounds from Salegentibacter sp. T436, an Arctic sea ice bacterium. Taxonomy, fermentation, isolation and biological activities. J Antibiot 60(5):301–308. doi:10.1038/ja.2007.38. Additional file 4: Spectral data of Fonsecin B (5). Two charts (chart 33-34) containing the mass (EI MS) and NMR (1HNMR) spectral data of Fonsecin B (5). 9. Namikoshi M, Akano K, Kobayashi H, Koike Y, Kitazawa A, Rondonuwu AB, Pratasik SB (2002) Distribution of marine filamentous fungi associated with marine sponges in coral reefs of Palau and Bunaken Island, Indonesia. J Tokyo Univ Fish 88:15–20 Additional file 5: Spectral data of Aurasperone A (6). Two charts (chart 35-36) containing the mass (EI MS) and NMR (1HNMR) spectral data of Aurasperone A (6) 10. Lange L (1996) Microbial metabolites–an infinite source of novel chemistry. Pure Appl Chem 68(2):745–748 Additional file 6: Spectral data of Aurasperone B (7). Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 12. Abdelazim A (2004) Secondary metabolites of marine-derived fungi: natural product chemistry and biological activity. PhD Thesis, Rheinischen Friedrich- University, Bonn, Germany 12. Abdelazim A (2004) Secondary metabolites of marine-derived fungi: natural product chemistry and biological activity. PhD Thesis, Rheinischen Friedrich- University, Bonn, Germany 12. Abdelazim A (2004) Secondary metabolites of marine-derived fungi: natural product chemistry and biological activity. PhD Thesis, Rheinischen Friedrich- University, Bonn, Germany 13. Feofilova EP (2001) The kingdom fungi: heterogeneity of physiological and biochemical properties and relationships with plants, animals, and prokaryotes (Review). Appl Biochem Microbiol 37(2):124–137. (Translated from Prikladnaya Biokhimiya i Mikrobiologiya 2001, 37(2):141-155). doi:10.1023/A:1002863311534. 13. Feofilova EP (2001) The kingdom fungi: heterogeneity of physiological and biochemical properties and relationships with plants, animals, and prokaryotes (Review). Appl Biochem Microbiol 37(2):124–137. (Translated from Prikladnaya Biokhimiya i Mikrobiologiya 2001, 37(2):141-155). doi:10.1023/A:1002863311534. 14. James GG, Zhang HJ, Susan LP, Bernard DS, Andrew DM, Fernando C, Norman RF (2004) Antimycobacterial naphthopyrones from Senna obliqua. J Nat Prod 67(2):225–227. doi:10.1021/np030348i. 14. James GG, Zhang HJ, Susan LP, Bernard DS, Andrew DM, Fernando C, Norman RF (2004) Antimycobacterial naphthopyrones from Senna obliqua. J Nat Prod 67(2):225–227. doi:10.1021/np030348i. 15. Masoud W, Kaltoft CH (2006) The effects of yeasts involved in the fermentation of Coffea arabica in East Africa on growth and ochratoxin A (OTA) production by Aspergillus ochraceus. Int J Food Microbiol 106(2):229–234. doi:10.1016/j.ijfoodmicro.2005.06.015. 15. Masoud W, Kaltoft CH (2006) The effects of yeasts involved in the fermentation of Coffea arabica in East Africa on growth and ochratoxin A (OTA) production by Aspergillus ochraceus. Int J Food Microbiol 106(2):229–234. doi:10.1016/j.ijfoodmicro.2005.06.015. 16. Bibani MAF, Baake M, Lovisetto B, Laatsch H, Helmke E, Weyland H (1998) Marine bacteria. X. Anthranilamides: new antimicroalgal active substances from a marine Streptomyces sp. J Antibiot 51(3):333–340. doi:10.7164/ antibiotics.51.333. 17. Barnett HL (1972) Illustrated genera of imperfect fungi. Burgess Publishing Company, Minneapolis, 2 18. Barnes CL, Steiner JR, Torres E, Pacheco R, Marquez H (1990) Structure and absolute configuration of pyrophen, a novel pyrone derivative of L- phenylalanine from Aspergillus niger. Int J Pept Protein Res 36(3):292–296 19. Varoglu M, Crews P (2000) Biosynthetically diverse compounds from a saltwater culture of sponge-derived Aspergillus niger. J Nat Prod 63(1):41–43. doi:10.1021/np9902892. 20. Abd-Alla HI, Shaaban M, Shaaban KA, Abu-Gabal NS, Shalaby NMM, Laatsch H (2009) New bioactive compounds from Aloe hijazensis. Nat Prod Res 23(11):1035–1049. doi:10.1080/14786410802242851. 21. Additional material Three charts (chart 37-39) containing the mass (HRESI, EI MS) and NMR (1HNMR) spectral data of Aurasperone B (7) 11. Hawksworth DL, Rossman AY (1997) Where are all the undescribed fungi? Phytopathology 87(9):888–891. doi:10.1094/PHYTO.1997.87.9.888. 11. Hawksworth DL, Rossman AY (1997) Where are all the undescribed fungi? Phytopathology 87(9):888–891. doi:10.1094/PHYTO.1997.87.9.888. Page 8 of 8 Page 8 of 8 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6 http://www.orgmedchemlett.com/content/2/1/6 Sakurai M, Kohno J, Yamamoto K, Okuda T, Nishio M, Kawano K, Ohnuki T (2002) TMC-256A1 and C1, new inhibitors of IL-4 signal transduction produced by Aspergillus niger var niger TC 1629. J Antibiot 55(8):685–692. doi:10.7164/antibiotics.55.685. 22. Priestap HA (1984) New naphthopyrones from Aspergillus fonsecaeus. Tetrahedron 40(19):3617–3624. doi:10.1016/S0040-4020(01)88792-5. 23. Tanaka H, Wang P, Namiki M (1972) Structure of Aurasperone C. Agric Biol Chem 36(13):2511–2517. doi:10.1271/bbb1961.36.2511. Chem 36(13):2511–2517. doi:10.1271/bbb1961.36.2511. 24. Bouras N, Mathieu F, Coppel Y, Lebrihi A (2005) Aurasperone F–a new member of the naphtho-gamma-pyrone class isolated from a cultured microfungus, Aspergillus niger C-433. Nat Prod Res 19(7):653–659. doi:10.1080/14786410412331286955. 24. Bouras N, Mathieu F, Coppel Y, Lebrihi A (2005) Aurasperone F–a new member of the naphtho-gamma-pyrone class isolated from a cultured microfungus, Aspergillus niger C-433. Nat Prod Res 19(7):653–659. doi:10.1080/14786410412331286955. 25. Zhang Y, Ling S, Fang Y, Zhu T, Gu Q, Zhu WM (2008) Isolation, structure elucidation, and antimycobacterial properties of dimeric naphtho-γ-pyrones from the marine-derived fungus Aspergillus carbonarius. Chem Biodivers 5(1):93–100. doi:10.1002/cbdv.200890017. 25. Zhang Y, Ling S, Fang Y, Zhu T, Gu Q, Zhu WM (2008) Isolation, structure elucidation, and antimycobacterial properties of dimeric naphtho-γ-pyrones from the marine-derived fungus Aspergillus carbonarius. Chem Biodivers 5(1):93–100. doi:10.1002/cbdv.200890017. 26. Collins CH, Lyne PM (1985) Microbiological methods. Butterworth and Co. Publishers Ltd., London, 5 pp 167–181 26. Collins CH, Lyne PM (1985) Microbiological methods. Butterworth and Co. Publishers Ltd., London, 5 pp 167–181 27. Takahashi A, Kurasawa S, Ikeda D, Okami Y, Takeuchi T (1989) Altemicidin, a new acaricidal and antitumor substance. I. Taxonomy, fermentation, isolation and physico-chemical and biological properties. J Antibiot 42(11):1556–1561. doi:10.7164/antibiotics.42.1556. 28. Sajid I, Fondja Yao CB, Shaaban KA, Hasnain S, Laatsch H (2009) Antifungal and antibacterial activities of indigenous Streptomyces isolates from saline farmlands: prescreening, ribotyping and metabolic diversity. World J Microbiol Biotechnol 25(4):601–610. doi:10.1007/s11274-008-9928-7. 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 doi:10.1186/2191-2858-2-6 Cite this article as: Shaaban et al.: Seven naphtho-g-pyrones from the marine-derived fungus Alternaria alternata: structure elucidation and biological properties. Organic and Medicinal Chemistry Letters 2012 2:6. Submit your manuscript to a journal and benefi t from:
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https://link.springer.com/content/pdf/10.1007/JHEP10(2018)047.pdf
English
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Angular analysis of B0 d → K∗μ+μ− decays in pp collisions at $$ \sqrt{s}=8 $$ TeV with the ATLAS detector
˜The œJournal of high energy physics/˜The œjournal of high energy physics
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Received: May 11, 2018 Revised: September 5, 2018 Accepted: September 27, 2018 Published: October 8, 2018 Received: May 11, 2018 Revised: September 5, 2018 Accepted: September 27, 2018 Published: October 8, 2018 JHEP10(2018)047 The ATLAS collaboration Abstract: An angular analysis of the decay B0 d →K∗µ+µ−is presented, based on proton- proton collision data recorded by the ATLAS experiment at the LHC. The study is us- ing 20.3 fb−1 of integrated luminosity collected during 2012 at centre-of-mass energy of √s = 8 TeV. Measurements of the K∗longitudinal polarisation fraction and a set of angu- lar parameters obtained for this decay are presented. The results are compatible with the Standard Model predictions. Keywords: Hadron-Hadron scattering (experiments) ArXiv ePrint: 1805.04000 https://doi.org/10.1007/JHEP10(2018)047 Contents 1 Introduction 1 2 Analysis method 2 3 The ATLAS detector, data, and Monte Carlo samples 4 4 Event selection 4 5 Maximum-likelihood fit 6 5.1 Signal model 7 5.2 Background modes 8 5.3 K∗cc control sample fits 10 5.4 Fitting procedure and validation 11 6 Results 11 7 Systematic uncertainties 18 8 Comparison with theoretical computations 21 9 Conclusion 23 A Correlation matrices 24 The ATLAS collaboration 30 Contents 1 Introduction 1 2 Analysis method 2 3 The ATLAS detector, data, and Monte Carlo samples 4 4 Event selection 4 5 Maximum-likelihood fit 6 5.1 Signal model 7 5.2 Background modes 8 5.3 K∗cc control sample fits 10 5.4 Fitting procedure and validation 11 6 Results 11 7 Systematic uncertainties 18 8 Comparison with theoretical computations 21 9 Conclusion 23 A Correlation matrices 24 The ATLAS collaboration 30 JHEP10(2018)047 1 Introduction Flavour-changing neutral currents (FCNC) have played a significant role in the construction of the Standard Model of particle physics (SM). These processes are forbidden at tree level and can proceed only via loops, hence are rare. An important set of FCNC processes involve the transition of a b-quark to an sµ+µ−final state mediated by electroweak box and penguin diagrams. If heavy new particles exist, they may contribute to FCNC decay amplitudes, affecting the measurement of observables related to the decay under study. Hence FCNC processes allow searches for contributions from sources of physics beyond the SM (hereafter referred to as new physics). This analysis focuses on the decay B0 d →K∗0(892)µ+µ−, where K∗0(892) →K+π−. Hereafter, the K∗0(892) is referred to as K∗and charge conjugation is implied throughout, unless stated otherwise. In addition to angular observables such – 1 – as the forward-backward asymmetry AFB,1 there is considerable interest in measurements of the charge asymmetry, differential branching fraction, isospin asymmetry, and ratio of rates of decay into dimuon and dielectron final states, all as a function of the invariant mass squared of the dilepton system q2. All of these observable sets can be sensitive to different types of new physics that allow for FCNCs at tree or loop level. The BaBar, Belle, CDF, CMS, and LHCb collaborations have published the results of studies of the angular distributions for B0 d →K∗µ+µ−[1–8]. The LHCb Collaboration has reported a potential hint, at the level of 3.4 standard deviations, of a deviation from SM calculations [3, 4] in this decay mode when using a parameterization of the angular distribution designed to minimise uncertainties from hadronic form factors. Measurements using this approach were also reported by the Belle and CMS Collaborations [6, 8] and they are consistent with the LHCb experiment’s results and with the SM calculations. This paper presents results following the methodology outlined in ref. [3] and the convention adopted by the LHCb Collaboration for the definition of angular observables described in ref. [9]. The results obtained here are compared with theoretical predictions that use the form factors computed in ref. [10]. JHEP10(2018)047 This article presents the results of an angular analysis of the decay B0 d →K∗µ+µ− with the ATLAS detector, using 20.3 fb−1 of pp collision data at a centre-of-mass energy √s = 8 TeV delivered by the Large Hadron Collider (LHC) [11] during 2012. 1The forward-backward asymmetry is given by the normalised difference between the number of positive muons going in the forward and backward directions with respect to the direction opposite to B0 d momentum in the dimuon rest frame. 1 Introduction Results are presented in six different bins of q2 in the range 0.04 to 6.0 GeV2, where three of these bins overlap. Backgrounds, including a radiative tail from B0 d →K∗J/ψ events, increase for q2 above 6.0 GeV2, and for this reason, data above this value are not studied. The operator product expansion used to describe the decay B0 d →K∗µ+µ−encodes short-distance contributions in terms of Wilson coefficients and long-distance contributions in terms of operators [12]. Global fits for Wilson coefficients have been performed using measurements of B0 d →K∗µ+µ−and other rare processes. Such studies aim to connect deviations from the SM predictions in several processes to identify a consistent pattern hinting at the structure of a potential underlying new-physics Lagrangian, see refs. [13–15]. The parameters presented in this article can be used as inputs to these global fits. 2This equation neglects possible Kπ S-wave contributions. The effect of an S-wave contribution is considered following the method used by LHCb in ref. [3]. 2 Analysis method Three angular variables describing the decay are defined according to convention described by the LHCb Collaboration in ref. [9]: the angle between the K+ and the direction opposite to the B0 d in the K∗centre-of-mass frame (θK); the angle between the µ+ and the direction opposite to the B0 d in the dimuon centre-of-mass frame (θL); and the angle between the two decay planes formed by the Kπ and the dimuon systems in the B0 d rest frame (φ). For B 0 d mesons the definitions are given with respect to the negatively charged particles. Figure 1 illustrates the angles used. 1The forward-backward asymmetry is given by the normalised difference between the number of positive muons going in the forward and backward directions with respect to the direction opposite to B0 d momentum in the dimuon rest frame. – 2 – φ B0 d µ+ µ− K+ π− θL θK Figure 1. An illustration of the B0 d →K∗µ+µ−decay showing the angles θK, θL and φ defined in the text. Angles are computed in the rest frame of the K∗, dimuon system and B0 d meson, respectively. Figure 1. An illustration of the B0 d →K∗µ+µ−decay showing the angles θK, θL and φ defined in the text. Angles are computed in the rest frame of the K∗, dimuon system and B0 d meson, respectively. Figure 1. An illustration of the B0 d →K∗µ+µ−decay showing the angles θK, θL and φ defined in the text. Angles are computed in the rest frame of the K∗, dimuon system and B0 d meson, respectively. JHEP10(2018)047 The angular differential decay rate for B0 d →K∗µ+µ−is a function of q2, cos θK, cos θL and φ, and can be written in several ways [16]. The form to express the differential decay amplitude as a function of the angular parameters uses coefficients that may be represented by the helicity or transversity amplitudes [17] and is written as2 1 dΓ/dq2 d4Γ dcosθLdcosθKdφdq2 = 9 32π " 3(1−FL) 4 sin2 θK +FL cos2 θK + 1−FL 4 sin2 θK cos2θL −FL cos2 θK cos2θL+S3 sin2 θK sin2 θL cos2φ +S4 sin2θK sin2θL cosφ+S5 sin2θK sinθL cosφ +S6 sin2 θK cosθL+S7 sin2θK sinθL sinφ +S8 sin2θK sin2θL sinφ+S9 sin2 θK sin2 θL sin2φ # . 3ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r, Φ) are used in the transverse plane, Φ being the azimuthal angle around the z-axis. The pseudorapidity is defined in terms of the polar angle θ as η = −ln tan(θ/2). 3 The ATLAS detector, data, and Monte Carlo samples The ATLAS experiment at the LHC is a general-purpose detector with a cylindrical ge- ometry and nearly 4π coverage in solid angle [19]. It consists of an inner detector (ID) for tracking, a calorimeter system and a muon spectrometer (MS). The ID consists of silicon pixel and strip detectors, with a straw-tube transition radiation tracker providing additional information for tracks passing through the central region of the detector.3 The ID has a coverage of |η| < 2.5, and is immersed in a 2T axial magnetic field generated by a superconducting solenoid. The calorimeter system, consisting of liquid argon and scintillator-tile sampling calorimeter subsystems, surrounds the ID. The outermost part of the detector is the MS, which employs several detector technologies in order to provide muon identification and a muon trigger. A toroidal magnet system is embedded in the MS. The ID, calorimeter system and MS have full azimuthal coverage. JHEP10(2018)047 The data analysed here were recorded in 2012 during Run 1 of the LHC. The centre- of-mass energy of the pp system was √s = 8 TeV. After applying data-quality criteria, the data sample analysed corresponds to an integrated luminosity of 20.3 fb−1. A number of Monte Carlo (MC) signal and background event samples were generated, with b-hadron production in pp collisions simulated with Pythia 8.186 [20, 21]. The AU2 set of tuned parameters [22] is used together with the CTEQ6L1 PDF set [23]. The EvtGen 1.2.0 program [24] is used for the properties of b- and c-hadron decays. The simulation included modelling of multiple interactions per pp bunch crossing in the LHC with Pythia soft QCD processes. The simulated events were then passed through the full ATLAS detector simulation program based on Geant 4 [25, 26] and reconstructed in the same way as data. The samples of MC generated events are described further in section 5. 2 Analysis method (2.1) −FL cos2 θK cos2θL+S3 sin2 θK sin2 θL cos2φ +S4 sin2θK sin2θL cosφ+S5 sin2θK sinθL cosφ +S6 sin2 θK cosθL+S7 sin2θK sinθL sinφ +S8 sin2θK sin2θL sinφ+S9 sin2 θK sin2 θL sin2φ # . (2.1) (2.1) Here FL is the fraction of longitudinally polarised K∗mesons and the Si are angular coefficients. These angular parameters are functions of the real and imaginary parts of the transversity amplitudes of B0 d decays into K∗µ+µ−. The forward-backward asymmetry is given by AFB = 3S6/4. The predictions for the S parameters depend on hadronic form factors which have significant uncertainties at leading order. It is possible to reduce the theoretical uncertainty in these predictions by transforming the Si using ratios constructed to cancel form factor uncertainties at leading order. These ratios are given by refs. [17, 18] as P1 = 2S3 1 −FL (2.2) P2 = 2 3 AFB 1 −FL (2.3) P3 = − S9 1 −FL (2.4) P ′ j=4,5,6,8 = Si=4,5,7,8 p FL(1 −FL) . (2.5) (2.2) (2.5) 2This equation neglects possible Kπ S-wave contributions. The effect of an S-wave contribution is considered following the method used by LHCb in ref. [3]. – 3 – All of the parameters introduced, FL, Si and P (′) j , may vary with q2 and the data are analysed in q2 bins to obtain an average value for a given parameter in that bin. 4 Event selection Several trigger signatures constructed from the MS and ID inputs are selected based on availability during the data-taking period, prescale factor and efficiency for signal iden- tification. Data are combined from 19 trigger chains where 21%, 89% or 5% of selected events pass one or more triggers with one, two, or at least three muons identified online in the MS, respectively. Of the events passing the requirement of at least two muons, the largest contribution comes from the chain requiring one muon with a transverse momentum pT > 4 GeV and the other muon with pT > 6 GeV. This combination of triggers ensures that the analysis remains sensitive to events down to the kinematic threshold of q2 = 4m2 µ, – 4 – where mµ is the muon mass. The effective average trigger efficiency for selected signal events is about 29%, determined from signal MC simulation. Muon track candidates are formed offline by combining information from both the ID and MS [27]. Tracks are required to satisfy |η| < 2.5. Candidate muon (kaon and pion) tracks in the ID are required to satisfy pT > 3.5 (0.5) GeV. Pairs of oppositely charged muons are required to originate from a common vertex with a fit quality χ2/NDF < 10. Candidate K∗mesons are formed using pairs of oppositely charged kaon and pion candidates reconstructed from hits in the ID. Candidates are required to satisfy pT(K∗) > 3.0 GeV. As the ATLAS detector does not have a dedicated charged-particle identification system, candidates are reconstructed with both possible Kπ mass hypothe- ses. The selection implicitly relies on the kinematics of the reconstructed K∗meson to determine which of the two tracks corresponds to the kaon. If both candidates in an event satisfy selection criteria, they are retained and one of them is selected in the next step following a procedure described below. The Kπ invariant mass is required to lie in a window of twice the natural width around the nominal mass of 896 MeV, i.e. in the range [846, 946] MeV. The charge of the kaon candidate is used to assign the flavour of the reconstructed B0 d candidate. JHEP10(2018)047 The B0 d candidates are reconstructed from a K∗candidate and a pair of oppositely charged muons. The four-track vertex is fitted and required to satisfy χ2/NDF < 2 to suppress background. 4 Event selection A significant amount of combinatorial, B0 d, B+, B0 s and Λb back- ground contamination remains at this stage. Combinatorial background is suppressed by requiring a B0 d candidate lifetime significance τ/στ > 12.5, where the decay time uncer- tainty στ is calculated from the covariance matrices associated with the four-track vertex fit and with the primary vertex fit. Background from final states partially reconstructed as B →µ+µ−X accumulates at invariant mass below the B0 d mass and contributes to the signal region. It is suppressed by imposing an asymmetric mass cut around the nominal B0 d mass, 5150 MeV < mKπµµ < 5700 MeV. The high-mass sideband is retained, as the parameter values for the combinatorial background shapes are extracted from the fit to data described in section 5. To further suppress background, it is required that the angle Θ, defined between the vector from the primary vertex to the B0 d candidate decay vertex and the B0 d candidate momentum, satisfies cos Θ > 0.999. Resolution effects on cos θK, cos θL and φ were found to have a negligible effect on the ATLAS B0 s →J/ψφ analysis [28]. It is assumed to also be the case for B0 d →K∗µ+µ−. On average 12% of selected events in the data have more than one reconstructed B0 d candidate. The fraction is 17% for signal MC samples and 2–10% for exclusive background MC samples. A two-step selection process is used for such events. For 4% of these events it is possible to select a candidate with the smallest value of the B0 d vertex χ2/NDF. However, the majority, about 96%, of multiple candidates arise from four-track combinations where the kaon and pion assignments are ambiguous. As these candidates have degenerate values for the B0 d candidate vertex χ2/NDF, a second selection step is required. The B0 d candidate reconstructed with the smallest value of |mKπ −mK∗|/σ(mKπ) is retained for analysis, where mKπ is the K∗candidate mass, σ(mKπ) is the per-event uncertainty in this quantity, and mK∗is the world average value of the K∗mass. – 5 – The selection procedure results in an incorrect flavour tag (mistag) for some signal events. The mistag probability of a B0 d (B 0 d) meson is denoted by ω (ω) and is determined from MC simulated events to be 0.1088 ± 0.0005 (0.1086 ± 0.0005). 4 Event selection The mistag probability varies slightly with q2 such that the difference ω −ω remains consistent with zero. Hence the average mistag rate ⟨ω⟩in a given q2 bin is used to account for this effect. If a candidate is mistagged, the values of cos θL, cos θK and φ change sign, while the latter two are also slightly shaped by the swapped hadron track mass hypothesis. Sign changes in these angles affect the overall sign of the terms multiplied by the coefficients S5, S6, S8 and S9 (similarly for the corresponding P (′) parameters) in equation (2.1). The corollary is that mistagged events result in a dilution factor of (1 −2⟨ω⟩) for the affected coefficients. JHEP10(2018)047 The region q2 ∈[0.98, 1.1] GeV2 is vetoed to remove any potential contamination from the φ(1020) resonance. The remaining data with q2 ∈[0.04, 6.0] GeV2 are analysed in order to extract the signal parameters of interest. Two K∗cc control regions are defined for B0 d decays into K∗J/ψ and K∗ψ(2S), respectively as q2 ∈[8, 11] and [12, 15] GeV2. The control samples are used to extract values for nuisance parameters describing the signal probability density function (pdf) from data as discussed in section 5.3. For q2 < 6 GeV2 the selected data sample consists of 787 events and is composed of signal B0 d →K∗µ+µ−decay events as well as background that is dominated by a combina- torial component that does not peak in mKπµµ and does not exhibit a resonant structure in q2. Other background contributions are considered when estimating systematic uncertain- ties. Above 6 GeV2 the background contribution increases significantly, including events coming from B0 d →K∗J/ψ with a radiative J/ψ →µ+µ−γ decay. Scalar Kπ contributions are neglected in the nominal fit and considered only when addressing systematic uncertain- ties. The data are analysed in the q2 bins [0.04, 2.0], [2.0, 4.0] and [4.0, 6.0] GeV2, where the bin width is chosen to provide a sample of signal events sufficient to perform an angular analysis. The width is much larger than the q2 resolution obtained from MC simulated signal events and observed in data for B0 d decays into K∗J/ψ and K∗ψ(2S). Additional overlapping bins [0.04, 4.0], [1.1, 6.0] and [0.04, 6.0] GeV2 are analysed in order to facilitate comparison with results of other experiments and with theoretical predictions. 5 Maximum-likelihood fit Extended unbinned maximum-likelihood fits of the angular distributions of the signal decay are performed on the data for each q2 bin. The discriminating variables used in the fit are mKπµµ, the cosines of the helicity angles (cos θK and cos θL), and φ. The likelihood L for a given q2 bin is L = e−n N! N Y k=1 X l nlPkl(mKπµµ, cos θK, cos θL, φ; bp, bθ), (5.1) (5.1) where N is the total number of events, the sum runs over signal and background compo- nents, nl is the fitted yield for the lth component, n is the sum over nl, and Pkl is the pdf evaluated for event k and component l. In the nominal fit, l iterates only over one signal – 6 – and one background component. The bp are parameters of interest (FL, Si) and bθ are nui- sance parameters. The remainder of this section discusses the signal model (section 5.1), treatment of background (section 5.2), use of K∗cc decay control samples (section 5.3), fitting procedure and validation (section 5.4). 5.1 Signal model The signal mass distribution is modelled by a Gaussian distribution with the width given by the per-event uncertainty in the Kπµµ mass, σ(mKπµµ), as estimated from the track fit, multiplied by a unit-less scale factor ξ, i.e. the width given by ξ · σ(mKπµµ). The mean values of the B0 d candidate mass (m0) and ξ of the signal Gaussian pdf are determined from fits to data in the control regions as described in section 5.3. The simultaneous extraction of all coefficients using the full angular distribution of equation (2.1) requires a certain minimum signal yield and signal purity to avoid a pathological fit behaviour. A significant fraction of fits to ensembles of simulated pseudo-experiments do not converge using the full distribution. This is mitigated using trigonometric transformations to fold certain angular distributions and thereby simplify equation (2.1) such that only three parameters are extracted in one fit: FL, S3 and one of the other S parameters. For these folding schemes the angular parameters of interest, denoted by bp in equation (5.1), are (FL, S3, Si) where i = 4, 5, 7, 8. These translate into (FL, P1, P ′ j), where j = 4, 5, 6, 8, using equation (2.5). Following ref. [3], the transformations listed below are used: JHEP10(2018)047 FL, S3, S4, P ′ 4 :        φ →−φ for φ < 0 φ →π −φ for θL > π 2 θL →π −θL for θL > π 2 , (5.2) FL, S3, S5, P ′ 5 : ( φ →−φ for φ < 0 θL →π −θL for θL > π 2 , (5.3) FL, S3, S7, P ′ 6 :        φ →π −φ for φ > π 2 φ →−π −φ for φ < −π 2 θL →π −θL for θL > π 2 , (5.4) FL, S3, S8, P ′ 8 :              φ →π −φ for φ > π 2 φ →−π −φ for φ < −π 2 θL →π −θL for θL > π 2 θK →π −θK for θL > π 2 . 5.1 Signal model (5.5) FL, S3, S4, P ′ 4 :        φ →−φ for φ < 0 φ →π −φ for θL > π 2 θL →π −θL for θL > π 2 , (5.2) (5.2) (5.3) ( θL →π −θL for θL > π 2 , FL, S3, S7, P ′ 6 :        φ →π −φ for φ > π 2 φ →−π −φ for φ < −π 2 θL →π −θL for θL > π 2 , (5.4) FL, S3, S8, P ′ 8 :              φ →π −φ for φ > π 2 φ →−π −φ for φ < −π 2 θL →π −θL for θL > π 2 θK →π −θK for θL > π 2 . (5.5) (5.4) (5.5) On applying transformation (5.2), (5.3), (5.4), and (5.5), the angular variable ranges become cos θL ∈[0, 1], cos θK ∈[−1, 1] and φ ∈[0, π], cos θL ∈[0, 1], cos θK ∈[−1, 1] and φ ∈[0, π], cos θL ∈[0, 1], cos θK ∈[−1, 1] and φ ∈[−π/2, π/2], cos θL ∈[0, 1], cos θK ∈[−1, 1] and φ ∈[−π/2, π/2], – 7 – – 7 – respectively. A consequence of using the folding schemes is that S6 (AFB) and S9 cannot be extracted from the data. The values and uncertainties of FL and S3 obtained from the four fits are consistent with each other and the results reported are those found to have the smallest systematic uncertainty. Three MC samples are used to study the signal reconstruction and acceptance. Two of them follow the SM prediction for the decay angle distributions taken from ref. [29], with separate samples generated for B0 d and B0 d decays. The third MC sample has FL = 1/3 and the angular distributions are generated uniformly in cos θK, cos θL and φ. The samples are used to study the effect of potential mistagging and reconstruction differences between particle and antiparticle decays and for determination of the acceptance. The acceptance function is defined as the ratio of reconstructed and generated distributions of cos θK, cos θL, φ, i.e. it is compensating for the bias in the angular distributions resulting from triggering, reconstruction and selection of events. Pkl = ε(cos θK)ε(cos θL)ε(φ)g(cos θK, cos θL, φ) · G(mKπµµ), Pkl = ε(cos θK)ε(cos θL)ε(φ)g(cos θK, cos θL, φ) · G(mKπµµ), where g(cos θK, cos θL, φ) is an angular differential decay rate resulting from one of the four folding schemes applied to equation (2.1) and G(mKπµµ) is the signal mass distribution. The MC sample generated with uniform cos θK, cos θL and φ distributions is used to determine the nominal acceptance functions for each of the transformed variables defined in equations (5.2)–(5.5). The other samples are used to estimate the related systematic uncertainty. Among the angular variables the cos θL distribution is the most affected by the acceptance. This is a result of the minimum transverse momentum requirements on the muons in the trigger and the larger inefficiency to reconstruct low-momentum muons, such that large values of | cos θL| are inaccessible at low q2. As q2 increases, the acceptance effects become less severe. The cos θK distribution is affected by the ability to reconstruct the Kπ system, but that effect shows no significant variation with q2. There is no significant acceptance effect for φ. Figure 2 shows the acceptance functions used for cos θK and cos θL for two different q2 ranges for the nominal angular distribution given in equation (2.1). where g(cos θK, cos θL, φ) is an angular differential decay rate resulting from one of the four folding schemes applied to equation (2.1) and G(mKπµµ) is the signal mass distribution. The MC sample generated with uniform cos θK, cos θL and φ distributions is used to determine the nominal acceptance functions for each of the transformed variables defined in equations (5.2)–(5.5). The other samples are used to estimate the related systematic uncertainty. Among the angular variables the cos θL distribution is the most affected by the acceptance. This is a result of the minimum transverse momentum requirements on the muons in the trigger and the larger inefficiency to reconstruct low-momentum muons, such that large values of | cos θL| are inaccessible at low q2. As q2 increases, the acceptance effects become less severe. The cos θK distribution is affected by the ability to reconstruct the Kπ system, but that effect shows no significant variation with q2. There is no significant acceptance effect for φ. Figure 2 shows the acceptance functions used for cos θK and cos θL for two different q2 ranges for the nominal angular distribution given in equation (2.1). 5.1 Signal model It is described by sixth-order (second- order) polynomial distributions for cos θK and cos θL (φ) and is assumed to factorise for each angular distribution, i.e. using ε(cos θK, cos θL, φ) = ε(cos θK)ε(cos θL)ε(φ). A systematic uncertainty is assessed in order to account for this assumption. The acceptance function multiplies the angular distribution in the fit, i.e. the signal pdf is JHEP10(2018)047 5.2 Background modes The fit to data includes a combinatorial background component that does not peak in the mKπµµ distribution. It is assumed that the background pdf factorises into a product of one- dimensional terms. The mass distribution of this component is described by an exponential function and second-order Chebychev polynomials are used to model the cos θK, cos θL and φ distributions. The values of the nuisance parameters describing these shapes are obtained from fits to the data independently for each q2 bin. Inclusive samples of bb →µ+µ−X and cc →µ+µ−X decays and eleven exclusive B0 d, B0 s, B+ and Λb background samples are studied in order to identify contributions of interest – 8 – K θ cos 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 1 Probability Density 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 2 [0.04, 2.0] GeV ∈ 2 q 2 [4.0, 6.0] GeV ∈ 2 q ATLAS Simulation L θ cos 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 1 Probability Density 0 0.2 0.4 0.6 0.8 1 1.2 2 [0.04, 2.0] GeV ∈ 2 q 2 [4.0, 6.0] GeV ∈ 2 q ATLAS Simulation Figure 2. The acceptance functions for (left) cos θK and (right) cos θL for (solid) q2 ∈ [0.04, 2.0] GeV2 and (dashed) q2 ∈[4.0, 6.0] GeV2, that shape the angular decay rate of equa- tion (2.1). K θ cos 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 1 Probability Density 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 2 [0.04, 2.0] GeV ∈ 2 q 2 [4.0, 6.0] GeV ∈ 2 q ATLAS Simulation L θ cos 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 1 Probability Density 0 0.2 0.4 0.6 0.8 1 1.2 2 [0.04, 2.0] GeV ∈ 2 q 2 [4.0, 6.0] GeV ∈ 2 q ATLAS Simulation JHEP10(2018)047 Figure 2. The acceptance functions for (left) cos θK and (right) cos θL for (solid) q2 ∈ [0.04, 2.0] GeV2 and (dashed) q2 ∈[4.0, 6.0] GeV2, that shape the angular decay rate of equa- tion (2.1). to be included in the fit model, or to be considered when estimating systematic uncertain- ties. The relevant exclusive modes found to be of interest are discussed below. 5.2 Background modes Events with Bc decays are suppressed by excluding the q2 range containing the J/ψ and ψ(2S), and by charm meson vetoes discussed in section 7. The exclusive background decays considered for the signal mode are Λb →Λ(1520)µ+µ−, Λb →pK−µ+µ−, B+ →K(∗)+µ+µ−and B0 s →φµ+µ−. These background contributions are accounted for as systematic uncertain- ties estimated as described in section 7. Two distinct background contributions not considered above are observed in the cos θK and cos θL distributions. They are not accounted for in the nominal fit to data, and are treated as systematic effects. A peak is found in the cos θK distribution near 1.0 and appears to have contributions from at least two distinct sources. One of these arises from misreconstructed B+ decays, such as B+ →K+µµ and B+ →π+µµ. These decays can be reconstructed as signal if another track is combined with the hadron to form a K∗candidate in such a way that the event passes the reconstruction and selection. The second contribution comes from combinations of two charged tracks that pass the selection and are reconstructed as a K∗candidate. These fake K∗candidates accumulate around cos θK of 1.0 and are observed in the Kπ mass sidebands away from the K∗meson. They are distinct from the structure of expected S-, P- and D-wave Kπ decays resulting from a signal B0 d →Kπµµ transition. The origin of this source of background is not fully understood. The observed excess may arise from a statistical fluctuation, an unknown background process, or a combination of both. Systematic uncertainties are assigned to evaluate the effect of these two background contributions, as described in section 7. Another peak is found in the cos θL distribution near values of ±0.7. It is associated with partially reconstructed B decays into final states with a charm meson. This is studied using Monte Carlo simulated events for the decays D0 →K−π+, D+ →K−π+π+ and D+ s →K+K−π+. 5.2 Background modes Events with a B meson decaying via an intermediate charm meson D0, D+ or D+ s are found to pass the selection and are reconstructed in such a way that – 9 – [MeV] µ µ π K m 5000 5200 5400 5600 Events / 40 MeV 0 1000 2000 3000 4000 5000 ATLAS -1 = 8 TeV, 20.3 fb s Data Total Fit Model Signal Combinatorial Λ (2S) ψ → b Λ + (2S) K ψ → + B (2S) K* ψ → s B [MeV] µ µ π K m 5000 5200 5400 5600 Events / 40 MeV 0 10000 20000 30000 40000 50000 ATLAS -1 = 8 TeV, 20.3 fb s Data Total Fit Model Signal Combinatorial Λ ψ J/ → b Λ + K ψ J/ → + B K* ψ J/ → s B [MeV] µ µ π K m 5000 5200 5400 5600 Events / 40 MeV 0 1000 2000 3000 4000 5000 ATLAS -1 = 8 TeV, 20.3 fb s Data Total Fit Model Signal Combinatorial Λ (2S) ψ → b Λ + (2S) K ψ → + B (2S) K* ψ → s B Figure 3. Fits to the Kπµµ invariant mass distributions for the (left) K∗J/ψ and (right) K∗ψ(2S) control region samples. The data are shown as points and the total fit model as the solid lines. The dashed lines represent (black) signal, (red) combinatorial background, (green) Λb background, (blue) B+ background and (magenta) B0 s background components. [MeV] µ µ π K m 5000 5200 5400 5600 Events / 40 MeV 0 10000 20000 30000 40000 50000 ATLAS -1 = 8 TeV, 20.3 fb s Data Total Fit Model Signal Combinatorial Λ ψ J/ → b Λ + K ψ J/ → + B K* ψ J/ → s B JHEP10(2018)047 Figure 3. Fits to the Kπµµ invariant mass distributions for the (left) K∗J/ψ and (right) K∗ψ(2S) control region samples. The data are shown as points and the total fit model as the solid lines. The dashed lines represent (black) signal, (red) combinatorial background, (green) Λb background, (blue) B+ background and (magenta) B0 s background components. they accumulate around 0.7 in | cos θL|. These are removed from the data sample when estimating systematic uncertainties, as described in section 7. 5.4 Fitting procedure and validation A two-step fit process is performed for the different signal bins in q2. The first step is a fit to the Kπµ+µ−invariant mass distribution, using the event-by-event uncertainty in the reconstructed mass as a conditional variable. For this fit, the parameters m0 and ξ are fixed to the values obtained from fits to data control samples as described in section 5.3. A second step adds the (transformed) cos θK, cos θL and φ variables to the likelihood in order to extract FL and the S parameters along with the values for the nuisance parameters related to the combinatorial background shapes. Some nuisance parameters, namely m0, ξ, signal and background yields, and the exponential shape parameter for the background mass pdf, are fixed to the results obtained from the first step. JHEP10(2018)047 The fit procedure is validated using ensembles of simulated pseudo-experiments gen- erated with the FL and S parameters corresponding to those obtained from the data. The purpose of these experiments is to measure the intrinsic fit bias resulting from the likeli- hood estimator used to extract signal parameters. These ensembles are also used to check that the uncertainties extracted from the fit are consistent with expectations. Ensembles of simulated pseudo-experiments are performed in which signal MC events are injected into samples of background events generated from the likelihood. The signal yield determined from the first step in the fit process is found to be unbiased. The angular parameters ex- tracted from the nominal fits have biases with magnitudes ranging between 0.01 and 0.04, depending on the fit variation and q2 bin. A similar procedure is used to estimate the effect of neglecting S-wave contamination in the data sample. Neglecting the S-wave component in the fit model results in a bias between 0.00 and 0.02 in the angular parameters. Simi- larly, neglecting exclusive background contributions from Λb, B+ and B0 s decays that peak in mKπµµ near the B0 d mass results in a bias of less than 0.01 on the angular parameters. All these effects are included in the systematic uncertainties described in section 7. The P (′) parameters are obtained using the fit results and covariance matrices from the second fit along with equations (2.2)–(2.5). 5.3 K∗cc control sample fits The mass distribution obtained from the simulated samples for K∗cc decays, respectively as q2 ∈[8, 11] and [12, 15] GeV2, and the signal mode, in different bins of q2, are found to be consistent with each other. Values of m0 and ξ for B0 d →K∗J/ψ and B0 d →K∗ψ(2S) events are used for the signal pdf and extracted from fits to the data. An extended unbinned maximum-likelihood fit is performed in the two K∗cc control region samples. There are three exclusive backgrounds included: Λb →Λcc, B+ →K+cc and B0 s →K∗cc. The K∗cc pdf has the same form as the signal model, combinatorial background is described by an exponential distribution, and double and triple Gaussian pdfs determined from MC simulated events are used to describe the exclusive background contributions. A systematic uncertainty is evaluated by allowing for 0, 1, 2 and 3 exclusive background components. The control sample fit projections for the variant of the fit including all three exclusive backgrounds can be found in figure 3. The impact of the used exclusive background model on the peak position and scale factor of the signal pdf is negligible. From these fits the statistical and systematic uncertainties in the values of m0 and ξ are extracted for the B0 d component in order to be used in the B0 d →K∗µ+µ−fits. From the J/ψ control data it is determined that the values for the nuisance parameters describing the signal model pdf in the Kπµµ mass are m0 = 5276.6 ± 0.3 ± 0.4 MeV and ξ = 1.210 ± 0.004 ± 0.002, where the uncertainties are statistical and systematic, respectively. The ψ(2S) sample yields compatible results albeit with larger uncertainties. These results are similar to those obtained from the MC simulated samples, and the numbers derived from the K∗J/ψ data are used for the signal region fits. – 10 – 6 Results The event yields obtained from the fits are summarised in table 1 where only statistical un- certainties are reported. Figures 4 through 9 show for the different q2 bins the distributions of the variables used in the fit for the S5 folding scheme (corresponding to the transfor- mation of equation (5.3)) with the total, signal and background fitted pdfs superimposed. Similar sets of distributions are obtained for the three other folding schemes: S4, S7 and S8. The results of the angular fits to the data in terms of the Si and P (′) j can be found in tables 2 and 3. Statistical and systematic uncertainties are quoted in the tables. The distributions of FL and the Si parameters as a function of q2 are shown in figure 10 and those for P (′) j are shown in figure 11. The correlations between FL and the Si parameters and between FL and the P (′) j are given in appendix A. – 11 – q2 [GeV2] nsignal nbackground [0.04, 2.0] 128 ± 22 122 ± 22 [2.0, 4.0] 106 ± 23 113 ± 23 [4.0, 6.0] 114 ± 24 204 ± 26 [0.04, 4.0] 236 ± 31 233 ± 32 [1.1, 6.0] 275 ± 35 363 ± 36 [0.04, 6.0] 342 ± 39 445 ± 40 Table 1. The values of fitted signal, nsignal, and background, nbackground, yields obtained for different bins in q2. The uncertainties indicated are statistical. JHEP10(2018)047 Table 1. The values of fitted signal, nsignal, and background, nbackground, yields obtained for different bins in q2. The uncertainties indicated are statistical. Table 1. The values of fitted signal, nsignal, and background, nbackground, yields obtained for different bins in q2. The uncertainties indicated are statistical. 6 Results [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 10 20 30 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 2.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 2.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 2.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 2.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Figure 4. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[0.04, 2.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 2.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 2.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 10 20 30 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 2.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Events / 25 MeV π Events / 0.04 Events / 0.08 Events / 0.04 Figure 4. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[0.04, 2.0] GeV2. 6 Results The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. – 12 – [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [2.0, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 -1 = 8 TeV, 20.3 fb s ATLAS 2 [2.0, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [2.0, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 10 20 -1 = 8 TeV, 20.3 fb s ATLAS 2 [2.0, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Figure 5. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[2.0, 4.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 -1 = 8 TeV, 20.3 fb s ATLAS 2 [2.0, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 10 20 -1 = 8 TeV, 20.3 fb s ATLAS 2 [2.0, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Events / 25 MeV π Events / 0.04 JHEP10(2018)047 Events / 0.08 Events / 0.04 Figure 5. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[2.0, 4.0] GeV2. 6 Results The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. – 13 – [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 10 20 30 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [4.0, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [4.0, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 10 20 30 40 50 -1 = 8 TeV, 20.3 fb s ATLAS 2 [4.0, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [4.0, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Figure 6. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[4.0, 6.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [4.0, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 10 20 30 -1 = 8 TeV, 20.3 fb s ATLAS 2 [4.0, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background π Events / 0.04 JHEP10(2018)047 Events / 0.08 Events / 0.04 Figure 6. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[4.0, 6.0] GeV2. 6 Results The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. – 14 – [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 30 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 20 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 20 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Figure 7. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[0.04, 4.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. 6 Results [rad] φ 0 1 2 3 π Events / 0.04 0 10 20 30 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 20 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 20 40 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 4.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background π Events / 0.04 JHEP10(2018)047 Events / 0.04 Events / 0.08 Figure 7. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[0.04, 4.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. – 15 – [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 20 40 60 80 -1 = 8 TeV, 20.3 fb s ATLAS 2 [1.1, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [rad] φ 0 1 2 3 π Events / 0.04 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [1.1, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 20 40 60 80 -1 = 8 TeV, 20.3 fb s ATLAS 2 [1.1, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [1.1, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Figure 8. 6 Results The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[1.1, 6.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. [rad] φ 0 1 2 3 π Events / 0.04 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [1.1, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [1.1, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background π Events / 0.04 [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 20 40 60 80 -1 = 8 TeV, 20.3 fb s ATLAS 2 [1.1, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background JHEP10(2018)047 Events / 0.08 Events / 0.04 Figure 8. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[1.1, 6.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. 6 Results – 16 – [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 50 100 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [rad] φ 0 1 2 3 π Events / 0.04 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 20 40 60 80 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 20 40 60 80 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Figure 9. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[0.04, 6.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. [rad] φ 0 1 2 3 π Events / 0.04 0 20 40 60 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background L θ cos 0 0.2 0.4 0.6 0.8 1 Events / 0.04 0 20 40 60 80 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background [MeV] µ µ π K m 5200 5400 5600 Events / 25 MeV 0 50 100 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background K θ cos 1 − 0.5 − 0 0.5 1 Events / 0.08 0 20 40 60 80 -1 = 8 TeV, 20.3 fb s ATLAS 2 [0.04, 6.0] GeV ∈ 2 S5 fold, q Data Total Fit Model Signal Background Events / 25 MeV π Events / 0.04 JHEP10(2018)047 Events / 0.04 Events / 0.08 Figure 9. 6 Results The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom right) cos θL obtained for q2 ∈[0.04, 6.0] GeV2. The (blue) solid line is a projection of the total pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents the signal component. These plots are obtained from a fit using the S5 folding scheme. – 17 – q2 [GeV2] FL S3 S4 S5 S7 S8 [0.04,2.0] 0.44±0.08±0.07 −0.02±0.09±0.02 0.15±0.20±0.10 0.33±0.13±0.08 −0.09±0.10±0.02 −0.14±0.24±0.09 [2.0,4.0] 0.64±0.11±0.05 −0.15±0.10±0.07 −0.37±0.15±0.10 −0.16±0.15±0.06 0.15±0.14±0.09 0.52±0.20±0.19 [4.0,6.0] 0.42±0.13±0.12 0.00±0.12±0.07 0.32±0.16±0.09 0.13±0.18±0.09 0.03±0.13±0.07 −0.12±0.21±0.05 [0.04,4.0] 0.52±0.07±0.06 −0.05±0.06±0.04 −0.15±0.12±0.09 0.16±0.10±0.05 0.01±0.08±0.05 0.19±0.16±0.12 [1.1,6.0] 0.56±0.07±0.06 −0.04±0.07±0.03 0.03±0.11±0.07 0.00±0.10±0.04 0.02±0.08±0.06 0.11±0.14±0.10 [0.04,6.0] 0.50±0.06±0.04 −0.04±0.06±0.03 0.03±0.10±0.07 0.14±0.09±0.03 0.02±0.07±0.05 0.07±0.13±0.09 Table 2. The values of FL, and S3, S4, S5, S7 and S8 parameters obtained for different bins in q2. The uncertainties indicated are statistical and systematic, respectively. q2 [GeV2] P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 [0.04,2.0] −0.05±0.30±0.08 0.31±0.40±0.20 0.67±0.26±0.16 −0.18±0.21±0.04 −0.29±0.48±0.18 [2.0,4.0] −0.78±0.51±0.34 −0.76±0.31±0.21 −0.33±0.31±0.13 0.31±0.28±0.19 1.07±0.41±0.39 [4.0,6.0] 0.14±0.43±0.26 0.64±0.33±0.18 0.26±0.35±0.18 0.06±0.27±0.13 −0.24±0.42±0.09 [0.04,4.0] −0.22±0.26±0.16 −0.30±0.24±0.17 0.32±0.21±0.11 0.01±0.17±0.10 0.38±0.33±0.24 [1.1,6.0] −0.17±0.31±0.13 0.05±0.22±0.14 0.01±0.21±0.08 0.03±0.17±0.12 0.23±0.28±0.20 [0.04,6.0] −0.15±0.23±0.10 0.05±0.20±0.14 0.27±0.19±0.06 0.03±0.15±0.10 0.14±0.27±0.17 Table 3. The values of P1, P ′ 4, P ′ 5, P ′ 6 and P ′ 8 parameters obtained for different bins in q2. The uncertainties indicated are statistical and systematic, respectively. q2 [GeV2] FL S3 S4 S5 S7 S8 [0.04,2.0] 0.44±0.08±0.07 −0.02±0.09±0.02 0.15±0.20±0.10 0.33±0.13±0.08 −0.09±0.10±0.02 −0.14±0.24±0.09 [2.0,4.0] 0.64±0.11±0.05 −0.15±0.10±0.07 −0.37±0.15±0.10 −0.16±0.15±0.06 0.15±0.14±0.09 0.52±0.20±0.19 [4.0,6.0] 0.42±0.13±0.12 0.00±0.12±0.07 0.32±0.16±0.09 0.13±0.18±0.09 0.03±0.13±0.07 −0.12±0.21±0.05 [0.04,4.0] 0.52±0.07±0.06 −0.05±0.06±0.04 −0.15±0.12±0.09 0.16±0.10±0.05 0.01±0.08±0.05 0.19±0.16±0.12 [1.1,6.0] 0.56±0.07±0.06 −0.04±0.07±0.03 0.03±0.11±0.07 0.00±0.10±0.04 0.02±0.08±0.06 0.11±0.14±0.10 [0.04,6.0] 0.50±0.06±0.04 −0.04±0.06±0.03 0.03±0.10±0.07 0.14±0.09±0.03 0.02±0.07±0.05 0.07±0.13±0.09 Table 2. The values of FL, and S3, S4, S5, S7 and S8 parameters obtained for different bins in q The uncertainties indicated are statistical and systematic, respectively. JHEP10(2018)047 Table 3. The values of P1, P ′ 4, P ′ 5, P ′ 6 and P ′ 8 parameters obtained for different bins in q2. The uncertainties indicated are statistical and systematic, respectively. 7 Systematic uncertainties Systematic uncertainties in the parameter values obtained from the angular analysis come from several sources. The methods for determining these uncertainties are based either on a comparison of nominal and modified fit results, or on observed fit biases in modified pseudo-experiments. The systematic uncertainties are symmetrised. The most significant ones are described in the following, in decreasing order of importance. • A systematic uncertainty is assigned for the combinatorial Kπ (fake K∗) background peaking at cos θK values around 1.0 obtained by comparing results of the nominal fit to that where data above cos θK = 0.9 are excluded from the fit. • A systematic uncertainty is assigned for the combinatorial Kπ (fake K∗) background peaking at cos θK values around 1.0 obtained by comparing results of the nominal fit to that where data above cos θK = 0.9 are excluded from the fit. • A systematic uncertainty is derived to account for background arising from partially reconstructed B →D0/D+/D+ s X decays, that manifest in an accumulation of events at | cos θL| values around 0.7. Two-track or three-track combinations are formed from the signal candidate tracks, and are reconstructed assuming the pion or kaon mass hypothesis. A veto is then applied for events in which a track combination has a mass in a window of 30 MeV around the D0, D+ or D+ s meson mass. Similarly, a veto is implemented to reject B+ →K+µ+µ−and B+ →π+µ+µ−events that pass the event selection. Here B+ candidates are reconstructed from one of the hadrons from the K∗ candidate and the muons in the signal candidate. Signal candidates that have a three- track mass within 50 MeV of the B+ mass are excluded from the fit. A few percent of signal events are removed when applying these vetoes, with a corresponding effect on the acceptance distributions. The fit results obtained from the data samples with – 18 – vetoes applied are compared to those obtained from the nominal fit and the change in each result is taken as the systematic uncertainty from these backgrounds. This systematic uncertainty dominates the measurement of FL at higher values of q2. vetoes applied are compared to those obtained from the nominal fit and the change in each result is taken as the systematic uncertainty from these backgrounds. This systematic uncertainty dominates the measurement of FL at higher values of q2. 7 Systematic uncertainties • The combinatorial background pdf shape has an uncertainty arising from the choice of the model. For the mass distribution it is assumed that an exponential function model is adequate; however, for the angular variables the data are re-fitted using third-order Chebychev polynomials. The change from the nominal result is taken as the uncertainty from this source. JHEP10(2018)047 • The acceptance function is assumed to factorise into three separate components, for cos θK, cos θL and φ. To validate this assumption, the signal simulated events are fitted with the acceptance function obtained from that same MC sample. Differences in the fit results from expectation are small and taken as the uncertainty resulting from this assumption. • A systematic uncertainty is assigned for the angular pdf model for the background by comparing the nominal result to that with a reduced fit range of mKπµµ ∈ [5200, 5700] MeV, in particular to account for possible residues of the partially re- constructed B-decays. • A correction is applied to the data by shifting the track pT according to the uncer- tainties arising from biases in rapidity and momentum scale. The change in results obtained is ascribed to the uncertainty in the ID alignment and knowledge of the magnetic field. • The maximum-likelihood estimator used is intrinsically biased. Ensembles of MC simulated events are used in order to ascertain the bias in the extracted values of the parameters of interest. The bias is assigned as a systematic uncertainty. • The pT spectrum of B0 d candidates observed in data is not accurately reproduced by the MC simulation. This difference in the kinematics results in a slight modification of the acceptance functions. This is accounted for by reweighting signal MC simulated events to resemble the pT spectrum found in data. The change in fitted parameter values obtained due to the reweighting is taken as the systematic uncertainty resulting from this difference. • The signal decay mode is resonant K∗→Kπ decay, but scalar contributions from non-resonant Kπ transitions may also exist. The LHCb Collaboration reported an S- wave contribution at the level of 5% of the signal [4, 30]. Ensembles of MC simulated events are fitted with 5% of the signal being drawn from an S-wave sample of events and the remaining 95% from signal. The observed change in fit bias is assigned as the systematic uncertainty from this source. 7 Systematic uncertainties Any variation in S-wave content as a function of q2 would not significantly affect the results reported here. – 19 – • The values of the nuisance parameters of the fit model obtained from MC control samples and fits to the data mass distribution have associated uncertainties. These parameters include m0, ξ, the signal and background yields, the shape parameter of the combinatorial background mass distribution, and the parameters of the signal acceptance functions. The uncertainty in the value of each of these parameters is varied independently in order to assess the effect on parameters of interest. This source of uncertainty has a small effect on the measurements reported here. • Background from exclusive modes peaking in mKπµµ is neglected in the nominal fit. This may affect the fitted results and is accounted for by computing the fit bias obtained when embedding MC simulated samples of Λb →Λ(1520)µ+µ−, Λb →pK−µ+µ−, B+ →K(∗)+µ+µ−and B0 s →φµ+µ−into ensembles of pseudo- data generated from the fit model containing only combinatorial background and sig- nal components. The change in fit bias observed when adding exclusive backgrounds is taken as the systematic error arising from neglecting those modes in the fit. JHEP10(2018)047 • The difference from nominal results obtained when fitting the B0 d signal MC events with the acceptance function for B0 d is taken as an upper limit of the systematic error resulting from event migration due to mistagging the B0 d flavour. • The parameters S5 and S8, as well as the respective P (′) j parameters are affected by dilution and thus have a multiplicative scaling applied to them. This dilution factor depends on the kinematics of the K∗decay and has a systematic uncertainty associ- ated with it. The effect of data/MC differences in the pT spectrum of B0 d candidates on the mistag probability was studied and found to be negligible. The uncertainty due to the limited number of MC events is used to compute the statistical uncertainty of ω and ω. Studies of MC simulated events indicate that there is no significant difference between the mistag probability for B0 d and B0 d events and the analysis assumes that the average mistag probability provides an adequate description of this effect. The magnitude of the mistag probability difference, |ω −ω|, is included as a systematic uncertainty resulting from this assumption. 7 Systematic uncertainties The total systematic uncertainties of the fitted Si and P (′) j parameter values are presented in tables 2 and 3, where the dominant contributions for FL come from the modelling of the angular distributions of the combinatorial background and the partially reconstructed decays peaking in cos θK and cos θL. These contributions and in addition also ID alignment and magnetic field calibration affect S3 (P1). The largest systematic uncertainty contribu- tion to S3 (P1) comes from partially reconstructed decays entering the signal region. This also affects the measurement of S5 (P ′ 5) and S7 (P ′ 6). The partially reconstructed decays peaking in cos θL affect the measurement of S4 (P ′ 4) and S8 (P ′ 8), whereas the fake K∗back- ground in cos θK affects S4 (P ′ 4), S5 (P ′ 5), and S8 (P ′ 8). The parameterization of the signal acceptance is another significant systematic uncertainty source for S4 (P ′ 4). The systematic uncertainties are smaller than the statistical uncertainties for all parameters measured. – 20 – 0 2 4 6 8 10 ] 2 [GeV 2 q 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 L F Data CFFMPSV fit theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 3 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 4 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 5 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 7 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 8 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s Figure 10. 7 Systematic uncertainties The measured values of FL, S3, S4, S5, S7, S8 compared with predictions from the theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar the total uncertainty. 0 2 4 6 8 10 ] 2 [GeV 2 q 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 3 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 5 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 L F Data CFFMPSV fit theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 4 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 L F Data CFFMPSV fit theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s JHEP10(2018)047 0 2 4 6 8 10 ] 2 [GeV 2 q 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 4 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 8 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 7 S Data CFFMPSV fit theory DHMV ATLAS -1 = 8 TeV, 20.3 fb s Figure 10. The measured values of FL, S3, S4, S5, S7, S8 compared with predictions from the theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar the total uncertainty. 4This result uses the experimental convention of equations (2.2)–(2.5) following the LHCb Collaboration’s notation in ref. [3]. In the DHMV calculation, a different convention is used as explained by equation (16) in ref. [15]. 8 Comparison with theoretical computations The results of theoretical approaches of Ciuchini et al. (CFFMPSV) [31], Descotes-Genon et al. (DHMV) [32], and J¨ager and Camalich (JC) [33, 34] are shown in figure 10 for the S parameters, and in figure 11 for the P (′) parameters, along with the results presented here.4 4This result uses the experimental convention of equations (2.2)–(2.5) following the LHCb Collaboration’s notation in ref. [3]. In the DHMV calculation, a different convention is used as explained by equation (16) in ref. [15]. – 21 – 0 2 4 6 8 10 ] 2 [GeV 2 q 2 − 1.5 − 1 − 0.5 − 0 0.5 1 1.5 2 1 P Data theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 2 − 1.5 − 1 − 0.5 − 0 0.5 1 1.5 2 4 P' Data theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 1 − 0.5 − 0 0.5 1 1.5 2 5 P' Data CFFMPSV fit theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 1 − 0.5 − 0 0.5 1 1.5 2 6 P' Data theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 1 − 0.5 − 0 0.5 1 1.5 2 8 P' Data theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s Figure 11. The measured values of P1, P ′ 4, P ′ 5, P ′ 6, P ′ 8 compared with predictions from the theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar the total uncertainty. 8 Comparison with theoretical computations 0 2 4 6 8 10 ] 2 [GeV 2 q 2 − 1.5 − 1 − 0.5 − 0 0.5 1 1.5 2 1 P Data theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s JHEP10(2018)047 0 2 4 6 8 10 ] 2 [GeV 2 q 1 − 0.5 − 0 0.5 1 1.5 2 5 P' Data CFFMPSV fit theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s 0 2 4 6 8 10 ] 2 [GeV 2 q 2 − 1.5 − 1 − 0.5 − 0 0.5 1 1.5 2 4 P' Data theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s ] [ q 0 2 4 6 8 10 ] 2 [GeV 2 q 1 − 0.5 − 0 0.5 1 1.5 2 8 P' Data theory DHMV theory JC ATLAS -1 = 8 TeV, 20.3 fb s Figure 11. The measured values of P1, P ′ 4, P ′ 5, P ′ 6, P ′ 8 compared with predictions from the theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar the total uncertainty. QCD factorisation is used by DHMV and JC, where the latter focus on the impact of long-distance corrections using a helicity amplitude approach. The CFFMPSV group takes a different approach, using the QCD factorisation framework to perform compatibility checks of the LHCb data with theoretical predictions. This approach also allows informa- tion from a given experimentally measured parameter of interest to be excluded in order to make a fit-based prediction of the expected value of that parameter from the rest of the data. – 22 – With the exception of the P ′ 4 and P ′ 5 measurements in q2 ∈[4.0, 6.0] GeV2 and P ′ 8 in q2 ∈[2.0, 4.0] GeV2 there is good agreement between theory and measurement. The P ′ 4 and P ′ 5 parameters have statistical correlation of 0.37 in the q2 ∈[4.0, 6.0] GeV2 bin. The ob- served deviation from the SM prediction of P ′ 4 and P ′ 5 is for both parameters approximately 2.7 standard deviations (local) away from the calculation of DHMV for this bin. 8 Comparison with theoretical computations The devi- ations are less significant for the other calculation and the fit approach. All measurements are found to be within three standard deviations of the range covered by the different pre- dictions. Hence, including experimental and theoretical uncertainties, the measurements presented here are found to agree with the predicted SM contributions to this decay. JHEP10(2018)047 9 Conclusion The results of an angular analysis of the rare decay B0 d →K∗µ+µ−are presented. This flavour-changing neutral current process is sensitive to potential new-physics contributions. The B0 d →K∗µ+µ−analysis presented here uses a total of 20.3 fb−1 of √s = 8 TeV pp col- lision data collected by the ATLAS experiment at the LHC in 2012. An extended unbinned maximum-likelihood fit of the angular distribution of the signal decay is performed in order to extract the parameters FL, Si and P (′) j in six bins of q2. Three of these bins overlap in order to report results in ranges compatible with other experiments and phenomenology studies. All measurements are found to be within three standard deviation of the range covered by the different predictions. The results are also compatible with the results of the LHCb, CMS and Belle collaborations. A Correlation matrices Four folding schemes are applied to the data in order to extract FL, S3, S4, S5, S7 and S8 from four separate fits. The P (′) parameters are subsequently derived from the fit results using equations (2.2)–(2.5). It is not possible to extract a full correlation matrix between fitted parameters obtained from different fits. In order to reconstruct the correlation ma- trix, ensembles of pseudo-experiments are simulated using the pdf corresponding to the nominal angular distributions. Each simulated ensemble has the four folding schemes ap- plied to it and four fits are performed on the resulting samples. The distributions obtained for pairs of parameters obtained from fits to these ensembles are used to compute Pearson correlation coefficients for those pairs. Correlation matrices for FL and the S parameters are reconstructed from all possible pairings for a given q2 bin. A similar method is used to extract the correlation matrices for the P (′) parameters. This procedure is repeated for each q2 bin studied in order to obtain correlation matrices given in the remainder of this appendix. The correlation matrices are statistical only. Contributions from systematic uncertainties are not included, since the measurement precision is statistically limited. • Table 4 (5) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [0.04, 2.0] GeV2. • Table 6 (7) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [2.0, 4.0] GeV2. • Table 8 (9) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [4.0, 6.0] GeV2. • Table 10 (11) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [0.04, 4.0] GeV2. • Table 10 (11) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [0.04, 4.0] GeV2. • Table 12 (13) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [1.1, 6.0] GeV2. • Table 12 (13) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [1.1, 6.0] GeV2. • Table 14 (15) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [0.04, 6.0] GeV2. Acknowledgments We thank CERN for the very successful operation of the LHC, as well as the support staff from our institutions without whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Aus- tralia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DRF/IRFU, France; SRNSFG, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, ERDF, FP7, Horizon 2020 and Marie Sk lodowska-Curie Actions, European Union; In- vestissements d’Avenir Labex and Idex, ANR, R´egion Auvergne and Fondation Partager – 23 – le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; CERCA Programme Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leverhulme Trust, United Kingdom. The crucial computing support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (U.K.) and BNL (U.S.A.), the Tier-2 facilities worldwide and large non-WLCG resource providers. Ma- jor contributors of computing resources are listed in ref. [35]. JHEP10(2018)047 Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2 A Correlation matrices • Table 14 (15) shows the statistical correlation matrix for FL and S (P (′)) parameters for the q2 bin [0.04, 6.0] GeV2. – 24 – FL S3 S4 S5 S7 S8 FL 1.00 0.11 −0.13 0.03 0.16 0.24 S3 1.00 0.31 0.28 0.73 0.45 S4 1.00 0.58 0.19 0.22 S5 1.00 0.14 0.28 S7 1.00 0.59 S8 1.00 Table 4. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 2.0] GeV2. FL S3 S4 S5 S7 S8 FL 1.00 0.11 −0.13 0.03 0.16 0.24 S3 1.00 0.31 0.28 0.73 0.45 S4 1.00 0.58 0.19 0.22 S5 1.00 0.14 0.28 S7 1.00 0.59 S8 1.00 Table 4. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 2.0] GeV2. Table 4. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 2.0] GeV2. JHEP10(2018)047 P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 P1 1.00 0.04 0.05 0.62 0.32 P ′ 4 1.00 0.53 −0.08 −0.06 P ′ 5 1.00 0.00 0.22 P ′ 6 1.00 0.55 P ′ 8 1.00 Table 5. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 2.0] GeV2. able 5. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 2.0] GeV2. Table 5. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 2.0] GeV2. FL S3 S4 S5 S7 S8 FL 1.00 0.27 0.35 −0.04 −0.15 −0.37 S3 1.00 −0.08 −0.44 −0.09 −0.20 S4 1.00 0.60 −0.02 −0.12 S5 1.00 −0.11 −0.20 S7 1.00 0.63 S8 1.00 Table 6. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [2.0, 4.0] GeV2. Table 6. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [2.0, 4.0] GeV2. Table 6. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [2.0, 4.0] GeV2. P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 P1 1.00 −0.12 −0.21 0.05 0.05 P ′ 4 1.00 0.51 0.08 0.03 P ′ 5 1.00 −0.23 0.22 P ′ 6 1.00 0.66 P ′ 8 1.00 Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. Table 7. A Correlation matrices Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. – 25 – FL S3 S4 S5 S7 S8 FL 1.00 0.33 −0.18 0.04 0.22 0.28 S3 1.00 0.15 0.23 0.60 0.05 S4 1.00 0.52 0.03 0.01 S5 1.00 0.28 0.27 S7 1.00 0.60 S8 1.00 Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [4.0, 6.0] GeV2. FL S3 S4 S5 S7 S8 FL 1.00 0.33 −0.18 0.04 0.22 0.28 S3 1.00 0.15 0.23 0.60 0.05 S4 1.00 0.52 0.03 0.01 S5 1.00 0.28 0.27 S7 1.00 0.60 S8 1.00 Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [4.0, 6.0] GeV2. Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [4.0, 6.0] GeV2. Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [4.0, 6.0] GeV2. JHEP10(2018)047 P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 P1 1.00 0.11 0.34 0.41 0.16 P ′ 4 1.00 0.37 0.06 0.04 P ′ 5 1.00 0.39 0.33 P ′ 6 1.00 0.62 P ′ 8 1.00 Table 9. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[4.0, 6.0] GeV2. P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 P1 1.00 0.11 0.34 0.41 0.16 P ′ 4 1.00 0.37 0.06 0.04 P ′ 5 1.00 0.39 0.33 P ′ 6 1.00 0.62 P ′ 8 1.00 Table 9. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[4.0, 6.0] GeV2. Table 9. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[4.0, 6.0] GeV2. FL S3 S4 S5 S7 S8 FL 1.00 0.08 0.05 0.01 0.18 0.14 S3 1.00 −0.04 0.03 0.29 −0.16 S4 1.00 0.79 0.08 0.03 S5 1.00 0.03 −0.02 S7 1.00 0.60 S8 1.00 Table 10. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 4.0] GeV2. Table 10. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 4.0] GeV2. Table 10. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 4.0] GeV2. A Correlation matrices P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 P1 1.00 −0.07 0.00 0.21 0.12 P ′ 4 1.00 0.78 0.08 0.02 P ′ 5 1.00 0.03 −0.04 P ′ 6 1.00 0.59 P ′ 8 1.00 Table 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 P1 1.00 −0.07 0.00 0.21 0.12 P ′ 4 1.00 0.78 0.08 0.02 P ′ 5 1.00 0.03 −0.04 P ′ 6 1.00 0.59 P ′ 8 1.00 Table 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. able 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. Table 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. – 26 – FL S3 S4 S5 S7 S8 FL 1.00 0.05 0.00 0.07 0.18 −0.03 S3 1.00 0.41 0.46 0.32 −0.01 S4 1.00 0.60 0.09 0.03 S5 1.00 0.17 0.24 S7 1.00 0.67 S8 1.00 Table 12. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [1.1, 6.0] GeV2. Table 12. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [1.1, 6.0] GeV2. JHEP10(2018)047 P1 P ′ 4 P ′ 5 P ′ 6 P ′ 8 P1 1.00 0.23 −0.09 0.08 −0.07 P ′ 4 1.00 0.53 0.15 0.08 P ′ 5 1.00 0.28 0.24 P ′ 6 1.00 0.67 P ′ 8 1.00 Table 13. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[1.1, 6.0] GeV2. able 13. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[1.1, 6.0] GeV2. Table 13. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[1.1, 6.0] GeV2. FL S3 S4 S5 S7 S8 FL 1.00 0.03 0.01 −0.10 0.13 0.06 S3 1.00 −0.02 −0.09 0.32 −0.01 S4 1.00 0.68 0.00 0.04 S5 1.00 −0.05 0.03 S7 1.00 0.65 S8 1.00 Table 14. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 6.0] GeV2. Table 14. Statistical correlation matrix for the FL and S parameters obtained for q2 [0.04, 6.0] GeV2. Table 14. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈ [0.04, 6.0] GeV2. 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Black25, , , j g , , , , Blair6, T. Blazek28a, I. Bloch44, C. Blocker26, A. Blue55, W. Blum97,*, U. Blumenschein90, j g R.E. Blair6, T. Blazek28a, I. Bloch44, C. Blocker26, A. Blue55, W. Blum97,*, U. Blumenschei , , , , , , Dr. Blunier144a, G.J. Bobbink118, V.S. Bobrovnikov120b,120a, S.S. Bocchetta94, A. Bocci47, , , , , , , , lunier144a, G.J. Bobbink118, V.S. Bobrovnikov120b,120a, S.S. Bocchetta94, A. Bocci47, C. Bock112, M. Boehler50, D. Boerner180, D. Bogavac112, A.G. Bogdanchikov120b,120a, C. Bohm43a, V. Boisvert91, P. Bokan170, T. Bold81a, A.S. Boldyrev111, A.E. Bolz59b, M. Bomben132, M. Bona90, M. Boonekamp142, A. Borisov140, G. Borissov87, J. Bortfeldt3 M. Bomben132, M. Bona90, M. Boonekamp142, A. Borisov140, G. Borissov87, J. Bortfeldt35, rtoletto131, V. Bortolotto61a,61b,61c, D. Boscherini23b, M. Bosman14, J.D. Bossio Sola30, D. Bortoletto131, V. Bortolotto61a,61b,61c, D. Boscherini23b, M. Bosman14, J.D. Bossio Sola3 J. Boudreau135, J. Bouffard2, E.V. Bouhova-Thacker87, D. Boumediene37, C. Bourdarios128, J. Boudreau135, J. Bouffard2, E.V. Bouhova-Thacker87, D. Boumediene37, C. Bourdarios128, O. Brandt59a, U. Bratzler162, B. Brau100, J.E. Brau127, W.D. Breaden Madden55, O. Brandt59a, U. Bratzler162, B. Brau100, J.E. Brau127, W.D. Breaden Madden55, K. Brendlinger44, A.J. Brennan102, L. Brenner118, R. Brenner170, S. Bressler178, D Brendlinger44, A.J. Brennan102, L. Brenner118, R. Brenner170, S. Bressler178, D.L. Briglin21, JHEP10(2018)047 JHEP10(2018)047 T.M. Bristow48, D. Britton55, D. The ATLAS collaboration Britzger44, I. Brock24, R. Brock104, G. Brooijmans38 T.M. Bristow48, D. Britton55, D. Britzger44, I. Brock24, R. Brock104, G. Brooijmans38, Brooks91, W.K. Brooks144b, J. Brosamer18, E. Brost119, J.H Broughton21, T. Brooks91, W.K. Brooks144b, J. Brosamer18, E. Brost119, J.H Broughton21 P.A. Bruckman de Renstrom82, D. Bruncko28b, A. Bruni23b, G. Bruni23b, L.S. Bruni118, P.A. Bruckman de Renstrom82, D. Bruncko28b, A. Bruni23b, G. Bruni23b, L.S. Bruni118, B.H. Brunt31, M. Bruschi23b, N. Bruscino24, P. Bryant36, L. Bryngemark44, T. Buanes17, Q. Buat149, P. Buchholz148, A.G. Buckley55, I.A. Budagov77, M.K. Bugge130, F. B¨uhrer50, O. Bulekov110, D. Bullock8, T.J. Burch119, S. Burdin88, C.D. Burgard50, A.M. Burger5, B. Burghgrave119, K. Burka82, S. Burke141, I. Burmeister45, J.T.P. Burr131, E. Busato37, D. B¨uscher50, V. B¨uscher97, P. Bussey55, J.M. Butler25, C.M. Buttar55, J.M. Butterworth92, P. Butti35, W. Buttinger29, A. Buzatu15b, A.R. Buzykaev120b,120a, S. Cabrera Urb´an172, D. Caforio138, V.M.M. Cairo40b,40a, O. Cakir4a, N. Calace52, P. Calafiura18, A. Calandri99 G. Calderini132, P. Calfayan63, G. Callea40b,40a, L.P. Caloba78b, S. Calvente Lopez96, D. Calvet37, S. Calvet37, T.P. Calvet99, R. Camacho Toro36, S. Camarda35, P. Camarri71a,71b, D. Cameron130, R. Caminal Armadans171, C. Camincher56, S. Campana35, M. Campanelli92, A. Camplani66a,66b, A. Campoverde148, V. Canale67a,67b, M. Cano Bret58c, J. Cantero125, , , , p , p A. Camplani66a,66b, A. Campoverde148, V. Canale67a,67b, M. Cano Bret58c, J. Cantero125, p , p , , , , T. Cao159, M.D.M. Capeans Garrido35, I. Caprini27b, M. Caprini27b, M. Capua40b,40a, R.M. Carbone38, R. Cardarelli71a, F.C. Cardillo50, I. Carli139, T. Carli35, G. Carlino67a, B.T. Carlson135, L. Carminati66a,66b, R.M.D. Carney43a,43b, S. Caron117, E. Carquin144b, S. Carr´a66a,66b, G.D. Carrillo-Montoya35, J. Carvalho136a, D. Casadei21, M.P. Casado14,g, M. Casolino14, D.W. Casper169, R. Castelijn118, V. Castillo Gimenez172, N.F. Castro136a, A. Catinaccio35, J.R. Catmore130, A. Cattai35, J. Caudron24, V. Cavaliere171, E. Cavallaro14, D. Cavalli66a, M. Cavalli-Sforza14, V. Cavasinni69a,69b, E. Celebi12b, F. Ceradini72a,72b, L. Cerda Alberich172, A.S. Cerqueira78a, A. Cerri153, L. Cerrito71a,71b, F. Cerutti18, A. Cervelli20, S.A. Cetin12b, A. Chafaq34a, D Chakraborty119, S.K. Chan57, W.S. Chan118, Y.L. Chan61a, P. Chang171, J.D. Chapman31, D.G. Charlton21, C.C. Chau165, C.A. Chavez Barajas153, S. Che122, S. Cheatham64a,64c, A. Chegwidden104, S. Chekanov6, S.V. Chekulaev166a, G.A. Chelkov77,av, M.A. Chelstowska35, C.H. Chen76, H. Chen29, J. Chen58a, S. Chen161, S.J. Chen15c, X. Chen15b,au, Y. Chen80, H.C. Cheng103, H.J. Cheng15 A. Cheplakov77, E. Cheremushkina140, R. Cherkaoui El Moursli34e, E. Cheu7, K. Cheung62, L. Chevalier142, V. Chiarella49, G. Chiarelli69a, G. Chiodini65a, A.S. Chisholm35, A. Chitan27b, L. Chevalier142, V. Chiarella49, G. Chiarelli69a, G. Chiodini65a, A.S. Chisholm35, A. The ATLAS collaboration De Sanctis71a,71b, De Lorenzi76, A. De Maria51,u, D. De Pedis70a, A. De Salvo70a, U. De Sanctis71a,71b, A. De Santo153, K. De Vasconcelos Corga99, J.B. De Vivie De Regie128, W.J. Dearnaley De Santo153, K. De Vasconcelos Corga99, J.B. De Vivie De Regie128, W.J. Dearnaley87, R. Debbe29, C. Debenedetti143, D.V. Dedovich77, N. Dehghanian3, I. Deigaard118, . Debbe29, C. Debenedetti143, D.V. Dedovich77, Debbe29, C. Debenedetti143, D.V. Dedovich77, N. Dehghanian3, I. Deigaard118, R. Debbe , C. Debenedetti , D.V. Dedovich , N. Dehghanian , I. Deigaard , M. Del Gaudio40b,40a, J. Del Peso96, D. Delgove128, F. Deliot142, C.M. Delitzsch52, , , , g , g , M. Del Gaudio40b,40a, J. Del Peso96, D. Delgove128, F. Deliot142, C.M. Delitzsch52, Del Gaudio40b,40a, J. Del Peso96, D. Delgove128, F. Deliot142, C.M. Delitzsch52, Della Pietra67a,67b, D. Della Volpe52, A. Dell’Acqua35, L. Dell’Asta25, M. Dell’Orso69a,69b, M. Della Pietra67a,67b, D. Della Volpe52, A. Dell’Acqua35, L. Dell’Asta25, M. Dell’Ors M. Delmastro5, C. Delporte128, P.A. Delsart56, D.A. DeMarco165, S. Demers181, M. elmastro5, C. Delporte128, P.A. Delsart56, D.A. DeMarco165, S. Demers181, M. Demichev77, A. Demilly132, S.P. Denisov140, D. Denysiuk142, L. D’Eramo132, D. Derendarz82, Demilly132, S.P. Denisov140, D. Denysiuk142, L. D’Eramo132, D. Derendarz82, J.E. Derkaoui34d, F. Derue132, P. Dervan88, K. Desch24, C. Deterre44, K. Dette45, Derkaoui34d, F. Derue132, P. Dervan88, K. Desch24, C. Deterre44, K. Dette45, M.R. Devesa30, P.O. Deviveiros35, A. Dewhurst141, S. Dhaliwal26, F.A. Di Bello52, A. Di Ciaccio71a,71b, L. Di Ciaccio5, W.K. Di Clemente133, C. Di Donato67a,67b, , , , , A. Di Girolamo35, B. Di Girolamo35, B. Di Micco72a,72b, R. Di Nardo35, K.F. Di Petrillo57, , , , , Girolamo35, B. Di Girolamo35, B. Di Micco72a,72b, R. Di Nardo35, K.F. Di Petrillo57, A. Di Simone50, R. Di Sipio165, D. Di Valentino33, C. Diaconu99, M. Diamond165, F.A. Dias3 A. Di Simone50, R. Di Sipio165, D. Di Valentino33, C. Diaconu99, M. Diamond165, F.A. Dias39 Dita27b, S. Dita27b, F. Dittus35, F. Djama99, T. Djobava157b, J.I. Djuvsland59a, P. Dita27b, S. Dita27b, F. Dittus35, F. Djama99, T. Djobava157b, J.I. Djuvsland59a, M.A.B. Do Vale78c, D. Dobos35, M. Dobre27b, C. Doglioni94, J. Dolejsi139, Z. Dolezal139, M. Donadelli78d, S. Donati69a,69b, P. Dondero68a,68b, J. Donini37, M. D’Onofrio88, J. Dopke A. Doria67a, M.T. Dova86, A.T. Doyle55, E. Drechsler51, M. Dris10, Y. Du58b, arte-Campderros159, A. Dubreuil52, E. Duchovni178, G. Duckeck112, A. Ducourthial132, J. Duarte-Campderros159, A. Dubreuil52, E. Duchovni178, G. Duckeck112, A. Ducourthial132 O.A. Ducu107,x, D. Duda118, A. Dudarev35, A.C. Dudder97, E.M. Duffield18, L. Duflot128, M. D¨uhrssen35, M. Dumancic178, A.E. Dumitriu27b,e, A.K. The ATLAS collaboration Ch Y H Chi 174 M V Chi h 77 K Ch i63 A R Ch t37 S Ch id 160 , , , , , V. Christodoulou92, D. Chromek-Burckhart35, M.C. Chu61a, J. Chudoba137, A.J. Chuinard101, J.J. Chwastowski82, L. Chytka126, A.K. Ciftci4a, D. Cinca45, V. Cindro89, I.A. Cioar˘a24, C. Ciocca23b,23a, A. Ciocio18, F. Cirotto67a,67b, Z.H. Citron178, M. Citterio66a, M. Ciubancan27b 52 57 38 48 18 43 43b A. Clark52, B.L. Clark57, M.R. Clark38, P.J. Clark48, R.N. Clarke18, C. Clement43a,43b, Y. Coadou99, M. Cobal64a,64c, A. Coccaro52, J. Cochran76, L. Colasurdo117, B. Cole38, A.P. Colijn118, J. Collot56, T. Colombo169, P. Conde Mui˜no136a,136b, E. Coniavitis50, S.H. Connell32b, I.A. Connelly98, S. Constantinescu27b, G. Conti35, F. Conventi67a,ax, – 31 – M. Cooke18, A.M. Cooper-Sarkar131, F. Cormier173, K.J.R. Cormier165, M. Corradi70a,70b, Cooke18, A.M. Cooper-Sarkar131, F. Cormier173, K.J.R. Cormier165, M. Corradi70a,70b, , p , , , , F. Corriveau101,af, A. Cortes-Gonzalez35, G. Cortiana113, G. Costa66a, M.J. Costa172, rriveau101,af, A. Cortes-Gonzalez35, G. Cortiana113, G. Costa66a, M.J. Costa172, , , , , , D. Costanzo146, G. Cottin31, G. Cowan91, B.E. Cox98, K. Cranmer121, S.J. Crawley55, , , , , , Costanzo146, G. Cottin31, G. Cowan91, B.E. Cox98, K. Cranmer121, S.J. Crawley55, R.A. Creager133, G. Cree33, S. Cr´ep´e-Renaudin56, F. Crescioli132, W.A. Cribbs43a,43b, A. Creager133, G. Cree33, S. Cr´ep´e-Renaudin56, F. Crescioli132, W.A. Cribbs43a,43b, Cristinziani24, V. Croft117, G. Crosetti40b,40a, A. Cueto96, T. Cuhadar Donszelmann146, M. Cristinziani24, V. Croft117, G. Crosetti40b,40a, A. Cueto96, T. Cuhadar Donszelmann146, A.R. Cukierman150, J. Cummings181, M. Curatolo49, J. C´uth97, P. Czodrowski35, M.J. Da Cunha Sargedas De Sousa136a,136b, C. Da Via98, W. Dabrowski81a, T. Dado28a,y, M.J. Da Cunha Sargedas De Sousa1 g , , , , T. Dai103, O. Dale17, F. Dallaire107, C. Dallapiccola100, M. Dam39, G. D’amen23b,23a, T. Dai103, O. Dale17, F. Dallaire107, C. Dallapiccola100, M. Dam39, G. D’amen23b,23a, y , , g , , , g M. Dano Hoffmann142, V. Dao152, G. Darbo53b, S. Darmora8, J. Dassoulas3, A. Dattagupta127, M. Dano Hoffmann142, V. Dao152, G. Darbo53b, S. Darmora8, J. Dassoulas3, A. Dattagupta127, T. Daubney44, S. D’Auria55, W. Davey24, C. David44, T. Davidek139, D.R. Davis47, JHEP10(2018)047 JHEP10(2018)047 P. Davison92, E. Dawe102, I. Dawson146, K. De8, R. De Asmundis67a, A. De Benedetti124, P. Davison92, E. Dawe102, I. Dawson146, K. De8, R. De Asmundis67a, A. De Benedetti124, S. De Castro23b,23a, S. De Cecco132, N. De Groot117, P. de Jong118, H. De la Torre104, F. De Lorenzi76, A. De Maria51,u, D. De Pedis70a, A. De Salvo70a, U. The ATLAS collaboration Duncan55, M. Dunford59a, H. Duran Yildiz4a, M. D¨uren54, A. Durglishvili157b, D. Duschinger46, B. Dutta44, D. Duvn M. Dyndal44, B.S. Dziedzic82, C. Eckardt44, K.M. Ecker113, R.C. Edgar103, T. Eifert35, G. Eigen17, K. Einsweiler18, T. Ekelof170, M. El Kacimi34c, R. El Kosseifi99, V. Ellajosyula99 M. Ellert170, S. Elles5, F. Ellinghaus180, A.A. Elliot174, N. Ellis35, J. Elmsheuser29, M. Elsing35 M. Ellert 70, S. Elles5, F. Ellinghaus 80, A.A. Elliot 7 , N. Ellis35, J. Elmsheuser 9, M. Elsing3 D. Emeliyanov141, Y. Enari161, O.C. Endner97, J.S. Ennis176, J. Erdmann45, A. Ereditato20, D. Emeliyanov141, Y. Enari161, O.C. Endner97, J.S. Ennis176, J. Erdmann45, A. Ereditato20, M. Ernst29, S. Errede171, M. Escalier128, C. Escobar172, B. Esposito49, O. Estrada Pastor172, A.I. Etienvre142, E. Etzion159, H. Evans63, A. Ezhilov134, M. Ezzi34e, F. Fabbri23b,23a, J. Faltova35, Y. Fang15a, M. Fanti66a,66b, A. Farbin8, A. Farilla72a, C. Farina135, M. Farina68a,68b, T. Farooque104, S. Farrell18, S.M. Farrington176, P. Farthouat35, F. Fassi34e, P. Fassnacht35, D. Fassouliotis9, M. Faucci Giannelli91, A. Favareto53b,53a, W.J. Fawcett131, L. Fayard128, O.L. Fedin134,q, W. Fedorko173, S. Feigl130, L. Feligioni99, C. Feng58b, E.J. Feng3 L. Fayard 8, O.L. Fedin 3 ,q, W. Fedorko 73, S. Feigl 30, L. Feligioni99, C. Feng58 , E.J. Feng35 H. Feng103, M.J. Fenton55, A.B. Fenyuk140, L. Feremenga8, P. Fernandez Martinez172, y , , , g , g , g H. Feng103, M.J. Fenton55, A.B. Fenyuk140, L. Feremenga8, P. Fernandez Martinez1 S. Fernandez Perez14, J. Ferrando44, A. Ferrari170, P. Ferrari118, R. Ferrari68 D.E. Ferreira de Lima59b, A. Ferrer172, D. Ferrere52, C. Ferretti103, F. Fiedler97, M. Filipuzzi44 – 32 – A. Filipˇciˇc89, F. Filthaut117, M. Fincke-Keeler174, K.D. Finelli154, M.C.N. Fiolhais136a,136c,b, L. Fiorini172, A. Fischer2, C. Fischer14, J. Fischer180, W.C. Fisher104, N. Flaschel44, I. Fleck148 , , , , , , P. Fleischmann103, R.R.M. Fletcher133, T. Flick180, B.M. Flierl112, L.R. Flores Castillo61a, M.J. Flowerdew113, G.T. Forcolin98, A. Formica142, F.A. F¨orster14, A.C. Forti98, A.G. Foster21 D. Fournier128, H. Fox87, S. Fracchia146, P. Francavilla132, M. Franchini23b,23a, S. Franchino59a D. Francis35, L. Franconi130, M. Franklin57, M. Frate169, M. Fraternali68a,68b, D. Freeborn92, S.M. Fressard-Batraneanu35, B. Freund107, D. Froidevaux35, J.A. Frost131, C. Fukunaga162, T. Fusayasu114, J. Fuster172, C. Gabaldon56, O. Gabizon158, A. Gabrielli23b,23a, A. Gabrielli18, B. Galhardo136a,136c, E.J. Gallas131, B.J. Gallop141, P. Gallus138, G. Galster39, K.K. Gan122, S. Ganguly37, Y. Gao88, Y.S. Gao150,m, C. Garc´ıa172, J.E. Garc´ıa Navarro172, JHEP10(2018)047 JHEP10(2018)047 J.A. Garc´ıa Pascual15a, M. Garcia-Sciveres18, R.W. Gardner36, N. Garelli150, V. Garonne130, A. Gascon Bravo44, K. Gasnikova44, C. Gatti49, A. Gaudiello53b,53a, G. Gaudio68a, I.L. The ATLAS collaboration Gavrilenko108, C. Gay173, G. Gaycken24, E.N. Gazis10, C.N.P. Gee141, J. Geisen51 M. Geisen97, M.P. Geisler59a, K. Gellerstedt43a,43b, C. Gemme53b, M.H. Genest56, C. Geng103 S. Gentile70a,70b, C. Gentsos160, S. George91, D. Gerbaudo14, A. Gershon159, G. Gessner45, S. Gentile70a,70b, C. Gentsos160, S. George91, D. Gerbaudo14, A. Gershon159, G. Gessner45, S Gh 148 M Gh 24 B G bb 23b S G 70a 70b N G 23b 23a S. Ghasemi148, M. Ghneimat24, B. Giacobbe23b, S. Giagu70a,70b, N. Giangiacomi23b,23a, P. Giannetti69a, S.M. Gibson91, M. Gignac173, M. Gilchriese18, D. Gillberg33, G. Gilles180, D.M. Gingrich3,aw, N. Giokaris9,*, M.P. Giordani64a,64c, F.M. Giorgi23b, P.F. Giraud142, P. Giromini57, D. Giugni66a, F. Giuli131, C. Giuliani113, M. Giulini59b, B.K. Gjelsten130, S. Gkaitatzis160, I. Gkialas9,k, E.L. Gkougkousis143, P. Gkountoumis10, L.K. Gladilin111, C. Glasman96, J. Glatzer14, P.C.F. Glaysher44, A. Glazov44, M. Goblirsch-Kolb26, J. Godlewski82, S. Goldfarb102, T. Golling52, D. Golubkov140, A. Gomes136a,136b,136d R. Goncalves Gama78b, J. Goncalves Pinto Firmino Da Costa142, R. Gon¸calo136a, G. Gonella50 L. Gonella21, A. Gongadze77, S. Gonz´alez de la Hoz172, S. Gonzalez-Sevilla52, L. Goossens35, L. Gonella21, A. Gongadze77, S. Gonz´alez de la Hoz172, S. Gonzalez-Sevilla52, L. Goossens35, P.A. Gorbounov109, H.A. Gordon29, I. Gorelov116, B. Gorini35, E. Gorini65a,65b, A. Goriˇsek89, L. Gonella , A. Gongadze , S. Gonzalez de la Hoz , S. Gonzalez Sevilla , L. Goossens P.A. Gorbounov109, H.A. Gordon29, I. Gorelov116, B. Gorini35, E. Gorini65a,65b, A. Goriˇsek A.T. Goshaw47, C. G¨ossling45, M.I. Gostkin77, C.A. Gottardo24, C.R. Goudet128 D. Goujdami34c, A.G. Goussiou145, N. Govender32b,c, E. Gozani158, L. Graber51, j , , , , , I. Grabowska-Bold81a, P.O.J. Gradin170, J. Gramling169, E. Gramstad130, S. Grancagnolo19, 134 27f 55 18 93 k 24 V. Gratchev134, P.M. Gravila27f, C. Gray55, H.M. Gray18, Z.D. Greenwood93,ak, C. Grefe24, K. Gregersen92, I.M. Gregor44, P. Grenier150, K. Grevtsov5, J. Griffiths8, A.A. Grillo143, K. Grimm87, S. Grinstein14,z, Ph. Gris37, J.-F. Grivaz128, S. Groh97, E. Gross178, J. Grosse-Knetter51, G.C. Grossi93, Z.J. Grout92, A. Grummer116, L. Guan103, W. Guan17 J. Guenther74, F. Guescini166a, D. Guest169, O. Gueta159, B. Gui122, E. Guido53b,53a, T. Guillemin5, S. Guindon2, U. Gul55, C. Gumpert35, J. Guo58c, W. Guo103, Y. Guo58a,t, R. Gupta41, S. Gupta131, G. Gustavino70a,70b, P. Gutierrez124, N.G. Gutierrez Ortiz92, C. Gutschow92, C. Guyot142, M.P. Guzik81a, C. Gwenlan131, C.B. Gwilliam88, A. Haas121, C. Haber18, H.K. Hadavand8, N. Haddad34e, A. Hadef99, S. Hageb¨ock24, M. Hagihara167, H. Hakobyan182,*, M. Haleem44, J. Haley125, G. Halladjian104, G.D. Hallewell99, K. Hamacher180, P. Hamal126, K. Hamano174, A. Hamilton32a, G.N. Hamity146, P.G. Hamnett44, K. Hamacher180, P. Hamal126, K. The ATLAS collaboration Hamano174, A. Hamilton32a, G.N. Hamity146, P.G. Hamnet L. Han58a, S. Han15d, K. Hanagaki79,w, K. Hanawa161, M. Hance143, B. Haney133, P. Hanke59 J.B. Hansen39, J.D. Hansen39, M.C. Hansen24, P.H. Hansen39, K. Hara167, A.S. Hard17 T. Harenberg180, F. Hariri128, S. Harkusha105, R.D. Harrington48, P.F. Harrison176, N.M. Hartmann112, M. Hasegawa80, Y. Hasegawa147, A. Hasib48, S. Hassani142, S. Haug20, R. Hauser104, L. Hauswald46, L.B. Havener38, M. Havranek138, C.M. Hawkes21, R.J. Hawkings g D. Hayakawa163, D. Hayden104, C.P. Hays131, J.M. Hays90, H.S. Hayward88, S.J. Haywood141, S.J. Head21, T. Heck97, V. Hedberg94, L. Heelan8, S. Heer24, K.K. Heidegger50, S. Heim4 T. Heim18, B. Heinemann44,ar, J.J. Heinrich112, L. Heinrich121, C. Heinz54, J. Hejbal137, – 33 – L. Helary35, A. Held173, S. Hellman43a,43b, C. Helsens35, R.C.W. Henderson87, Y. Heng179, S. Henkelmann173, A.M. Henriques Correia35, S. Henrot-Versille128, G.H. Herbert19, H. Herde2 V. Herget175, Y. Hern´andez Jim´enez32c, H. Herr97, G. Herten50, R. Hertenberger112, L. Hervas g g T.C. Herwig133, G.G. Hesketh92, N.P. Hessey166a, J.W. Hetherly41, S. Higashino79, E. Hig´on-Rodriguez172, K. Hildebrand36, E. Hill174, J.C. Hill31, K.H. Hiller44, S.J. Hillier21, M. Hils46, I. Hinchliffe18, M. Hirose50, D. Hirschbuehl180, B. Hiti89, O. Hladik137, X. Hoad48, J. Hobbs152, N. Hod166a, M.C. Hodgkinson146, P. Hodgson146, A. Hoecker35, M.R. Hoeferkamp116, F. Hoenig112, D. Hohn24, T.R. Holmes36, M. Homann45, S. Honda167, T. Honda79, T.M. Hong135, B.H. Hooberman171, W.H. Hopkins127, Y. Horii115, A.J. Horton149 J Y H h 56 S H 155 A H d 34a J H h98 J H 86 M H b k 126 g J-Y. Hostachy56, S. Hou155, A. Hoummada34a, J. Howarth98, J. Hoya86, M. Hrabovsky126, J. Hrdinka35, I. Hristova19, J. Hrivnac128, A. Hrynevich106, T. Hryn’ova5, P.J. Hsu62, JHEP10(2018)047 JHEP10(2018)047 S.-C. Hsu145, Q. Hu58a, S. Hu58c, Y. Huang15a, Z. Hubacek138, F. Hubaut99, F. Huegging24, 131 38 87 35 152 77 h T.B. Huffman131, E.W. Hughes38, G. Hughes87, M. Huhtinen35, P. Huo152, N. Huseynov77,a J. Huston104, J. Huth57, G. Iacobucci52, G. Iakovidis29, I. Ibragimov148, L. Iconomidou-Fayard128, Z. Idrissi34e, P. Iengo35, O. Igonkina118,ac, T. Iizawa177, Y. Ikegami79 M. Ikeno79, Y. Ilchenko11, D. Iliadis160, N. Ilic150, G. Introzzi68a,68b, P. Ioannou9,*, M. Iodice72 M. Ikeno79, Y. Ilchenko11, D. Iliadis160, N. Ilic150, G. Introzzi68a,68b, P. Ioannou9, , M. Iodice72a K I d id 38 V I lit 57 M F I 170 N I hiji 129 M I hi 161 M I hit k 163 K. Iordanidou38, V. Ippolito57, M.F. Isacson170, N. Ishijima129, M. Ishino161, M. Ishitsuka163, C. Issever131, S. Istin12c, F. Ito167, J.M. Iturbe Ponce61a, R. The ATLAS collaboration Iuppa73a,73b, H. Iwasaki79, J.M. Izen42, V. Izzo67a, S. Jabbar3, P. Jackson1, R.M. Jacobs24, V. Jain2, K.B. Jakobi97, K. Jakobs50, S. Jakobsen74, T. Jakoubek137, D.O. Jamin125, D.K. Jana93, R. Jansky52, J. Janssen24, M. Janus51, P.A. Janus81a, G. Jarlskog94, N. Javadov77,ah, T. Jav˚urek50, M. Javurkova50, F. Jeanneau142, L. Jeanty18, J. Jejelava157a,ai, A. Jelinskas176, P. Jenni50,d, y j C. Jeske176, S. J´ez´equel5, H. Ji179, J. Jia152, H. Jiang76, Y. Jiang58a, Z. Jiang150,r, S. Jiggins92 J. Jimenez Pena172, S. Jin15a, A. Jinaru27b, O. Jinnouchi163, H. Jivan32c, P. Johansson146, K.A. Johns7, C.A. Johnson63, W.J. Johnson145, K. Jon-And43a,43b, R.W.L. Jones87, K.A. Johns7, C.A. Johnson63, W.J. Johnson145, K. Jon-And43a,43b, R.W.L. Jones87, S.D. Jones153, S. Jones7, T.J. Jones88, J. Jongmanns59a, P.M. Jorge136a,136b, J. Jovicevic166a, Jo s , C Jo so , W J Jo so , Jo d , R W Jo es , D. Jones153, S. Jones7, T.J. Jones88, J. Jongmanns59a, P.M. Jorge136a,136b, J. Jovicevic166a, X. Ju179, A. Juste Rozas14,z, A. Kaczmarska82, M. Kado128, H. Kagan122, M. Kagan150, S.J. Kahn99, T. Kaji177, E. Kajomovitz47, C.W. Kalderon94, A. Kaluza97, S. Kama41, A. Kamenshchikov140, N. Kanaya161, L. Kanjir89, V.A. Kantserov110, J. Kanzaki79, B. Kaplan121, L.S. Kaplan179, D. Kar32c, K. Karakostas10, N. Karastathis10, M.J. Kare E. Karentzos10, S.N. Karpov77, Z.M. Karpova77, K. Karthik121, V. Kartvelishvili87, A.N. Karyukhin140, K. Kasahara167, L. Kashif179, R.D. Kass122, A. Kastanas151, Y. K ato161, A. Katre52, J. Katzy44, K. Kawade80, K. Kawagoe85, T. Kawamoto161, C. Kato161, A. Katre52, J. Katzy44, K. Kawade80, K. Kawagoe85, T. Kawamoto161, y g awamura51, E.F. Kay88, V.F. Kazanin120b,120a, R. Keeler174, R. Kehoe41, J.S. Keller33, G. Kawamura51, E.F. Kay88, V.F. Kazanin120b,120a, R. Keeler174, R. Kehoe41, J.S. Keller33 J.J. Kempster91, J. Kendrick21, H. Keoshkerian165, O. Kepka137, S. Kersten180, B.P. Kerˇsev Kempster91, J. Kendrick21, H. Keoshkerian165, O. Kepka137, S. Kersten180, B.P. Kerˇsevan89, R.A. Keyes101, M. Khader171, F. Khalil-Zada13, A. Khanov125, A.G. Kharlamov120b,120a, Keyes101, M. Khader171, F. Khalil-Zada13, A. Khanov125, A.G. Kharlamov120b,120a, harlamova120b,120a, A. Khodinov164, T.J. Khoo52, V. Khovanskiy109,*, E. Khramov77, J. Khubua157b, S. Kido80, C.R. Kilby91, H.Y. Kim8, S.H. Kim167, Y.K. Kim36, N. Kimura160, ubua157b, S. Kido80, C.R. Kilby91, H.Y. Kim8, S.H. Kim167, Y.K. Kim36, N. Kimura160, O.M. Kind19, B.T. King88, D. Kirchmeier46, J. Kirk141, A.E. Kiryunin113, T. Kishimoto161, M. Kind19, B.T. King88, D. Kirchmeier46, J. Kirk141, A.E. Kiryunin113, T. Kishimoto161, * D. Kisielewska81a, V. Kitali44, K. Kiuchi167, O. Kivernyk5, E. Kladiva28b,*, D. Kisielewska81a, V. Kitali44, K. Kiuchi167, O. Kivernyk5, E. Kladiva28b,*, g P. Klimek119, A. Klimentov29, R. Klingenberg45,*, T. Klingl24, T. Klioutchnikova35, P. The ATLAS collaboration Kluit118, P. Klimek119, A. Klimentov29, R. Klingenberg45,*, T. Klingl24, T. Klioutchnikova35, P. Kluit118, S. Kluth113, E. Kneringer74, E.B.F.G. Knoops99, A. Knue113, A. Kobayashi161, D. Kobayashi16 T. Kobayashi161, M. Kobel46, M. Kocian150, P. Kodys139, T. Koffas33, E. Koffeman118, M.K. K¨ohler178, N.M. K¨ohler113, T. Koi150, M. Kolb59b, I. Koletsou5, A.A. Komar108,*, Y. Komori161, T. Kondo79, N. Kondrashova58c, K. K¨oneke50, A.C. K¨onig117, T. Kono79,aq R. Konoplich121,am, N. Konstantinidis92, R. Kopeliansky63, S. Koperny81a, A.K. Kopp50, K. Korcyl82, K. Kordas160, A. Korn92, A.A. Korol120b,120a,ap, I. Korolkov14, E.V. Korolkova146 – 34 – O. Kortner113, S. Kortner113, T. Kosek139, V.V. Kostyukhin24, A. Kotwal47, A. Koulouris10, , , , y , , A. Kourkoumeli-Charalampidi68a,68b, C. Kourkoumelis9, E. Kourlitis146, V. Kouskoura29, p , , , , A.B. Kowalewska82, R. Kowalewski174, T.Z. Kowalski81a, C. Kozakai161, W. Kozanecki142, A.S. Kozhin140, V.A. Kramarenko111, G. Kramberger89, D. Krasnopevtsev110, M.W. Krasny1 A. Krasznahorkay35, D. Krauss113, J.A. Kremer81a, J. Kretzschmar88, K. Kreutzfeldt54, P. Krieger165, K. Krizka36, K. Kroeninger45, H. Kroha113, J. Kroll137, J. Kroll133, J. Kroseber J. Krstic16, U. Kruchonak77, H. Kr¨uger24, N. Krumnack76, M.C. Kruse47, T. Kubota102, H. Kucuk92, S. Kuday4b, J.T. Kuechler180, S. Kuehn35, A. Kugel59a, F. Kuger175, T. Kuhl44, T. Kunigo83, A. Kupco137, T. Kupfer45, O. Kuprash159, H. Kurashige80, L.L. Kurchaninov166a, Kunigo83, A. Kupco137, T. Kupfer45, O. Kupras T. Kunigo , A. Kupco , T. Kupfer , O. Kuprash , H. Kurashige , L.L. Kurchaninov Y.A. Kurochkin105, M.G. Kurth15d, V. Kus137, E.S. Kuwertz174, M. Kuze163, J. Kvita126, JHEP10(2018)047 JHEP10(2018)047 T. Kwan174, D. Kyriazopoulos146, A. La Rosa113, J.L. La Rosa Navarro78d, L. La Rotonda40b,40a, F. La Ruffa40b,40a, C. Lacasta172, F. Lacava70a,70b, J. Lacey44, H. Lacker19, D. Lacour132, E. Ladygin77, R. Lafaye5, B. Laforge132, S. Lai51, S. Lammers63, W. Lampl7, E. Lan¸con29, U. Landgraf50, M.P.J. Landon90, M.C. Lanfermann52, V.S. Lang59a, J.C. Lange14, R.J. Langenberg35, A.J. Lankford169, F. Lanni29, K. Lantzsch24, A. Lanza68a, g g A. Lapertosa53b,53a, S. Laplace132, J.F. Laporte142, T. Lari66a, F. Lasagni Manghi23b,23a, M. Lassnig35, P. Laurelli49, W. Lavrijsen18, A.T. Law143, P. Laycock88, T. Lazovich57, M. Lazzaroni66a,66b, B. Le102, O. Le Dortz132, E. Le Guirriec99, E.P. Le Quilleuc142, M. LeBlanc174, T. LeCompte6, F. Ledroit-Guillon56, C.A. Lee29, G.R. Lee141,i, L. Lee57, S.C. Lee155, B. Lefebvre101, G. Lefebvre132, M. Lefebvre174, F. Legger112, C. Leggett18, G. Lehmann Miotto35, X. Lei7, W.A. Leight44, M.A.L. Leite78d, R. Leitner139, D. Lellouch178, B. Lemmer51, K.J.C. Leney92, T. Lenz24, B. Lenzi35, R. Leone7, S. Leone69a, C. Leonidopoulos48, G. Lerner153, C. Leroy107, A.A.J. Lesage142, C.G. Lester31, M. Levchenko134, J. Levˆeque5, D. The ATLAS collaboration Levin103, L.J. Levinson178, M. Levy21, D. Lewis90, B. Li58a,t, C-Q. Li58a,al, H. Li152, L. Li58c , , y , , , Q , , Q. Li15d, S. Li47, X. Li58c, Y. Li148, Z. Liang15a, B. Liberti71a, A. Liblong165, K. Lie61c, J. Liebal24, W. Liebig17, A. Limosani154, S.C. Lin156, T.H. Lin97, R.A. Linck63, B.E. Lindquist152, A.L. Lionti52, E. Lipeles133, A. Lipniacka17, M. Lisovyi59b, T.M. Liss171,at, A. Lister173, A.M. Litke143, B. Liu155,ae, H.B. Liu29, H. Liu103, J.B. Liu58a, J.K.K. Liu131, J. Liu58b, K. Liu99, L. Liu171, M. Liu58a, Y.L. Liu58a, Y.W. Liu58a, M. Livan68a,68b, A. Lleres5 b 44 J. Llorente Merino15a, S.L. Lloyd90, C.Y. Lo61b, F. Lo Sterzo155, E.M. Lobodzinska44, P. Loch7, 98 26 181 * 19 146 137 F.K. Loebinger98, K.M. Loew26, A. Loginov181,*, T. Lohse19, K. Lohwasser146, M. Lokajicek B.A. Long25, J.D. Long171, R.E. Long87, L. Longo65a,65b, K.A. Looper122, J.A. Lopez144b, g , g , g , g , p , p , D. Lopez Mateos57, I. Lopez Paz14, A. Lopez Solis132, J. Lorenz112, N. Lorenzo Martinez5, M. Losada22, P.J. L¨osel112, A. L¨osle50, X. Lou15a, A. Lounis128, J. Love6, P.A. Love87, H. L , , , , , , , , N. Lu103, Y.J. Lu62, H.J. Lubatti145, C. Luci70a,70b, A. Lucotte56, C. Luedtke50, F. Luehring63, W. Lukas74, L. Luminari70a, O. Lundberg43a,43b, B. Lund-Jensen151, M.S. Lutz100, P.M. Luzi1 g D. Lynn29, R. Lysak137, E. Lytken94, F. Lyu15a, V. Lyubushkin77, H. Ma29, L.L. Ma58b, D. Madaffari172, R. Madar37, W.F. Mader46, A. Madsen44, J. Maeda80, S. Maeland17, y g A.A. Maier113, T. Maier112, A. Maio136a,136b,136d, O. Majersky28a, S. Majewski127, Y. Makida R. Mantifel101, M. Mantoani51, S. Manzoni66a,66b, L. Mapelli35, G. Marceca30, L. March L. Marchese131, G. Marchiori132, M. Marcisovsky137, M. Marjanovic37, D.E. Marley103, – 35 – F. Marroquim78b, S.P. Marsden98, Z. Marshall18, M.U.F Martensson170, S. Marti-Garcia172, C.B. Martin122, T.A. Martin176, V.J. Martin48, B. Martin dit Latour17, M. Martinez14,z, , , , , , V.I. Martinez Outschoorn171, S. Martin-Haugh141, V.S. Martoiu27b, A.C. Martyniuk92, A. Marzin35, L. Masetti97, T. Mashimo161, R. Mashinistov108, J. Masik98, A.L. Maslennikov120b,120a, L. Massa71a,71b, P. Mastrandrea5, A. Mastroberardino40b,40a, T. Masubuchi161, P. M¨attig180, J. Maurer27b, B. Maˇcek89, S.J. Maxfield88, D.A. Maximov120b,120a, R. Mazini155, I. Maznas160, S.M. Mazza66a,66b, N.C. Mc Fadden , , , , , G. Mc Goldrick165, S.P. Mc Kee103, A. McCarn103, R.L. McCarthy152, T.G. McCarthy113, G. Mc Goldrick165, S.P. Mc Kee103, A. McCarn103, R.L. McCarthy152, T.G. The ATLAS collaboration McCarthy113, L I McClymont92 E F McDonald102 J A Mcfayden92 G Mchedlidze51 S J McMahon141 P.C. McNamara102, R.A. McPherson174,af, S. Meehan145, T.M. Megy50, S. Mehlha A. Mehta88, T. Meideck56, B. Meirose42, D. Melini172,h, B.R. Mellado Garcia32c, JHEP10(2018)047 JHEP10(2018)047 J.D. Mellenthin51, M. Melo28a, F. Meloni20, A. Melzer24, S.B. Menary98, L. Meng88, X.T. Meng103, A. Mengarelli23b,23a, S. Menke113, E. Meoni40b,40a, S. Mergelmeyer19, P. Mermod52, L. Merola67a,67b, C. Meroni66a, F.S. Merritt36, A. Messina70a,70b, J. Metc A.S. Mete169, C. Meyer133, J. Meyer118, J-P. Meyer142, H. Meyer Zu Theenhausen59a, , y , y , y , y , F. Miano153, R.P. Middleton141, S. Miglioranzi53b,53a, L. Mijovi´c48, G. Mikenberg178, F. Miano153, R.P. Middleton141, S. Miglioranzi53b,53a, L. Mijovi´c48, G. Mikenberg178, 137 89 102 165 36 48 M. Mikestikova137, M. Mikuˇz89, M. Milesi102, A. Milic165, D.W. Miller36, C. Mills48, A. Mil Mikestikova137, M. Mikuˇz89, M. Milesi102, A. Milic165, D.W. Miller36, C. Mills48, A. Milov178, D.A. Milstead43a,43b, A.A. Minaenko140, Y. Minami161, I.A. Minashvili157b, A.I. Mincer121 A. Milstead43a,43b, A.A. Minaenko140, Y. Minami161, I.A. Minashvili157b, A.I. Mincer121, B. Mindur81a, M. Mineev77, Y. Minegishi161, Y. Ming179, L.M. Mir14, K.P. Mistry133, i177, J. Mitrevski112, V.A. Mitsou172, A. Miucci20, P.S. Miyagawa146, A. Mizukami79, T. Mitani177, J. Mitrevski112, V.A. Mitsou172, A. Miucci20, P.S. Miyagawa146, A. Mizukami79, J.U. Mj¨ornmark94, T. Mkrtchyan182, M. Mlynarikova139, T. Moa43a,43b, K. Mochizuki107, Mogg50, S. Mohapatra38, S. Molander43a,43b, R. Moles-Valls24, R. Monden83, P. Mogg50, S. Mohapatra38, S. Molander43a,43b, R. Moles-Valls24, R. Monden83, M.C. Mondragon104, K. M¨onig44, J. Monk39, E. Monnier99, A. Montalbano152, J. Montejo Berlingen35, F. Monticelli86, S. Monzani66a, R.W. Moore3, N. Morange128, D. Moreno22, M. Moreno Ll´acer35, P. Morettini53b, S. Morgenstern35, D. Mori149, T. Mori1 2, P. Moschovakos10, M. Mosidze157b, H.J. Moss146, J. Moss150,n, K. Motohashi163, L. Morvaj152, P. Moschovakos10, M. Mosidze157b, H.J. Moss146, J. Moss150,n, K. Motohashi R. Mount150, E. Mountricha29, E.J.W. Moyse100, S. Muanza99, F. Mueller113, J. Mueller135, R.S.P. Mueller112, D. Muenstermann87, P. Mullen55, G.A. Mullier20, F.J. Munoz Sanchez98, W.J. Murray176,141, H. Musheghyan35, M. Muˇskinja89, A.G. Myagkov140,an, M. Myska138, B.P. Nachman18, O. Nackenhorst52, K. Nagai131, R. Nagai79,aq, K. Nagano79, Y. Nagasaka60 167 50 99 35 115 79 K. Nagata167, M. Nagel50, E. Nagy99, A.M. Nairz35, Y. Nakahama115, K. Nakamura79, T. Nakamura161, I. Nakano123, R.F. Naranjo Garcia44, R. Narayan11, D.I. Narrias Villar59a, I. Naryshkin134, T. Naumann44, G. Navarro22, R. Nayyar7, H.A. Neal103,*, P.Y. Nechaeva108, T.J. Neep142, A. Negri68a,68b, M. Negrini23b, S. Nektarijevic117, C. Nellist128, A. Nelson169, M.E. Nelson131, S. Nemecek137, P. Nemethy121, M. Nessi35,f, M.S. Neubauer171, M. Neuman P.R. The ATLAS collaboration Pozo Astigarraga35, P. Pralavorio99, A. Pranko18, S. Prell76, D P i 98 M P i 65a S P i 101 N P kl 110 K P k fi 61c F P k hi 144b T. Poulsen94, J. Poveda35, M.E. Pozo Astigarraga35, P. Pralavorio99, A. Pranko18, S. Prell76, g g D. Price98, M. Primavera65a, S. Prince101, N. Proklova110, K. Prokofiev61c, F. Prokoshin144 S. Protopopescu29, J. Proudfoot6, M. Przybycien81a, A. Puri171, P. Puzo128, J. Qian103, V. Radeka29, V. Radescu131, S.K. Radhakrishnan152, P. Radloff127, P. Rados102, F. Ragusa66a,66b, G. Rahal95, J.A. Raine98, S. Rajagopalan29, C. Rangel-Smith170, T. Rashid12 S. Raspopov5, M.G. Ratti66a,66b, D.M. Rauch44, F. Rauscher112, S. Rave97, I. Ravinovich178, J.H. Rawling98, M. Raymond35, A.L. Read130, N.P. Readioff56, M. Reale65a,65b, g , y , , , , D.M. Rebuzzi68a,68b, A. Redelbach175, G. Redlinger29, R. Reece143, R.G. Reed32c, K. Reeves42, 7 d 7 D.M. Rebuzzi68a,68b, A. Redelbach175, G. Redlinger29, R. Reece143, R.G. Reed3 L. Rehnisch19, J. Reichert133, A. Reiss97, C. Rembser35, H. Ren15d, M. Rescigno70a, S. Resconi66a, E.D. Resseguie133, S. Rettie173, E. Reynolds21, O.L. Rezanova120b,120a, S. Resconi , E.D. Resseguie , S. Rettie , E. Reynolds , O.L. Rezanova , P Reznicek139 R Rezvani107 R Richter113 S Richter92 E Richter Was81b O Ricken2 P. Reznicek139, R. Rezvani107, R. Richter113, S. Richter92, E. Richter-Was81b, O. Ricken24, M. Ridel132, P. Rieck113, C.J. Riegel180, J. Rieger51, O. Rifki124, M. Rijssenbeek152, A. Rimoldi68a,68b, M. Rimoldi20, L. Rinaldi23b, G. Ripellino151, B. Risti´c35, E. Ritsch35, I. Riu14, f F. Rizatdinova125, E. Rizvi90, C. Rizzi14, R.T. Roberts98, S.H. Robertson101,af, A. Robichaud-Veronneau101, D. Robinson31, J.E.M. Robinson44, A. Robson55, E. Rocco97, C. Roda69a,69b, Y. Rodina99,aa, S. Rodriguez Bosca172, A. Rodriguez Perez14, D. Rodriguez Rodriguez172, S. Roe35, C.S. Rogan57, O. Røhne130, J. Roloff57, A. Romaniouk11 M Romano23b,23a S M Romano Sae 37 E Romero Adam172 N Rompotis88 M Ron ani50 D. Rodriguez Rodriguez172, S. Roe35, C.S. Rogan57, O. Røhne130, J. Roloff57, A. Romaniouk110, Rod gue Rod gue , S Roe , C S Roga , O Rø e , J Ro o , Ro a ou , M. Romano23b,23a, S.M. Romano Saez37, E. Romero Adam172, N. Rompotis88, M. Ronzani50, L. Roos132, S. Rosati70a, K. Rosbach50, P. Rose143, N-A. Rosien51, E. Rossi67a,67b, L.P. Rossi53b, J.H.N. Rosten31, R. Rosten145, M. Rotaru27b, J. Rothberg145, D. Rousseau128, A. Rozanov99, Y. Rozen158, X. Ruan32c, F. Rubbo150, F. R¨uhr50, A. Ruiz-Martinez33, Z. Rurikova50, N.A. Rusakovich77, H.L. Russell101, J.P. Rutherfoord7, N. Ruthmann35, Y.F. Ryabov134, M. Rybar171, G. Rybkin128, S. Ryu6, A. The ATLAS collaboration Newman21, T.Y. Ng61c, T. Nguyen Manh107, R.B. Nickerson131, R. Nicolaidou142, J. Nielsen143, V. Nikolaenko140,an, I. Nikolic-Audit132, K. Nikolopoulos21, J.K T. Nobe161, Y. Noguchi83, M. Nomachi129, I. Nomidis33, M g j g K. Ntekas169, E. Nurse92, F. Nuti102, F.G. Oakham33,aw, H. Oberlack113, T. Obermann24 g j g K. Ntekas169, E. Nurse92, F. Nuti102, F.G. Oakham33,aw, H. O – 36 – M. Ouchrif34d, F. Ould-Saada130, A. Ouraou142, K.P. Oussoren118, Q. Ouyang15a, M. Owen55, M. Ouchrif , F. Ould Saada , A. Ouraou , K.P. Oussoren , Q. Ouyang , M. Owen , R E Owen21 V E Ozcan12c N Ozturk8 K Pachal149 A Pacheco Pages14 y g R.E. Owen21, V.E. Ozcan12c, N. Ozturk8, K. Pachal149, A. Pacheco Pages14, , , , , g , L. Pacheco Rodriguez142, C. Padilla Aranda14, S. Pagan Griso18, M. Paganini181, F. Paige2 g , , g , g G. Palacino63, S. Palazzo40b,40a, S. Palestini35, M. Palka81b, D. Pallin37, E.St. Panagiotopoulou10, I. Panagoulias10, C.E. Pandini132, J.G. Panduro Vazquez91, P S. Panitkin29, D. Pantea27b, L. Paolozzi52, T.D. Papadopoulou10, K. Papageorgiou9,k, A. Paramonov6, D. Paredes Hernandez181, A.J. Parker87, K.A. Parker44, M.A. Parker31, F. Parodi53b,53a, J.A. Parsons38, U. Parzefall50, V.R. Pascuzzi165, J.M.P. Pasner143, B. Pearson113, S. Pedraza Lopez172, R. Pedro136a,136b, S.V. Peleganchuk120b,120a, O. Penc137 C. Peng15d, H. Peng58a, J. Penwell63, B.S. Peralva78a, M.M. Perego142, D.V. Perepelitsa29, JHEP10(2018)047 JHEP10(2018)047 F. Peri19, L. Perini66a,66b, H. Pernegger35, S. Perrella67a,67b, R. Peschke44, V.D. Peshekhonov77,*, K. Peters44, R.F.Y. Peters98, B.A. Petersen35, T.C. Pe , , , , , A. Petridis1, C. Petridou160, P. Petroff128, E. Petrolo70a, M. Petrov131, F. Petrucci72a,72b Petridis1, C. Petridou160, P. Petroff128, E. Petrolo70a, M. Petrov131, F. Petrucci72a,72b, E. Pettersson100, A. Peyaud142, R. Pezoa144b, F.H. Phillips104, P.W. Phillips141, N.E. Pettersson100, A. Peyaud142, R. Pezoa144b, F.H. Phillips104, P.W. Phillips141, Piacquadio152, E. Pianori176, A. Picazio100, E. Piccaro90, M.A. Pickering131, R. Piegaia30, G. Piacquadio152, E. Pianori176, A. Picazio100, E. Piccaro90, M.A. Pickering131, R. Piegaia3 J.E. Pilcher36, A.D. Pilkington98, A.W.J. Pin98, M. Pinamonti71a,71b, J.L. Pinfold3, H. Pirumov44, M. Pitt178, L. Plazak28a, M.-A. Pleier29, V. Pleskot97, E. Plotnikova77, D. Pluth76, P. Podberezko120b,120a, R. Poettgen43a,43b, R. Poggi68a,68b, L. Poggioli128, D. Pohl2 G. Polesello68a, A. Poley44, A. Policicchio40b,40a, R. Polifka35, A. Polini23b, C.S. Pollard55, V. Polychronakos29, K. Pomm`es35, D. Ponomarenko110, L. Pontecorvo70a, G.A. Popeneciu27 A. Poppleton35, S. Pospisil138, K. Potamianos18, I.N. Potrap77, C.J. Potter31, G. Poulard35, A. Poppleton , S. Pospisil , K. Potamianos , I.N. Potrap , C.J. Potter , G. Poulard , T. Poulsen94, J. Poveda35, M.E. The ATLAS collaboration Schopf24, M. Schott97, J.F.P. Schouwenberg117, J. Schovancova35, S. Schramm52, N. Schuh97, A. Schulte97, M.J. Schultens24, H-C. Schultz-Coulon59a, H. Schulz19, M. Schumacher50, B.A. Schumm143, Ph. Schune142, A. Schwartzman150, T.A. Schwarz103, H. Schweiger98, Ph. Schwemling142, R. Schwienhorst104, A. Sciandra24, G. Sciolla26, M. Scornajenghi40b,40a, F. Scuri69a, F. Scutti102, J. Searcy103, P. Seema24, S.C. Seidel116, A. Seiden143, J.M. Seixas78b, G. Sekhniaidze67a, K. Sekhon103, S.J. Sekula41, N. Semprini-Cesari23b,23a, S. Senkin37, C. Serfon130, L. Serin128, L. Serkin64a,64 M. Sessa72a,72b, R. Seuster174, H. Severini124, F. Sforza35, A. Sfyrla52, E. Shabalina51, N.W. Shaikh43a,43b, L.Y. Shan15a, R. Shang171, J.T. Shank25, M. Shapiro18, P.B. Shatalov1 , , g , , p , , K. Shaw64a,64b, S.M. Shaw98, A. Shcherbakova43a,43b, C.Y. Shehu153, Y. Shen124, N. Sherafati3 P. Sherwood92, L. Shi155,as, S. Shimizu80, C.O. Shimmin181, M. Shimojima114, I.P.J. Shipsey131, Shiyakova77, J. Shlomi178, A. Shmeleva108, D. Sh S. Shojaii66a, D.R. Shope124, S. Shrestha122, E. Shulga110, M.A. Shupe7, P. Sicho137, A.M. Sickles171, P.E. Sidebo151, E. Sideras Haddad32c, O. Sidiropoulou175, A. Sidoti23b,23a F. Siegert46, Dj. Sijacki16, J. Silva136a,136d, S.B. Silverstein43a, V. Simak138, L. Simic16, S. Simion128, E. Simioni97, B. Simmons92, M. Simon97, P. Sinervo165, N.B. Sinev127, M. Sioli23b,23a, G. Siragusa175, I. Siral103, S.Yu. Sivoklokov111, J. Sj¨olin43a,43b, M.B. S P. Skubic124, M. Slater21, T. Slavicek138, M. Slawinska82, K. Sliwa168, R. Slovak139, V. Smakhtin178, B.H. Smart5, J. Smiesko28a, N. Smirnov110, S.Yu. Smirnov110, Y. Sm H. Smart5, J. Smiesko28a, N. Smirnov110, S.Yu. L.N. Smirnova111, O. Smirnova94, J.W. Smith51, M.N.K. Smith38, R.W. Smith38, M. Smizanska87, K. Smolek138, A.A. Snesarev108, I.M. Snyder127, S. Snyder29, R. Sobie174,af, F. Socher46, A. Soffer159, A. Søgaard48, D.A. Soh155, G. Sokhrannyi89, C.A. Solans Sanchez35, M. Solar138, E.Yu. Soldatov110, U. Soldevila172, A.A. Solodkov140, A. Soloshenko77, O.V. Solovyanov140, V. Solovyev134, P. Sommer50, H. Son168, A. Sopczak138, D. Sosa59b, C.L. Sotiropoulou69a,69b, R. Soualah64a,64c,j, A.M. Soukharev120b,120a, D. South44, B.C. Sowden91, S. Spagnolo65a,65b, M. Spalla69a,69b, M. Spangenberg176, F. Span`o91 Sperlich19, F. Spettel113, T.M. Spieker59a, R. Spighi23b, G. Spigo35, L.A. Spiller102, Spousta139, R.D. St. Denis55,*, A. Stabile66a,66b, R. Stamen59a, S. Stamm19, E. Stanecka82, p , p , p , p g , p g , p , M. Spousta139, R.D. St. Denis55,*, A. Stabile66a,66b, R. Stamen59a, S. Stamm19, E. Stanecka82, R.W. Stanek6, C. Stanescu72a, M.M. Stanitzki44, B. Stapf118, S. Stapnes130, E.A. Starchenko1 G.H. Stark36, J. Stark56, S.H Stark39, P. Staroba137, P. Starovoitov59a, S. St¨arz35, R. Staszewski82, P. Steinberg29, B. Stelzer149, H.J. Stelzer35, O. Stelzer-Chilton166a, H. S G.A. Stewart55, M.C. Stockton127, M. Stoebe101, G. Stoicea27b, P. Stolte51, S. Stonjek113, A.R. Stradling8, A. The ATLAS collaboration Ryzhov140, G.F. Rzehorz51, A.F. Saavedra154, G. Sabato118, S. Sacerdoti30, H.F-W. Sadrozinski143, R. Sadykov77, F. Safai Tehrani70a, P. Saha119, M. Sahinsoy59a, M. Saimpert44, M. Saito161, T. Saito161, H. Sakamoto161, M. Romano23b,23a, S.M. Romano Saez37, E. Romero Adam172, N. Rompotis88, M. Ronzani50, L. Roos132, S. Rosati70a, K. Rosbach50, P. Rose143, N-A. Rosien51, E. Rossi67a,67b, L.P. Rossi53b, J.H.N. Rosten31, R. Rosten145, M. Rotaru27b, J. Rothberg145, D. Rousseau128, A. Rozanov99, Y Rozen158 X Ruan32c F Rubbo150 F R¨uhr50 A Ruiz-Martinez33 Z Rurikova50 , , , p , , L. Roos132, S. Rosati70a, K. Rosbach50, P. Rose143, N-A. Rosien51, E. Rossi67a,67b, L.P. Rossi53b , , , , , , J.H.N. Rosten31, R. Rosten145, M. Rotaru27b, J. Rothberg145, D. Rousseau128, A. Rozanov99, , , , g , , , Y. Rozen158, X. Ruan32c, F. Rubbo150, F. R¨uhr50, A. Ruiz-Martinez33, Z. Rurikova50, N A Rusakovich77 H L Russell101 J P Rutherfoord7 N Ruthmann35 Y F Ryabov134 , , , g , , Y. Rozen158, X. Ruan32c, F. Rubbo150, F. R¨uhr50, A. Ruiz-Martinez33, Z. Rurikova50, M. Rybar171, G. Rybkin128, S. Ryu6, A. Ryzhov140, G.F. Rzehorz51, A.F. Saavedra15 G. Sabato118, S. Sacerdoti30, H.F-W. Sadrozinski143, R. Sadykov77, F. Safai Tehrani70 P. Saha119, M. Sahinsoy59a, M. Saimpert44, M. Saito161, T. Saito161, H. Sakamoto161 – 37 – Y. Sakurai177, G. Salamanna72a,72b, J.E. Salazar Loyola144b, D. Salek118, P.H. Sales De Bruin17 , , y , , , D. Salihagic113, A. Salnikov150, J. Salt172, D. Salvatore40b,40a, F. Salvatore153, , , y , , D. Salihagic113, A. Salnikov150, J. Salt172, D. Salvatore40b,40a, F. Salvatore153, g , , , , , A. Salvucci61a,61b,61c, A. Salzburger35, D. Sammel50, D. Sampsonidis160, D. Sampsonidou16 J. S´anchez172, V. Sanchez Martinez172, A. Sanchez Pineda64a,64c, H. Sandaker130, R.L. Sandbach90, C.O. Sander44, M. Sandhoff180, C. Sandoval22, D.P.C. Sankey141, M. Sannino53b,53a, Y. Sano115, A. Sansoni49, C. Santoni37, H. Santos136a, I. Santoyo Castillo153 A. Sapronov77, J.G. Saraiva136a,136d, B. Sarrazin24, O. Sasaki79, K. Sato167, E. Sauvan5, p , , , , , , G. Savage91, P. Savard165,aw, N. Savic113, C. Sawyer141, L. Sawyer93,ak, J. Saxon36, C. Sbarra23b, P. Schacht113, B.M. Schachtner112, D. Schaefer35, L. Schaefer133, R. Schaefer44, J. Schaeffer97, S. Schaepe24, S. Schaetzel59b, U. Sch¨afer97, A.C. Schaffer128, D. Schaile112, R.D. Schamberger1 JHEP10(2018)047 JHEP10(2018)047 V.A. Schegelsky134, D. Scheirich139, M. Schernau169, C. Schiavi53b,53a, S. Schier143, L.K. Schildgen24, C. Schillo50, M. Schioppa40b,40a, S. Schlenker35, K.R. Schmidt-Sommerfeld113 g , , pp , , K. Schmieden35, C. Schmitt97, S. Schmitt44, S. Schmitz97, U. Schnoor50, L. Schoeffel142, A. Schoening59b, B.D. Schoenrock104, E. The ATLAS collaboration Straessner46, M.E. Stramaglia20, J. Strandberg151, S. Strandberg43a,43b M. Strauss124, P. Strizenec28b, R. Str¨ohmer175, D.M. Strom127, R. Stroynowski41, A. Strubig48 – 38 – S.A. Stucci29, B. Stugu17, N.A. Styles44, D. Su150, J. Su135, S. Suchek59a, Y. Sugaya129, , g , y , , , , g y , M. Suk138, V.V. Sulin108, D.M.S. Sultan73a,73b, S. Sultansoy4c, T. Sumida83, S. Sun57, X. Su , g , y , , , , g y , M. Suk138, V.V. Sulin108, D.M.S. Sultan73a,73b, S. Sultansoy4c, T. Sumida83, S. Sun57, X. Sun3 K. Suruliz153, C.J.E. Suster154, M.R. Sutton153, S. Suzuki79, M. Svatos137, M. Swiatlowski36, S.P. Swift2, I. Sykora28a, T. Sykora139, D. Ta50, K. Tackmann44,ab, J. Taenzer159, A. Taffard16 R. Tafirout166a, N. Taiblum159, H. Takai29, R. Takashima84, E.H. Takasugi113, T. Takeshita147 Y. Takubo79, M. Talby99, A.A. Talyshev120b,120a, J. Tanaka161, M. Tanaka163, R. Tanak S. Tanaka79, R. Tanioka80, B.B. Tannenwald122, S. Tapia Araya144b, S. Tapprogge97, S. Tarem158, G.F. Tartarelli66a, P. Tas139, M. Tasevsky137, T. Tashiro83, E. Tassi40b,40a, W. Taylor166b, P. Teixeira-Dias91, D. Temple149, H. Ten Kate35, P.K. Teng155, J.J. Teoh129, F. Tepel180, S. Terada79, K. Terashi161, J. Terron96, S. Terzo14, M. Testa49, R.J. Teuscher165,af JHEP10(2018)047 JHEP10(2018)047 T. Theveneaux-Pelzer99, F. Thiele39, J.P. Thomas21, J. Thomas-Wilsker91, A.S. Thompson55, P.D. Thompson21, L.A. Thomsen181, E. Thomson133, M.J. Tibbetts18, R.E. Ticse Torres99, V.O. Tikhomirov108,ao, Yu.A. Tikhonov120b,120a, S. Timoshenko110, P. Tipton181, S. Tisserant9 K. Todome163, S. Todorova-Nova5, S. Todt46, J. Tojo85, S. Tok´ar28a, K. Tokushuku79, 122, L. Tomlinson98, M. Tomoto115, L. Tompkins150,r, K. Toms116, B. Tong57, E. Tolley122, L. Tomlinson98, M. Tomoto115, L. Tompkins150,r, K. Toms116, B. Tong57, E. Tolley122, L. Tomlinson98, M. Tomoto115, L. Tompkins150,r, K. Toms116, B. Tong57, 50 127 149 145 99 d 9 P. Tornambe50, E. Torrence127, H. Torres149, E. Torr´o Pastor145, J. Toth99,ad, F. Touchard D.R. Tovey146, C.J. Treado121, T. Trefzger175, F. Tresoldi153, A. Tricoli29, I.M. Trigger166a, S. Trincaz-Duvoid132, M.F. Tripiana14, W. Trischuk165, B. Trocm´e56, A. Trofymov44, C. Troncon66a, M. Trottier-McDonald18, M. Trovatelli174, L. Truong32b, M. Trzebinski82, A. Trzupek82, K.W. Tsang61a, J.C-L. Tseng131, P.V. Tsiareshka105, G. Tsipolitis10, N. Tsirintanis9, S. Tsiskaridze14, V. Tsiskaridze50, E.G. Tskhadadze157a, K.M. Tsui61a, I.I. Tsukerman109, V. Tsulaia18, S. Tsuno79, D. Tsybychev152, Y. Tu61b, A. Tudorache27b, I.I. Tsukerman109, V. Tsulaia18, S. Tsuno79, D. Tsybychev152, Y. Tu61b, A. Tudorache27b, V. Tudorache27b, T.T. Tulbure27a, A.N. Tuna57, S.A. Tupputi23b,23a, S. Turchikhin77, D. Turgeman178, I. Turk Cakir4b,v, R. Turra66a, P.M. Tuts38, G. Ucchielli23b,23a, I. Ueda7 D. Turgeman178, I. Turk Cakir4b,v, R. Turra66a, P.M. Tuts38, G. Ucchielli23b,23a, I. Ueda79 Y. The ATLAS collaboration Weidberg131, B. Weinert63, J. Weingarten51, M. Weirich97, C. Weiser50, H. Weits118, , , , , , , A.R. Weidberg131, B. Weinert63, J. Weingarten51, M. Weirich97, C. Weiser50, H. Weits118 g g P.S. Wells35, T. Wenaus29, T. Wengler35, S. Wenig35, N. Wermes24, M.D. Werner76, P. W S. Wells35, T. Wenaus29, T. Wengler35, S. Wenig35, N. Wermes24, M.D. Werner76, P. Werner35, Wessels59a, K. Whalen127, N.L. Whallon145, A.M. Wharton87, A.S. White103, A. White8, M. Wessels59a, K. Whalen127, N.L. Whallon145, A.M. Wharton87, A.S. White103, A. White J. White1, R. White144b, D. Whiteson169, B.W. Whitmore87, F.J. Wickens141, M.J. White1, R. White144b, D. Whiteson169, B.W. Whitmore87, F.J. Wickens141, g F. Wilk98, H.G. Wilkens35, H.H. Williams133, S. Williams31, C. Willis104, S. The ATLAS collaboration Watts145, S. Watts98, B.M. Waugh92, A.F. Webb11, S. Webb97, M.S. Weber20, S.A. Weber33, S.W. Weber175, J.S. Webster6, A.R. Weidberg131, B. Weinert63, J. Weingarten51, M. Weirich97, C. Weiser50, H. Weits118, P.S. Wells35, T. Wenaus29, T. Wengler35, S. Wenig35, N. Wermes24, M.D. Werner76, P. Werner35, M. Wessels59a, K. Whalen127, N.L. Whallon145, A.M. Wharton87, A.S. White103, A. White8, M.J. White1, R. White144b, D. Whiteson169, B.W. Whitmore87, F.J. Wickens141, W. Wiedenmann179, M. Wielers141, C. Wiglesworth39, L.A.M. Wiik-Fuchs50, A. Wildauer113, F. Wilk98, H.G. Wilkens35, H.H. Williams133, S. Williams31, C. Willis104, S. Willocq100, J.A. Wilson21, I. Wingerter-Seez5, E. Winkels153, F. Winklmeier127, O.J. Winston153, B.T. Winter24, M. Wittgen150, M. Wobisch93, T.M.H. Wolf118, R. Wolff99, M.W. Wolter82, H. Wolters136a,136c, V.W.S. Wong173, S.D. Worm21, B.K. Wosiek82, J. Wotschack35, K.W. Wo´zniak82, M. Wu36, S.L. Wu179, X. Wu52, Y. Wu103, T.R. Wyatt98, B.M. Wynne48, S. Xella39, Z. Xi103, L. Xia15b, D. Xu15a, L. Xu29, T. Xu142, B. Yabsley154, S. Yacoob32a, D. Yamaguchi163, Y. Yamaguchi129, A. Yamamoto79, S. Yamamoto161, T. Yamanaka161, M. Yamatani161, K. Yamauchi115, Y. Yamazaki80, Z. Yan25, H.J. Yang58c,58d, H.T. Yang18, Y. Yang155, Z. Yang17, W-M. Yao18, Y.C. Yap132, Y. Yasu79, E. Yatsenko5, K.H. Yau Wong24, J. Ye41, S. Ye29, I. Yeletskikh77, E. Yigitbasi25, E. Yildirim97, K. Yorita177, K. Yoshihara133, C.J.S. Young35, C. Young150, J. Yu8, J. Yu76, S.P.Y. Yuen24, I. Yusuff31,a, B. Zabinski82, G. Zacharis10, R. Zaidan14, A.M. Zaitsev140,an, N. Zakharchuk44, J. Zalieckas17, A. Zaman152, S. Zambito57, D. Zanzi102, C. Zeitnitz180, G. Zemaityte131, A. Zemla81a, J.C. Zeng171, Q. Zeng150, O. Zenin140, D. Zerwas128, D. Zhang103, F. Zhang179, G. Zhang58a,ag, H. Zhang15c, J. Zhang6, L. Zhang50, L. Zhang58a, M. Zhang171, P. Zhang15c, R. Zhang58a,e, R. Zhang24, X. Zhang58b, Y. Zhang15d, Z. Zhang128, X. Zhao41, Y. Zhao58b,128,aj, Z. Zhao58a, A. Zhemchugov77, B. Zhou103, C. Zhou179, L. Zhou41, M.S. Zhou15d, M. Zhou152, N. Zhou15b, C.G. Zhu58b, H. Zhu15a, J. Zhu103, Y. Zhu58a, X. Zhuang15a, K. Zhukov108, A. Zibell175, D. Zieminska63, N.I. Zimine77, C. Zimmermann97, S. Zimmermann50, Z. Zinonos113, M. Zinser97, M. Ziolkowski148, G. Zobernig179, A. Zoccoli23b,23a, R. Zou36, M. Zur Nedden19, L. Zwalinski35 P.M. Watkins21, A.T. Watson21, M.F. Watson21, G. Watts145, S. Watts98, B.M. Waugh9 M. Watkins21, A.T. Watson21, M.F. Watson21, G. Watts145, S. Watts98, B.M. Waugh92, , , , , , g , F. Webb11, S. Webb97, M.S. Weber20, S.A. Weber33, S.W. Weber175, J.S. Webster6, , , , , , g A.F. Webb11, S. Webb97, M.S. Weber20, S.A. Weber33, S.W. Weber175, J.S. Webster6, , , , , , , R. The ATLAS collaboration Unno79, C. Unverdorben112, J. Urban28b, P. Urquijo102, P. Urrejola97, G. Usai8, J. Usui79, Y. Unno79, C. Unverdorben112, J. Urban28b, P. Urquijo102, P. Urrejola97, G. Usai8, J. Usui79, L. Vacavant99, V. Vacek138, B. Vachon101, K.O.H. Vadla130, A. Vaidya92, C. Valderanis112, E. Valdes Santurio43a,43b, S. Valentinetti23b,23a, A. Valero172, L. Val´ery14, S. Valkar139, A. Vallier5, J.A. Valls Ferrer172, W. Van Den Wollenberg118, H. Van der Graaf118, P. Van Gemmeren6, J. Van Nieuwkoop149, I. Van Vulpen118, M.C. van Woerden118, dia71a,71b, W. Vandelli35, A. Vaniachine164, P. Vankov118, G. Vardanyan182, R. Vari70a, M. Vanadia71a,71b, W. Vandelli35, A. Vaniachine164, P. Vankov118, G. Vardanyan182, rnes7, C. Varni53b,53a, T. Varol41, D. Varouchas128, A. Vartapetian8, K.E. Varvell154, E.W. Varnes7, C. Varni53b,53a, T. Varol41, D. Varouchas128, A. Vartapetian8, K.E. Varvell154, , , , , p , , G.A. Vasquez144b, J.G. Vasquez181, F. Vazeille37, T. Vazquez Schroeder101, J. Veatch51, G.A. Vasquez144b, J.G. Vasquez181, F. Vazeille37, T. Vazquez Schroeder101, J. Veatch51, q , q , , q , , V. Veeraraghavan7, L.M. Veloce165, F. Veloso136a,136c, S. Veneziano70a, A. Ventura65a,65b, M. Venturi174, N. Venturi35, A. Venturini26, V. Vercesi68a, M. Verducci72a,72b, W. Verkerke118, A.T. Vermeulen118, J.C. Vermeulen118, M.C. Vetterli149,aw, N. Viaux Maira144b, O. Viazlo94, I. Vichou171,*, T. Vickey146, O.E. Vickey Boeriu146, G.H.A. Viehhauser131, S. Viel18, L. Vigani131, M. Villa23b,23a, M. Villaplana Perez66a,66b, E. Vilucchi49, M.G. Vincter33, V.B. Vinogradov77, A. Vishwakarma44, C. Vittori23b,23a, I. Vivarelli153, S. Vlachos10, g , , p , , , K. Vorobev110, M. Vos172, R. Voss35, J.H. Vossebeld88, N. Vranjes16, M. Vranjes Milosavljevic1 V. Vrba138, M. Vreeswijk118, T. ˇSfiligoj89, R. Vuillermet35, I. Vukotic36, T. ˇZeniˇs28a, V. Vrba138, M. Vreeswijk118, T. ˇSfiligoj89, R. Vuillermet35, I. Vukotic36, T. ˇZeniˇs28a, L. ˇZivkovi´c16, P. Wagner24, W. Wagner180, J. Wagner-Kuhr112, H. Wahlberg86, S. Wahrmund46, J. Wakabayashi115, J. Walder87, R. Walker112, W. Walkowiak148, V. Wallangen43a,43b, C. Wang15c, C. Wang58b,e, F. Wang179, H. Wang18, H. Wang3, J. Wang154, J. Wang44, Q. Wang124, R. Wang6, S.M. Wang155, T. Wang38, W. Wang155,p, W.X. Wang58a,ag, Z. Wang58c, C. Wanotayaroj127, A. Warburton101, C.P. Ward31, D.R. Wardrope92, A. Washbrook48, L. ˇZivkovi´c16, P. Wagner24, W. Wagner180, J. Wagner-Kuhr112, H. Wahlberg86, S. Wahrmund J. Wakabayashi115, J. Walder87, R. Walker112, W. Walkowiak148, V. Wallangen43a,43b, C. Wang15c, C. Wang58b,e, F. Wang179, H. Wang18, H. Wang3, J. Wang154, J. Wang44, g , g , g , g , g , g , g , Q. Wang124, R. Wang6, S.M. Wang155, T. Wang38, W. Wang155,p, W.X. Wang58a,ag, Z. Wa – 39 – P.M. Watkins21, A.T. Watson21, M.F. Watson21, G. 1 Department of Physics, University of Adelaide, Adelaide, Australia The ATLAS collaboration Willocq100, JHEP10(2018)047 JHEP10(2018)047 1 Department of Physics, University of Adelaide, Adelaide, Australi 3 Department of Physics, University of Alberta, Edmonton AB, Canada 4 (a)Department of Physics, Ankara University, Ankara; (b)Istanbul Aydin University, Istanbul; (c)Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 4 (a)Department of Physics, Ankara University, Ankara; (b)Istanbul Aydin University, Istanbul; (c) f O f A (c)Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5 LAPP, Universit´e Grenoble Alpes, Universit´e Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of America 7 Department of Physics, University of Arizona, Tucson AZ, United Sta 9 Physics Department, National and Kapodistrian University of Athens, 9 Physics Department, National and Kapodistrian University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Department of Physics, University of Texas at Austin, Austin TX, United States of America 12 (a)Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul; (b)Istanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul; (c)Department of Physics, Bogazici University, Istanbul; (d)Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14 Institut de F´ısica d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, Barcelona, Spain 15 (a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b)Physics Department, Tsinghua University, Beijing; (c)Department of Physics, Nanjing University, Nanjing; (d)University of Chinese Academy of Science (UCAS), Beijing, China 15 (a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b)Physics Department, Tsinghua University, Beijing; (c)Department of Physics, Nanjing University, Nanjing; (d)University of Chinese Academy of Science (UCAS), Beijing, China – 40 – 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 17 Department for Physics and Technology, University of Bergen, Bergen, Norway 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 19 Institut f¨ur Physik, Humboldt Universit¨at zu Berlin, Berlin, Germany Institut f¨ur Physik, Humboldt Universit¨at zu Berlin 20 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 21 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 22 Centro de Investigaci´ones, Universidad Antonio Nari˜no, Bogota, Colombia ( ) (b) 23 (a)Dipartimento di Fisica e Astronomia, Universit`a di Bologna, Bologna; (b)INFN Sezione di Bologna, Italy 24 Physikalisches Institut, Universit¨at Bonn, Bonn, Germany JHEP10(2018)047 JHEP10(2018)047 25 Department of Physics, Boston University, Boston MA, United States of America 27 (a)Transilvania University of Brasov, Brasov; (b)Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest; (c)Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi; (d)National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca; (e)University Politehnica Bucharest, Bucharest; (f)West University in Timisoara, Timisoara, Romania 27 (a)Transilvania University of Brasov, Brasov; (b)Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest; (c)Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi; (d)National Institute for Research and Development of Isotopic and Molecular Technologies, Ph i D t t Cl j N (e)U i it P lit h i B h t B h t (f)W t Iasi; (d)National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca; (e)University Politehnica Bucharest, Bucharest; (f)West University in Timisoara, Timisoara, Romania 28 (a)Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava; (b)Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 29 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 30 Departamento de F´ısica, Universidad de Buenos Aires, Buenos Aires, Argentina 31 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 32 (a)Department of Physics, University of Cape Town, Cape Town; (b)Department of Mechanical Engineering Science, University of Johannesburg, Johannesburg; (c)School of Physics, University of the Witwatersrand, Johannesburg, South Africa 32 (a)Department of Physics, University of Cape Town, Cape Town; (b)Department of Mechanical Engineering Science, University of Johannesburg, Johannesburg; (c)School of Physics, University o the Witwatersrand, Johannesburg, South Africa the Witwatersrand, Johannesburg, South Africa 33 Department of Physics, Carleton University, Ottawa ON, Canada 33 Department of Physics, Carleton University, Otta 34 (a)Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca; (b)Centre National de l’Energie des Sciences Techniques Nucleaires (CNESTEN), Rabat; (c)Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech; (d)Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e)Facult´e des sciences, Universit´e Mohammed V, Rabat, Morocco 34 (a)Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca; (b)Centre National de l’Energie des Sciences Techniques LPHEA-Marrakech; (d)Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; ( ) 35 CERN, Geneva, Switzerland 36 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 36 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America Nevis Laboratory, Columbia University, Irvington NY, United States of America 38 Nevis Laboratory, Columbia University, Irvington NY, United States of America 39 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark ( ) ( ) 40 (a)Dipartimento di Fisica, Universit`a della Calabria, Rende; (b)INFN Gruppo Collegato di Cosenz Laboratori Nazionali di Frascati, Italy 41 Physics Department, Southern Methodist University, Dallas TX, United States of America 42 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 43 ( ) (b) 44 Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 45 Lehrstuhl f¨ur Experimentelle Physik IV, Technische Universit¨at Dortmund, Dor 45 Lehrstuhl fur Experimentelle Physik IV, Technische Universitat Dortmund, Dortmund, Germany 46 Institut f¨ur Kern- und Teilchenphysik, Technische Universit¨at Dresden, Dresden, Germany 46 Institut f¨ur Kern- und Teilchenphysik, Technische Universit¨at Dresden, Dresden, Germany 47 Department of Physics, Duke University, Durham NC, United States of America 48 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, Un 48 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy SUPA School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 50 Physikalisches Institut, Albert-Ludwigs-Universit¨at Freiburg, Freiburg, Germany 51 II. The ATLAS collaboration Physikalisches Institut, Georg-August-Universit¨at G¨ottingen, G¨ottingen, Germany – 41 – 52 D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland 52 D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland 53 ( ) (b) 53 (a)Dipartimento di Fisica, Universit`a di Genova, Genova; (b)INFN Sezione di Genova, Italy 54 II. The ATLAS collaboration Physikalisches Institut, Justus-Liebig-Universit¨at Giessen, Giessen, Germany 55 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56 LPSC, Universit´e Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge MA, United States of America 58 (a)Department of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Hefei; (b)Institute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao; (c)School of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai; (d)Tsung-Dao Lee Institute, Shanghai, China Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao; (c)School of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai; (d)Tsung-Dao Lee Institute, Shanghai, China JHEP10(2018)047 JHEP10(2018)047 59 (a)Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 59 (a)Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)Ph ik li h I tit t R ht K l U i it¨t H id lb H id lb G 59 (a)Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; KirchhoffInstitut fur Physik, Ruprecht Karls Universitat Heidelberg, Heidelberg; (b)Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 60 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Jap 61 (a)Department of Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b)Department of Physics, University of Hong Kong, Hong Kong; (c)Department of Physics and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay Kowloon, Hong Kong, China 62 Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 64 (a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b)ICTP, Trieste; (c)Dipartim di Chimica Fisica e Ambiente Universit`a di Udine Udine Italy 64 (a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b)ICTP, Trieste; (c)Dipartimento 64 (a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy 65 (a)INFN Sezione di Lecce; (b)Dipartimento di Matematica e Fisica, Universit`a del Salento, Lecce, Italy 66 (a)INFN Sezione di Milano; (b)Dipartimento di Fisica, Universit`a di Milano, Milano, Italy 67 (a)INFN Sezione di Napoli; (b)Dipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 68 (a)INFN Sezione di Pavia; (b)Dipartimento di Fisica, Universit`a di Pavia, Pavia, Italy N Sezione di Pisa; (b)Dipartimento di Fisica E. The ATLAS collaboration Ferm 69 (a)INFN Sezione di Pisa; (b)Dipartimento di Fisica E. The ATLAS collaboration Fermi, Universit`a di Pisa, Pisa, Italy 70 (a)INFN Sezione di Roma; (b)Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 71 (a)INFN Sezione di Roma Tor Vergata; (b)Dipartimento di Fisica, Universit`a di Roma Tor Verga Roma, Italy 72 (a)INFN Sezione di Roma Tre; (b)Dipartimento di Matematica e Fisica, Universit`a Roma Tre, Roma, Italy 73 (a)INFN-TIFPA; (b)Universit`a degli Studi di Trento, Trento, Italy 74 Institut f¨ur Astro- und Teilchenphysik, Leopold-Franzens-Universit¨at, Innsbruck, Austria versity of Iowa, Iowa City IA, United States of Ame 76 Department of Physics and Astronomy, Iowa State University, Ames IA 76 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of America epartment of Physics and Astronomy, Iowa State Un 77 Joint Institute for Nuclear Research, Dubna, Russia 78 (a)Departamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora; (b)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (c)Universidade Federal de S˜ao Jo˜ao del Rei (UFSJ), S˜ao Jo˜ao del Rei; (d)Instituto de F´ısica, Universidade de S˜ao Paulo, S˜ao Paulo, Brazil 79 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 80 Graduate School of Science, Kobe University, Kobe, Japan uate School of Science, Kobe University, Kobe, Japan 81 (a)AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, 81 (a)AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 82 Krakow; (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 82 I tit t f N l Ph i P li h A d f S i K k P l d Krakow; (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 82 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 83 Faculty of Science, Kyoto University, Kyoto, Japan 83 Faculty of Science, Kyoto University, Kyoto, Japan 84 Kyoto University of Education, Kyoto, Japan 85 Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fukuoka, Japan – 42 – 86 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 86 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 87 Physics Department, Lancaster University, Lancaster, United Kingdom Instituto de Fısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 87 Physics Department Lancaster University Lancaster United Kingdom 87 Physics Department, Lancaster University, Lancaster, United Kingdom 88 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 89 Department of Experimental Particle Physics, Joˇzef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia University of Ljubljana, Ljubljana, Slovenia 90 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom f y y, Q y y f , , g 91 Department of Physics, Royal Holloway University of London, Egham, United Kingdom 92 Department of Physics and Astronomy, University College London, London, United Kingdom 93 Louisiana Tech University, Ruston LA, United States of America 94 Fysiska institutionen, Lunds universitet, Lund, Sweden 95 Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3), Villeurbanne, France JHEP10(2018)047 JHEP10(2018)047 96 Departamento de F´ısica Teorica C-15 and CIAFF, Universidad Aut´onoma de Madrid, Madrid, Spain 97 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 97 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 98 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 98 School of Physics and Astronomy, University of Manchester, Manc 99 CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 100 Department of Physics, University of Massachusetts, Amherst MA, United States of America 100 Department of Physics, University of Massachusetts, Amherst MA, U 101 Department of Physics, McGill University, Montreal QC, Canada 102 School of Physics, University of Melbourne, Victoria, Australia 103 Department of Physics, University of Michigan, Ann Arbor MI, United States of America 4 104 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of America 105 B.I. The ATLAS collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 106 Research Institute for Nuclear Problems of Byelorussian State University Minsk Belarus 105 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 106 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 107 Group of Particle Physics, University of Montreal, Montreal QC, Canada 108 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 109 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 110 National Research Nuclear University MEPhI, Moscow, Russia Research Nuclear University MEPhI, Moscow, Russi 111 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 112 Fakult¨at f¨ur Physik, Ludwig-Maximilians-Universit¨at M¨unchen, M¨unchen, Germany 113 Max-Planck-Institut f¨ur Physik (Werner-Heisenberg-Institut), M¨unchen, Germany 114 Nagasaki Institute of Applied Science, Nagasaki, Japan 115 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 116 116 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 117 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 118 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 119 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 119 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 120 (a)Budker Institute of Nuclear Physics, SB RAS, Novosibirsk; (b)Novosibirsk State University Novosibirsk, Russia 120 (a)Budker Institute of Nuclear Physics, SB RAS, Novosibirsk; (b)Novosibirsk State University Novosibirsk, Russia 121 Department of Physics, New York University, New York NY, United States of America 122 121 Department of Physics, New York University, New York NY, United State 122 Ohio State University, Columbus OH, United States of America te University, Columbus OH, United States of Ameri 123 Faculty of Science, Okayama University, Okayama, Japan 124 Homer L. The ATLAS collaboration Petersburg, Russia 135 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America 136 (a)Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas - LIP; (b)Departamento de F´ısica, Faculdade de Ciˆencias, Universidade de Lisboa, Lisboa; (c)Departamento de F´ısica, Universidade de Coimbra, Coimbra; (d)Centro de F´ısica Nuclear da Universidade de Lisboa, Lisboa; (e)Departamento de F´ısica, Universidade do Minho, Braga; (f)Departamento de F´ısica Teorica y del Cosmos, Universidad de Granada, Granada (Spain); (g)Dep F´ısica and CEFITEC of Faculdade de Ciˆencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal JHEP10(2018)047 JHEP10(2018)047 (e)Departamento de F´ısica, Universidade do Minho, Braga; (f)Departamento de F´ısica Teorica y del Cosmos, Universidad de Granada, Granada (Spain); (g)Dep F´ısica and CEFITEC of Faculdade de Ciˆencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 137 Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 138 Czech Technical University in Prague, Prague, Czech Republic es University, Faculty of Mathematics and Physics, P 140 State Research Center Institute for High Energy Physics, NRC KI, Protvino, Russia 141 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 142 IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 143 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of America 144 (a)Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b)Departamento F´ısica, Universidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Chile 144 (a)Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b)Departamento de F´ i U i id d T´ i F d i S t M ´ V l ´ Chil 144 (a)Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b)Departamento de ´ d d ´ d S ´ l ´ Ch l sidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Ch 145 Department of Physics, University of Washington, Seattle WA, United States of America 146 Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 147 Department of Physics, Shinshu University, Nagano, Japan 148 Department Physik, Universit¨at Siegen, Siegen, Germany 149 Department of Physics, Simon Fraser University, Burnaby BC, Canada 150 SLAC National Accelerator Laboratory, Stanford CA, United States of America 151 Physics Department, Royal Institute of Technology, Stockholm, Sweden 152 152 Departments of Physics and Astronomy, Stony Brook University, Stony Brook NY, United States America 153 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 154 School of Physics, University of Sydney, Sydney, Australia 155 Institute of Physics, Academia Sinica, Taipei, Taiwan 156 Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan 157 (a)E. The ATLAS collaboration Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America 125 Department of Physics, Oklahoma State University, Stillwater OK, United States of America 126 Palack´y University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Republic 127 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America – 43 – 128 LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France 129 Graduate School of Science, Osaka University, Osaka, Japan 130 Department of Physics, University of Oslo, Oslo, Norway 131 Department of Physics, Oxford University, Oxford, United Kingdom 132 LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France 133 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America , , , / , , 133 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 133 Department of Physics, University of Pennsylvania, Philadelphia PA, Unite 134 Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNP St. The ATLAS collaboration Javakhishvili Tbilisi Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia ics Institute, Tbilisi State University, Tbilisi, Georgi 158 Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 159 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 160 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 161 International Center for Elementary Particle Physics and Department of Physics, University of Tokyo, Tokyo, Japan 162 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 163 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 164 Tomsk State University, Tomsk, Russia 164 Tomsk State University, Tomsk, Russia 165 Department of Physics, University of Toronto, Toronto ON, Canada 166 (a)TRIUMF, Vancouver BC; (b)Department of Physics and Astronomy, York University, Toronto ON, Canada – 44 – 167 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 168 Department of Physics and Astronomy, Tufts University, Medford MA, United States of America f y y f y f f 169 Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of America 170 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 171 Department of Physics, University of Illinois, Urbana IL, United States of America 172 171 Department of Physics, University of Illinois, Urbana IL, United States of America 172 Instituto de F´ısica Corpuscular (IFIC), Centro Mixto Universidad de Valencia - CSIC, Valencia, Spain 172 Instituto de F´ısica Corpuscular (IFIC), Centro Mixto Universidad de Valencia - CSIC, Valencia, Spain 173 Department of Physics, University of British Columbia, Vancouver BC, Canada 174 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada JHEP10(2018)047 JHEP10(2018)047 175 Fakult¨at f¨ur Physik und Astronomie, Julius-Maximilians-Universit¨at W¨urzburg, W¨urzburg, Germany 176 Department of Physics, University of Warwick, Coventry, United Kingdom 176 Department of Physics, University of Warwick, Coventry, United Kingdom 177 Waseda University, Tokyo, Japan 177 Waseda University, Tokyo, Japan 177 Waseda University, Tokyo, Japan 178 Department of Particle Physics, Weizmann Institute of Science, Rehovot, Israel 179 Department of Physics, University of Wisconsin, Madison WI, United States of America 180 180 Fakult¨at f¨ur Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universit¨at Wuppertal, Wuppertal, Germany 181 Department of Physics, Yale University, New Haven CT, United States of America 182 Yerevan Physics Institute, Yerevan, Armenia a Also at Department of Physics, University of Malaya, Kuala Lumpur, Malaysia b Also at Borough of Manhattan Community College, City University of New York, NY, United States of America c Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa d Also at CERN, Geneva, Switzerland e Also at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France f Also at D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland g Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain h Also at Departamento de F´ısica Teorica y del Cosmos, Universidad de Granada, Granada (Spain), Spain g Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain h Also at Departamento de F´ısica Teorica y del Cosmos, Universidad de Granada, Granada (Spain) Spain i Also at Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago, Chile j Also at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United Arab Emirates j Also at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United Arab Emirates k Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece k Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece l Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY, United States of America l Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY, United States of America m Also at Department of Physics, California State University, Fresno CA, United States of America n Also at Department of Physics, California State University, Sacramento CA, United States of America o Also at Department of Physics, King’s College London, London, United Kingdom p Also at Department of Physics, Nanjing University, Nanjing, China q Also at Department of Physics, St. The ATLAS collaboration Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbil Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 157 (a)E. Andronikashvili Institute of Physics, Iv. The ATLAS collaboration Petersburg State Polytechnical University, St. Petersburg, Russia r Also at Department of Physics, Stanford University, United States of America s Also at Department of Physics, University of Fribourg, Fribourg, Switzerland t Also at Department of Physics, University of Michigan, Ann Arbor MI, United States of America Also at Department of Physics, University of Michigan, Ann Arbor MI, United States of America u Also at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy u Also at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy u Also at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy – 45 – v Also at Giresun University, Faculty of Engineering, Giresun, Turkey w Also at Graduate School of Science, Osaka University, Osaka, Japan x Also at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania y Also at II. The ATLAS collaboration Physikalisches Institut, Georg-August-Universit¨at G¨ottingen, G¨ottingen, Germany z Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain ucio Catalana de Recerca i Estudis Avancats, ICREA aa Also at Institut de F´ısica d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, Barcelona, Spain ab Also at Institut f¨ur Experimentalphysik, Universit¨at Hamburg, Hamburg, Germany ac Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands ad Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary JHEP10(2018)047 JHEP10(2018)047 ae Also at Institute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao, China af Also at Institute of Particle Physics (IPP), Canada ag Also at Institute of Physics, Academia Sinica, Taipei, Taiwan ah Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan ai Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia aj Also at LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France ak Also at Louisiana Tech University, Ruston LA, United States of America al Also at LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France am Also at Manhattan College, New York NY, United States of America an Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia an Also at Moscow Institute of Physics and Technology State University, Dolgopru ao Also at National Research Nuclear University MEPhI, Moscow, Russia at National Research Nuclear University MEPhI, Mo ap Also at Novosibirsk State University, Novosibirsk, Russia aq Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan as Also at School of Physics, Sun Yat-sen University, Guangzhou, China ol of Physics, Sun Yat-sen University, Guangzhou, C at Also at The City College of New York, New York NY, United States of America au Also at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China au Also at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China av Also at Tomsk State University Tomsk and Moscow Institute of Physics and Technology State av Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia aw Also at TRIUMF, Vancouver BC, Canada ax Also at Universita di Napoli Parthenope, Napoli, Italy – 46 –
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The Influencing Factors of Taxpayer Compliance: Risk Preferences as a Moderating Variable
International journal of academic research in accounting, finance and management sciences
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Abstract This research was conducted to examine and determine the factors that influence taxpayer compliance with risk preference as a moderating variable. The independent variable used in this study is understanding of taxation regulations, tax sanctions, and tax rates. The population in this study was 1,613,317 taxpayers registered at SAMSAT Bekasi City. Sampling was done using the Accidental Sampling method on taxpayers registered at SAMSAT Bekasi City which produced 99 samples. The data analysis technique used is Structural Equation Modeling (SEM) using the Partial Least Square application with a significance level of 5%. The results of this study indicate that understanding of tax regulations, tax sanctions, and tax rates has a significant effect on the level of compliance of taxpayers, risk preference does not significantly influence the relationship between understanding of tax regulations on tax compliance levels and risk preference has a significant effect on the relationship between tax sanctions with taxpayer compliance level. Key words Understanding of Tax Regulations, Tax Sanctions, Tax Rates, Risk Preferences, Taxpayer Compliance Received: 26 Jan 2020 © The Authors 2019 Revised: 30 Jan 2020 Published by Human Resource Management Academic Research Society (www.hrmars.com) This article is published under the Creative Commons Attribution (CC BY 4.0) license. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this license may be seen at: http://creativecommons.org/licences/by/4.0/legalcode Accepted: 04 Feb 2020 Published Online: 07 Feb 2020 y words Understanding of Tax Regulations, Tax Sanctions, Tax Rates, Risk Preferences, Taxpayer Compliance http://dx.doi.org/10.6007/IJARAFMS/v9-i4/6845 (DOI: 10.6007/IJARAFMS/v9-i4/6845) The Influencing Factors of Taxpayer Compliance: Risk Preferences as a Moderating Variable The Influencing Factors of Taxpayer Compliance: Risk Preferences as a Moderating Variable Tresia Risda Saragih1, Khoirul Aswar2 Faculty of Economics and Business, Universitas Pembangunan Nasional Veteran Jakarta, Jl. RS Fatmawati, Pondok Labu, Jakarta, 1E-mail: tresiarisda1106@gmail.com, 2E-mail: aswar_law@yahoo.com Abstract This research was conducted to examine and determine the factors that influence taxpayer compliance with risk preference as a moderating variable. The independent variable used in this study is understanding of taxation regulations, tax sanctions, and tax rates. The population in this study was 1,613,317 taxpayers registered at SAMSAT Bekasi City. Sampling was done using the Accidental Sampling method on taxpayers registered at SAMSAT Bekasi City which produced 99 samples. The data analysis technique used is Structural Equation Modeling (SEM) using the Partial Least Square application with a significance level of 5%. The results of this study indicate that understanding of tax regulations, tax sanctions, and tax rates has a significant effect on the level of compliance of taxpayers, risk preference does not significantly influence the relationship between understanding of tax regulations on tax compliance levels and risk preference has a significant effect on the relationship between tax sanctions with taxpayer compliance level. Key words Understanding of Tax Regulations, Tax Sanctions, Tax Rates, Risk Preferences, Taxpayer Compliance Received: 26 Jan 2020 © The Authors 2019 Revised: 30 Jan 2020 Published by Human Resource Management Academic Research Society (www.hrmars.com) This article is published under the Creative Commons Attribution (CC BY 4.0) license. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this license may be seen at: http://creativecommons.org/licences/by/4.0/legalcode Accepted: 04 Feb 2020 blished Online: 07 Feb 2020 1. Introduction Tresia Risda Saragih1, Khoirul Aswar2 Tresia Risda Saragih1, Khoirul Aswar2 1,2Faculty of Economics and Business, Universitas Pembangunan Nasional Veteran Jakarta, Jl. RS Fatmawati, Pondok Labu, Jakarta, 1E-mail: tresiarisda1106@gmail.com, 2E-mail: aswar_law@yahoo.com 1,2Faculty of Economics and Business, Universitas Pembangunan Nasional Veteran Jakarta, Jl. RS Fatmawati, Pondok Labu, Jakarta, 1E-mail: tresiarisda1106@gmail.com, 2E-mail: aswar_law@yahoo.com Abstract 1. Introduction According to research results in the field the reason Bekasi residents are in arrears in tax payments is because lack of awareness of paying taxes and his financial condition (Tempo, 2019). Based on the above, the researcher wants to conduct a research related to tax compliance. One reason for the low morale of a tax is a tax rate that is too high so it is burdensome for the mandatory tax. In the study of Atawodi and Ojeka (2012) high tax rates is a factor the most important thing that causes non- compliance with taxes. Tax rate motorized vehicles are regulated in Law No. 28 of 2009 article 6, namely for first motor vehicle ownership is at least 1% (one percent) and the highest of 2% (two percent). There are a number of studies related to the effect of tax rates on taxpayer compliance such as research conducted by Cahyani and Naniek (2019), Tawas et al. (2016), Helhel and Helhel (2014), Atawodi and Ojeka (2012) where the results of the study stated that Tax rates have a positive effect on taxpayer compliance. Results of the study inversely proportional to the research conducted by Mas'ud et al. (2014) which the results of the study tax rates have a negative effect on taxpayer compliance, and research conducted by Mustofa et al. (2016), Yusro and Kriswanto (2014) which stated that the tax rate is not affect the compliance of taxpayers. In addition, an understanding of tax regulations is also a factor which affects taxpayer compliance. If taxpayers understand more about taxation regulations, the more taxpayers will understand the sanctions that will be accepted if they neglect their tax obligations (Ananda et al., 2015). There are a number of studies related to understanding regulations taxation of taxpayer compliance which is the result of the research understanding of tax regulations has a positive effect on mandatory compliance taxes (Cahyani and Naniek 2019, Mareti and Dwimulyani 2019, Asrianti, 2018, Utari and Setiawan 2019, Ananda et al. 2015, Adiasa 2013). The results of the study are inversely proportional to research by Syah and Krisidiyawati (2017), Andinata (2015), Mas'ud et al. (2014) in which the understanding of tax regulations has no effect taxpayer compliance. Another factor that is considered to affect tax compliance is tax sanctions. 1. Introduction In general, tax is compulsory contributions paid by the people to a country which useful for the benefit of government and society because tax is state revenue that will be used for the construction of public facilities. Therefore, taxes are important for state income. National development is one of the ongoing government activities continuously. State revenue in 2018 amounted to Rp1,942.3 trillion or 102.5% with the tax ratio reaches 11.5% of the increased Gross Domestic Product (GDP) by 0.8% from 2017 (Kemenkeu, 2019). Even so, the acceptance ratio tax in Indonesia is the lowest among the G-20 countries and countries emerging markets. To be equal to developing countries, Indonesia needs to have a tax ratio of 20% of GDP. To achieve this target the government seeks to reform taxation in various ways, one of which is building tax compliance and awareness. level of compliance Indonesian people in paying taxes are still very low compared to other countries, as a comparison of the tax ratio in Malaysia of 16%, the Philippines 14%, Thailand 17%, South Korea 25%, South Africa 27%, and Brazil 34% (Detik Finance, 2018). One of the local taxes levied by the local government from the people is tax Motorized Vehicles in 2018, the tax target in Bekasi will reach Rp. 928,833,000,000, but the realization was 35.13% or Rp. 326,292,255,850, with a total of 1,459,933 units of motorized vehicles. In April 2018, 546,922 units of vehicles have not been paid annual taxes motorized vehicles or 37.46% (Tribun, 2018). In January 2019, the number of motorized vehicles was at Bekasi as many as 1,613,317 units, while the number of motorized 269 International Journal of Academic Research in Accounting, Finance and Management Sciences Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) vehicles is as much 620,073 units are still in arrears on motor vehicle tax with the potential for mandatory receivables the tax is estimated to be more than IDR 186,000,000,000. According to research results in the field the reason Bekasi residents are in arrears in tax payments is because lack of awareness of paying taxes and his financial condition (Tempo, 2019). vehicles is as much 620,073 units are still in arrears on motor vehicle tax with the potential for mandatory receivables the tax is estimated to be more than IDR 186,000,000,000. 1. Introduction There are a number of studies related to the effect of tax sanctions on taxpayer compliance conducted by Cahyani and Naniek (2019), Mareti and Dwimulyani (2019), Asrianti (2018), Syah and Krisdiyawati (2017), Setiawan and Meliana (2017), Tawas et al. (2016), Helhel and Helhel (2014), Ardyanto and Utaminingsih (2014) where the results of the study stated that tax sanctions have a positive effect on taxpayer compliance. The results of this study are inversely proportional to that of the study conducted by Rahmawati and Yulianto (2018), Andinata (2015), Pranata et al. (2015) which states that tax sanctions do not affect the tax taxpayer compliance. According to Alabede et al. (2011) things that must be considered in taxpayers making tax payments is a risk that taxpayers must consider before making tax payments. Torgler (2003) conveys that decision a taxpayer can be influenced by his behavior towards the risks faced. Based on the description that has been stated above, the main problems are will be examined as follows: a) Does understanding of tax regulations affect on taxpayer compliance? b) Does the tax sanction affect on taxpayer compliance? c) Does the tax rate affect on taxpayer compliance? d) Does risk preference affect on taxpayer compliance? e) Does risk preference can moderate the relationship between understanding of tax regulations with taxpayer compliance? e) Does risk preference can moderate the relationship between understanding of tax regulations with taxpayer compliance? f) Does risk preference can moderate the relationship between tax sanctions with taxpayer compliance? 2.1. Prospect theory Prospect theory is a theory that explains how one takes decisions in uncertain conditions. Prospect theory was first developed by Daniel Kahneman and Amos Tversky in the early 1980s, where this theory is includes two scientific disciplines namely psychology and economics which is an analysis a person's behavior in making decisions between two choices. Related research uses prospect theory as a theoretical basis for examines taxpayer compliance with risk preferences as moderation, which is Mareti and 270 International Journal of Academic Research in Accounting, Finance and Management Sciences Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) Dwimulyani (2019), Irawati and Sari (2019), Tambun and Witriyanto (2018), Setiawan and Meliana (2017), Ardyanto and Utaminingsih (2014), Adustom (2013), Aryobimo and Cahyonowati (2012), Alabede et al. (2011), and Torgler (2003). The relationship of this research to prospect theory is to explain preferences risks can affect taxpayer compliance. If a taxpayer has high risk, the taxpayer will not necessarily pay the obligation the tax because if the taxpayer has the risk seeking nature though taxpayers have a high risk then it will not affect taxpayers to keep paying taxes, whereas taxpayers who have the risk aversion if taxpayers have low risk, the taxpayers will avoid it tax liability. 3.1. Effects of Understanding of Taxation Regulations on Taxpayer Compliance An understanding of taxation is needed so that taxpayers understand, understand, to be able to apply what is contained in the regulations listed. In research Cahyani and Naniek (2019), Mareti and Dwimulyani (2019), Utari and Setiawan (2019), Asrianti (2018), Mustofa (2016), Ananda et al. (2015), dan Adiasa (2013) examine the influence of understanding of regulations taxation of taxpayer compliance. The results of previous studies stated that understanding of tax regulations has a significant effect on taxpayer compliance. Based on the description above, a hypothesis can be formulated as follows: H1 = Understanding tax regulations has a significant on rates taxpayer compliance. 2.2. Theory Planned of Behavior Theory of planned behavior is a theory that emphasizes the rationality of human behavior is also in the belief that the target behavior is below control of individual consciousness. According to Ajzen (1991), individual behavior is influenced by individual's intention (behavioral intention) towards that particular behavior. There are three Components in Theory Planned Behavior, include: Behavioral beliefs, individual beliefs will result from a behavior and evaluation of these results (beliefs strength and outcome evaluation), such as taxpayer awareness. In addition, normative beliefs, namely beliefs about the normative expectations of others and motivation to meet these expectations (normative beliefs and motivation to comply), like understanding tax regulations. Next, Control beliefs about the existence of things that support or inhibit that behavior will be displayed (control beliefs), such as tax penalties and tax rates and perceptions about how strong the things that support and hinder the behavior (perceived power). Barriers that may arise when the behavior is displayed can come from within oneself and from the environment. This theory explains that taxpayers will comply or not in their obligations in the field of taxation by considering the benefits and effects of his actions in paying taxes. In addition the taxpayer will consider the information obtained from outside. Thus, taxpayers will be influenced by certain factors in His actions are control of trust. Research that uses theory of planned behavior as a theoretical basis for examining taxpayer compliance, Widanaputra and Ratnadi (2019) using the independent variable awareness of taxpayers, obligations moral, and environment, Novianti and Dewi (2017) with independent variables which used is tax amnesty, and Nugraheni and Purwanto (2015) with variables independent awareness of taxpayers, tax knowledge and understanding, sanctions tax, the tax authority, the quality of tax services, attributable justice, procedural justice, and interactional justice. 3. Development of Hypotheses and Research Models The development of hypotheses in this study is based on factors that are allegedly having an influence on the level of tax compliance, so that it can formulate as follows: 3.6. Effects of Risk Preference on the Relationship between Tax Sanctions with taxpayer com One of the goals of tax sanctions is to create compliance with payments tax. According to Elin and Susi (2019) tax sanctions are a deterrent to compulsory tax does not violate existing rules. When the taxpayer does not pay tax the vehicle will risk being netted in zebra operations carried out by the authorities the police, administrative fines. So with these risks it will affect taxpayer compliance. When taxpayers have jobs that are not still, taxpayers are aware of existing sanctions if they do not comply with existing regulations and will add to the burden because many risks will be faced. This is in line with prospect theory which the risk of financial condition is able to influence tax compliance that will result in tax penalties if you do not pay the tax. In the research Asrianti (2018) risk preferences affect the relationship of tax sanctions with taxpayer compliance. Therefore, researchers are interested re-test the hypothesis to find empirical evidence so that the hypothesis which was formulated in this research, include: H6 = Risk preference has a significant effect on the relationship between tax sanctions with taxpayer compliance H2 = Tax sanction has a significant effect on taxpayer compliance. 3.3. Effect of Tax Rates on Taxpayer Compliance The tax rate is the amount of value that must be paid by taxpayers to country through the fiscus. In the study of Atawodi and Ojeka (2012) stated that exorbitant tax rate cause taxpayer non-compliance. In the research of Cahyani and Naniek (2019), Tawas et al. (2016), Helhel and Helhel (2014), and Atawodi and Ojeka (2012) examine the tax rate on taxpayer compliance. The results of previous studies stated that the tax rate has significant effect taxpayer compliance. Based on the description above, it can be formulated hypothesis as follows: H3 = Tax rates has a significant effect on taxpayer compliance. 3.2. Effects of Tax Sanctions on Taxpayer Compliance Tax sanctions must be given as a form of punishment for taxpayers who are breaking the rules will be a deterrent so as not to repeat it again so creating an increase in tax compliance. In Cahyani and Naniek's research (2019), Mareti and Dwimulyani (2019), Asrianti (2018), Mustofa et al. (2016), Syah and Krisdiyawati (2017), Setiawan and Meliana (2017), Tawas et al. (2016), Helhel and Helhel (2014), Ardyanto and Utaminingsih (2014) tested tax sanctions for taxpayer compliance. The results of previous studies stated that tax sanctions have a significant effect on taxpayer compliance. Based on description above, then the hypothesis can be formulated as follows: 271 International Journal of Academic Research in Accounting, Finance and Management Sciences Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) 3.4. Effect of Risk Preference on Taxpayer Compliance According to Torgler (2003) Risk preferences can affect a person's decision taxpayers against the risks faced. The risk of being the one considered taxpayers in paying taxes (Alabede et al., 2011). When risk preferences have a strong relationship with taxpayer compliance, then the level of taxpayer compliance will be low. In the research of Dwi et al. (2019) as well Aryobimo and Cahyonowati (2012) risk preferences have a positive effect on taxpayer compliance. On the other hand in the research of Irawati and Sari (2019), Setiawan and Meliana (2017), Adiasa (2013) risk preference has no effect on compliance taxpayer. Based on the description, the hypothesis can be formulated as following: H4 = Risk preference has a significant effect on taxpayer compliance. 3.5. Effects of Risk Preference on Relationships Between Understanding of Tax Regulations with taxpayer compliance Risk preference as a moderating variable which means risk preferences can be strengthen or weaken the relationship between understanding of tax regulations with tax compliance. In dealing with risks that are happens every taxpayer must have a decision to face a risk. When the taxpayer has problems in his social life, such as lack information about taxation or changes in policy will affect taxpayer compliance. In the research of Dwi et al. (2019), Utari and Setiawan (2019), Asrianti (2018) Risk preferences can moderated understanding about tax regulations with taxpayer compliance. Therefore, researchers are interested re-test the hypothesis to find empirical evidence so that the hypothesis which was formulated in this research, include: H5 = Risk preference has a significant effect on the relationship between understanding of tax regulations with taxpayer compliance. 4. Methodology of research The method in this study used a questionnaire survey to taxpayers registered in SAMSAT Bekasi. A total of 99 questionnaires were returned. The hypothesis in this study was tested using Structural Equation 272 International Journal of Academic Research in Accounting, Finance and Management Sciences Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) Modeling (SEM) by the Smart Partial Least Square (PLS) application. The dependent variable in this study is taxpayer compliance measured using indicators adopted from research conducted by Rahmawati and Yulianto (2018). The independent variable in this study was to understanding of tax regulations measured using indicators adopted from Adiasa (2013), tax sanctions from Ardyanto and Utaminingsih (2014), and tax rates from Cahyani and Naniek (2019). In addition, the moderating variable in this study is risk preference measured using indicators adopted from Adiasa (2013). This study was measured using a Likert scale with 5 levels (1 = Strongly Disagree, 2 = Disagree, 3 = Neutral, 4 = Agree, 5 = Strongly Agree). Figure 1. Conceptual Framework H4 Figure 1. Conceptual Framework 5. Results and discussions Results Hypothesis Test Path & Bootstrapping Algorithm The results of testing this hypothesis use the structural equation modeling (SEM) model using smart PLS software version 3.0. Table 2 shows that understanding tax regulations (PPP, t = 2.230 and p-value = 0.026), tax penalties (SP, t = 5.119 and p-value = 0,000), and tax rates (TP, t = 2,346 and p- value = 0.019) affects taxpayer compliance (KP), which means H1, H2, and H3 are accepted. Meanwhile, risk preference (PR, t = 1,947 and p-value = 0.052), which means H4 is rejected. The risk preference in moderating the understanding of tax regulations (PR. PPP, t = 0.157 and p-value = 0.875) does not affect taxpayer compliance, which means H4 is rejected. Finally, risk preference in moderating tax sanctions (PR. SP, t = 2.045 and p-value = 0.041) affects taxpayer compliance. Understanding tax regulations has a significant effect on taxpayer compliance. Understanding tax regulations has a significant effect on the level of tax compliance. This shows that the higher the taxpayer understands the tax regulations that have been set; the taxpayer tends to be obedient in paying his obligations. The results of this study are supported by research of Cahyani and Naniek (2019), Elin and Susi (2019), Asrianti (2018), Mustofa et al. (2016), Post et al. (2015), and Adiasa (2013) who state that understanding of regulations taxation has a significant effect on taxpayer compliance. The results of this study are also in accordance with the theory of planned behavior whereby understanding tax regulations as normative beliefs that mean beliefs about the normative expectations of others and motivation to fulfill these expectations (normative beliefs and motivation to comply). In the relationship of tax sanctions to taxpayer compliance shows that tax sanctions significantly influence tax compliance. This shows that the application of sanctions imposed on violating taxpayers will cause a deterrent effect so as to increase taxpayer compliance. The results of this study are in line with research by Cahyani and Naniek (2019), Elin and Susi (2019), Asrianti (2018), Mustofa et al. (2016), Asrofi and Krisdiyawati (2017), Tawas et al. (2016), Helhel and Ahmed (2018) 2014), and Arif and Nanik (2014) which state that tax sanctions have a significant effect on taxpayer compliance The results of this study are in accordance with the theory of planned behavior where tax sanctions are linked as control beliefs. 5. Results and discussions Validity and reability tests are done before testing hypothesis. Test the validity by looking at the extracted average variance (AVE) with suggested values above 0.5 and reability test with see the value of Composite Reliability and Cronbach's Alpha. Composite reliability value for all constructs above 0.7 which means all constructs in this study, it is estimated that it meets the criteria and values suggested in Cronbach's Alpha is above 0.6 for all constructs (Ghozali, 2014). Besides that, in assessing a model with PLS will begin by looking at R- Square (R2) for each dependent latent variable (Ghozali, 2014). Test results validity and Reliability can be seen in the following table: Table 1. Validity and Reliability Test Results Table 1. Validity and Reliability Test Results Table 1. Validity and Reliability Test Results R2 = 0,612 Variabel AVE Composite Reliability Cronbach’s Alpha KP 0,575 0,841 0,743 PPP 0,534 0,821 0,709 PR 0,502 0,922 0,909 SP 0,631 0,872 0,803 TP 0,715 0,834 0,607 In the table above, the lowest AVE value is 0.502 at moderating variable is risk preference. Then it can be concluded that throughout variable is declared valid. Then, the lowest value in the Composite Reliability value is 0.821 for the variable understanding of tax regulations so it can be concluded that all variables in this study have good reliability of each construct and Cronbach’s value Alpha for the lowest is 0.607 at the tax rate so it can concluded that all variables in this study had a reliability good for each construct. R2 test results tax compliance is equal to 0.612 thus indicating that the variable is latent independent namely understanding tax regulations, tax sanctions, tax rates, and moderation variables is risk preferences, can explain latent variables dependent: taxpayer compliance of 61.2% and the remaining 38.8% explained by other factors outside this research such as socialization taxation, taxpayer awareness, and others. 273 International Journal of Academic Research in Accounting, Finance and Management Sciences Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) Table 2. Results Hypothesis Test Path & Bootstrapping Algorithm Hypothesis Variable Path Coefficient T Statistics P Values H1 PPP -> KP 0,206 2,230 0,026 H2 SP -> KP 0,458 5,119 0,000 H3 TP -> KP 0,222 2,346 0,019 H4 PR -> KP -0,119 1,947 0,052 H5 PR. PPP -> KP -0,015 0,157 0,875 H6 PR. SP -> KP 0,193 2,045 0,041 Table 2. 5. Results and discussions In this study, tax sanctions support the level of taxpayer compliance, effective and efficient tax sanctions will increase taxpayer compliance in meeting obligations because it can provide a deterrent effect to any non- compliant taxpayers. Implementation of strict sanctions will harm taxpayers so that taxpayers would prefer to carry out their obligations. In addition, the relationship of tax rates to taxpayer compliance shows that tax rates has a significant effect on taxpayer compliance. This shows that the application of tax rates in accordance with established regulations will increase taxpayer compliance. The results of this study were supported by Cahyani and Naniek (2019), Tawas et al. (2016), Helhel and Ahmed (2014), and Atawodi and Ojeka (2012) who stated that tax rates had a significant effect on tax compliance. The results of this study are consistent with the theory of planned behavior whereby the tax rate as a perception of behavioral control. Variable risk preference for taxpayer compliance shows that risk preference has no significant effect on taxpayer compliance. This shows that the taxpayers in the study sample accept the existing risks so that risk preferences do not affect taxpayer compliance. The results of this study are not in line with the research of Dwi et al. (2019) and Aryobimo and Cahyonowati (2012) risk preferences have a positive effect on taxpayer compliance. On the other hand, in the research of Irawati and Sari (2019), Setiawan and Meliana (2017), Adiasa (2013) risk preferences do not affect tax compliance. This is not in line with the prospect theory that even though the taxpayer has a high risk, it will not affect the taxpayer to continue paying taxes, while the taxpayer who has a risk aversion if the taxpayer has a low risk, the taxpayer will 274 International Journal of Academic Research in Accounting, Finance and Management Sciences Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) actually avoid his tax obligations. Then, the risk preference variable in moderating the relationship of understanding of tax regulations to taxpayer compliance shows that risk preferences cannot moderate the relationship of understanding of tax regulations towards taxpayer compliance with taxpayer compliance. This shows that the taxpayers sampled in this study understand the tax regulations but the existing risk cannot influence a taxpayer's decision to pay his taxes so that the risk preference cannot moderate the relationship of understanding the tax regulations to the level of tax compliance. 5. Results and discussions The results of this study are not in line with the research of Dwi et al. (2019), Utari and Setiawan (2019), and Asrianti (2018). Risk preferences can strengthen the relationship between understanding tax regulations and taxpayer compliance. However, this study is in line with the study of Adiasa (2013) which states that risk preferences cannot moderate the relationship of understanding tax regulations to taxpayer compliance. The results of this study are not in accordance with prospect theory, where taxpayers will make their own decisions in dealing with existing risks. So, risk preference has no effect on the relationship between understanding tax regulations and the level of tax compliance. Finally, risk preference in moderating the relationship of tax sanctions to taxpayer compliance shows that risk preferences have a significant effect in moderating the relationship of tax sanctions to taxpayer compliance with taxpayer compliance. This shows that taxpayers who have a high risk will affect the relationship of tax sanctions to the level of tax compliance. The public will comply with the regulations if there are sanctions that are applied because sanctions as a regulatory tool so that taxpayers become more orderly because if they violate the rules set then the risk of being sanctioned will be higher. The results of this study are in line with research by Asrianti (2018) which states that risk preference influences the relationship of tax sanctions with taxpayer compliance. The results of this study are in accordance with prospect theory, where taxpayers will make their own decisions in dealing with existing risks depending on the perspective of the taxpayer to see whether the risk preferences will affect positive or negative. 6. Conclusions This research begins with the problem of taxpayer compliance in Indonesia which is still low and requires improvements to taxpayer compliance. Problems with taxpayer compliance also occur with motor vehicle tax at SAMSAT Bekasi. This study aims to examine and determine the factors that influence taxpayer compliance with risk preference as a moderating variable. The results of this study indicate that understanding of tax regulations, tax sanctions, and tax rates has a significant effect on tax compliance. Meanwhile, risk preference has no effect on taxpayer compliance. Then, risk preference has no effect on the relationship between understanding tax regulations and taxpayer compliance. However, risk preference influences the relationship of tax sanctions with taxpayer compliance. This study has a limitation that this study only uses taxpayer research objects that is registered in SAMSAT Bekasi and cannot be generalized because of the limited time and busyness of taxpayers. In addition, the majority of the sample in this study came from respondents with a senior high school education or less. Meanwhile, the sample with master degree (S2) or doctoral degree (S3) is very small. Suggestions for this research for SAMSAT Bekasi are expected to be more active in providing information to the whole community through tax socialization to improve tax knowledge and understanding both directly and through the mass media regarding the importance of paying taxes on time, increasing supervision with zebra operations, and improving services to minimize the existence of brokers. Then, for further research it is expected to be able to add other variables such as awareness of taxpayers, taxation socialization, service quality of focus and use the taxation understanding variable as a moderating variable and expand the object of research not only to one SAMSAT but to all SAMSAT in the Bekasi area so that research can be used universally References 1. Adinata, M.C. (2015). 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European Journal of Business and Management, 6(22), 48-58. 40. Yusro, H. W., & Kiswanto. (2013). Pengaruh tarif pajak, mekanisme pembayaran pajak dan kesadaran membayar pajak terhadap kepatuhan wajib pajak UMKM di Kabupaten Jepara, Accounting Analysis Journal, 3(4), 429-436. 277 International Journal of Academic Research in Accounting, Finance and Management Sciences Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) 41. Bachtiar, Y. (2018). Target pendpatan pajak kendaraan bermotor Kota Bekasi. Diakses 12 September 2019, dari https://jakarta.tribunnews.com/2018/05/07/samsat-targetkan-rp-928-miliar pendapatan-pajak-kendaraan-bermotor-kota-bekasi. 278
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Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation
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Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation Suvi Santala*, Matti Karp, Ville Santala Suvi Santala*, Matti Karp, Ville Santala Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland OPEN ACCESS Citation: Santala S, Karp M, Santala V (2014) Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation. PLoS ONE 9(12): e113786. doi:10. 1371/journal.pone.0113786 Editor: Guo-Qiang Chen, Tsinghua University, China Received: September 12, 2014 Accepted: October 30, 2014 Published: December 3, 2014 Copyright:  2014 Santala et al. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and repro- duction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The project was funded by Academy of Finland (project ProChassis 272602, URL: http:// www.aka.fi/fi/A/), and by Suomen Kulttuurirahasto (URL: http://www.skr.fi/) to MK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu- script. RESEARCH ARTICLE Abstract In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well- characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications. Engineered Synthetic Coculture for Improved Performance concentrations of substrates, intermediates, or other potentially harmful compounds. Common end-metabolites, such as organic acids, are rapidly converted to other compounds. Thus, tolerance to high concentrations of inhibitory compounds is not essential for individual members of a community. In biotechnological applications, however, engineered pure cultures are typically utilized as production platforms because of the easier bioprocess control and more straight-forward genetic engineering. Nevertheless, establishing a sustainable and economical bioprocess is a challenge, for which metabolic engineering and synthetic biology are seeking solutions [1–4]. For example, accumulation of unwanted side products and inefficient carbon and energy fluxes are common issues. Since Escherichia coli is a widely used host in bioprocesses, considerable effort is dedicated to controlling the overflow metabolism. In this phenomenon, carbon is excessively transported into the cells, and the capacity of tricarboxylic acid (TCA) cycle is exceeded. As a result, the metabolism is shifted to produce acetate from acetyl coenzyme A (CoA) [5]. The accumulation of acetate results in the induction of the stress response, growth inhibition, significant carbon loss, and eventually reduced productivity [6, 7]. Previous strategies to neglect the negative effects of acetate include both genetic engineering of E. coli to circumvent the acetate production and comprehensive optimization of fed-batch bioprocesses [8–10]. These approaches, however, potentially decrease the growth rate or require a substantial amount of optimization and engineering work. ‘‘Pure cocultures’’ typically consisting of two known strains are a more recent approach to improve the production platforms [11, 12], and a trend towards more controllable and tunable cocultures involving systems biology and synthetic biology solutions is growing stronger [13, 14]. For example, Eiteman et al. [15] have described the co-fermentation of hexoses and pentoses by a community of engineered E. coli. Tsai et al. [16] established a system for ethanol production from cellulose by a synthetic yeast community. Although the obtained ethanol yields are low, the study is an elegant example of the utilization of an engineered community for bioenergy production. Previous strategies to neglect the negative effects of acetate include both genetic engineering of E. coli to circumvent the acetate production and comprehensive optimization of fed-batch bioprocesses [8–10]. These approaches, however, ll d h h b l f The culture systems described above are based on exploiting strains of single species, but they represent the first steps towards consolidated processing of sustainable substrate to products by an engineered consortium. PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Introduction In a microbial ecosystem, bacterial species cooperate by producing metabolites that serve as substrates for other species of a community. Dynamic and symbiotic interactions between different species enable an efficient carbon and energy flow. Furthermore, the interactions balance rapid environmental changes and high Competing Interests: The authors have declared that no competing interests exist. 1 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Strains In the initial coculture, E. coli K12 BW25113 (from Yale E. coli Genetic Stock Center CGSC, Connecticut, USA) was used. The single gene knock-out strain of A. baylyi ADP1 (DSM 24193) (gene deletion high-affinity gluconate permease, gntT, ACIAD0544) was employed in the study (kindly provided by Dr. Veronique de Berardinis, Genoscope, France). In the single gene knock-out mutant, the target gene is replaced with a gene cassette containing a kanamycin resistance gene (Kanr) [18]. The strain ADP1Dgnt::Kanr/tdk was transformed with a construct pVKK81-T-lux, and designated as ABlux. In the first stage of GFP production, E. coli K12 BW25113 was transformed with sfGFP/pAK400c and designated as ECsf. In the second stage, both E. coli K12 BW25113 and A. baylyi ADP1Dgnt::Kanr/tdk were transformed with a plasmid pBAV1C-T5-GFP, resulting in strains designated as ECg and ABg, respectively. For monitoring the individual GFP production of E. coli, a control strain ADP1Dgnt::Kanr/tdk transformed with a plasmid pBAV1C- ara without the insert was constructed, and the strain was designated as ABc. Genetic constructs are described in the section ‘‘plasmid construction and transformations’’. The same strains without the plasmids were used as background control strains for fluorescence measurement. Engineered Synthetic Coculture for Improved Performance engineered to produce recombinant protein, and batch cocultures with different substrate concentrations were conducted to demonstrate the effects of accelerated growth to recombinant protein production. Finally, both strains were engineered to produce the same recombinant protein, and cocultivations in a bioreactor were carried out. Employing different strains of a single species broadens the engineering possibilities; however, the strains often possess very similar carbon utilization patterns, thus leaving unresolved the issues related to by-product accumulation and the inefficient carbon flow. On the other hand, genetic engineering is very challenging for mixed populations which efficiently degrade complex substrates and mixed sugars [17]. In the present study, the advantages of both mixed populations and readily engineered model strains are exploited; the well-characterized model organisms E. coli K12 and Acinetobacter baylyi ADP1 were employed for the construction of a synthetic and symbiotic microecosystem with directed carbon flow for improved culture performance. In the first stage of the study, the strains were cultured in a minimal medium batch cultivation to monitor the coculture compatibility, performance of individual strains, and carbon flow. Secondly, E. coli strain was PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 2 / 16 Engineered Synthetic Coculture for Improved Performance [19] using primers 59-AATAGCTAGCCTGTAGAAAAGAGGAAGGAAATAA- TAAATGGAGAAAAAAATCACTGGATATAC-39 and 59-TAATAGTGCACT- TACGCCCCGCCCTGCCAC-39 with restriction sites NheI and ApaLI. For expression of bacterial luciferase operon, a plasmid pVKK81-T-lux (V. Santala and M. Karp, unpublished data) containing the bacterial luciferase genes luxCDABE from Photorhabdus luminescens and tetracycline resistance gene (T) was used. For ADP1, the plasmids pVKK81-T-lux, pBAV1C-T5-GFP, and pBAV1C-ara without an insert [22] were naturally transformed by the method described previously [23], and the colonies were selected from Luria-Agar (LA) plates (1 g/l NaCl, 5 g/l yeast extract, 10 g/l tryptone, 15 g/l agar) containing tetracycline (10 mg/ml) and chloramphenicol (25 mg/ml), respectively. For E. coli, the plasmids sfGFP/pAK400c and pBAV1C-T5-GFP were transformed by electroporation and the colonies were selected from LA plates containing chloramphenicol (25 mg/ml). The constructs were verified by restriction analysis, fluorescence, or luminescence determination. Medium composition and cultivations For the initial coculture, minimal salts medium MA/9 described previously (9) was used. Glucose (,50 mM) was used as the initial carbon and energy source. The strains were cultivated in batch bottles (50 ml medium/250 ml Erlenmeyer flasks) at 37˚C and 300 rpm both individually and in cocultures for 12 h. Two parallel bottles were used for each culture. For study of the GFP production of E. coli both in monoculture and in coculture at variable substrate concentrations, the strains ECg and ABc were cultivated in 5 ml of a MA/9 medium supplemented with 50 mM, 100 mM, or 250 mM glucose and 100 mM IPTG at 30˚C and 300 rpm. The cultivations were performed both individually and in cocultures in two parallel tubes for 24 h. For 0.5-litre batch cultivations in a bioreactor, the monoculture (ECg) and the cocultures (ECg and ABg or ECg and ABc) were cultivated in a modified Luria- Bertani medium (1 g/l NaCl, 5 g/l yeast extract, 10 g/l tryptone) supplemented with 2 mM MgSO4, 0.5 mM CaCl2, 3 mM FeCl3 and 100 mM glucose, and 25 mg/ ml chloramphenicol. The cultivation was carried out in a 1-litre vessel (Sartorius Biostat B plus Twin System, Germany) with an online pH monitoring system at 30˚C for 10 h. The cultures were aerated by stirring (350 rpm) and supply of oxygen (O2 partial pressure set to 20%). Equal inoculum sizes for each strain were used. Control cultivations without glucose supplementation were conducted in 250 ml batch bottles in otherwise similar conditions. Luminescence, fluorescence, and biomass determination Plasmid construction and transformations For digestions and ligations, the enzymes and buffers were provided by Fermentas (Lithuania) and used according to the manufacturer’s instructions. The PCR reagents were provided by Finnzymes (Finland) (DNA polymerase PhusionTM and buffer) and Fermentas (nucleotides). The primers were obtained from ThermoFisher Scientific (Germany) with appropriate restriction sites. The superfolder GFP variant (from BBa_I746909) was amplified with primers 59-GAGTTCTAGAGAAGGAGATATACATATGCGTAAAGGCGAAGAGCTGTT- C-39 and 59-CTAGCAAGCTTAGTGGTGATGGTGATGATGTTTGTATAG-39 with restriction sites XbaI and HindIII, respectively, and cloned to pAK400c vector [19, 20] under the IPTG-inducible lactose promoter, resulting in construct sfGFP/ pAK400c. To construct the plasmid pBAV1C-T5-GFP with chloramphenicol resistance gene cat, the original plasmid pBAV1K-T5-GFP [21] was partially amplified with PCR using primers 59-TAATAGCTAGCTATTTAAAGATACC- CCAAGAAGCTAATTATAAC-39 and 59-TAATAGTGCACTCGCTTGGACTCC- TGTTGATAG-39. The product was ligated with cat gene amplified from pAK400c For digestions and ligations, the enzymes and buffers were provided by Fermentas (Lithuania) and used according to the manufacturer’s instructions. The PCR reagents were provided by Finnzymes (Finland) (DNA polymerase PhusionTM and buffer) and Fermentas (nucleotides). The primers were obtained from ThermoFisher Scientific (Germany) with appropriate restriction sites. The superfolder GFP variant (from BBa_I746909) was amplified with primers 59-GAGTTCTAGAGAAGGAGATATACATATGCGTAAAGGCGAAGAGCTGTT- C-39 and 59-CTAGCAAGCTTAGTGGTGATGGTGATGATGTTTGTATAG-39 with restriction sites XbaI and HindIII, respectively, and cloned to pAK400c vector [19, 20] under the IPTG-inducible lactose promoter, resulting in construct sfGFP/ pAK400c. To construct the plasmid pBAV1C-T5-GFP with chloramphenicol resistance gene cat, the original plasmid pBAV1K-T5-GFP [21] was partially amplified with PCR using primers 59-TAATAGCTAGCTATTTAAAGATACC- CCAAGAAGCTAATTATAAC-39 and 59-TAATAGTGCACTCGCTTGGACTCC- TGTTGATAG-39. The product was ligated with cat gene amplified from pAK400c 3 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Analysis for glucose and end-metabolite concentrations Glucose consumption and acetate accumulation/consumption in the cultivations were determined with high performance liquid chromatography (HPLC) as described previously [24]. Identification and quantification of glucose and end- metabolites were based on co-chromatography using external standards. The mediums were used as controls. Engineered Synthetic Coculture for Improved Performance measured immediately with a Victor 2 plate reader (Perkin Elmer Life Sciences, Finland). Finland). The total biomass in the bottles and in the bioreactor was determined by optical density measurement at the wavelength 600 nm every hour (OD600). Also, in the initial coculture and in the bioreactor experiments, the cells were plated every two hours (starting from 2 or 5 h) on LA plates with and without kanamycin (30 mg/ ml) in order to determine the individual cell numbers of E. coli and A. baylyi according to CFU (colony forming units). For bioreactor batch cultures, 40 ml biomass samples were taken at the end of the cultivation, centrifuged at 30,000 g for 30 min and freeze-dried for gravimetric dry cell weight (CDW) determination. For fluorescence determination, 1 ml samples were taken from all cultivations, centrifuged for 1 min at 10,000 g, and suspended in 1 ml 10 mM Tris-HCl buffer (pH 8.2) containing 150 mM NaCl. The samples for fluorescence measurements were diluted to linear range and measured as four 10–50 ml parallel samples with Fluoroskan Ascent FL (Thermo Labsystems, Finland) using the wavelengths 485 nm (excitation) and 538 nm (emission). The total biomass in the bottles and in the bioreactor was determined by optical density measurement at the wavelength 600 nm every hour (OD600). Also, in the initial coculture and in the bioreactor experiments, the cells were plated every two hours (starting from 2 or 5 h) on LA plates with and without kanamycin (30 mg/ ml) in order to determine the individual cell numbers of E. coli and A. baylyi according to CFU (colony forming units). For bioreactor batch cultures, 40 ml biomass samples were taken at the end of the cultivation, centrifuged at 30,000 g for 30 min and freeze-dried for gravimetric dry cell weight (CDW) determination. For fluorescence determination, 1 ml samples were taken from all cultivations, centrifuged for 1 min at 10,000 g, and suspended in 1 ml 10 mM Tris-HCl buffer (pH 8.2) containing 150 mM NaCl. The samples for fluorescence measurements were diluted to linear range and measured as four 10–50 ml parallel samples with Fluoroskan Ascent FL (Thermo Labsystems, Finland) using the wavelengths 485 nm (excitation) and 538 nm (emission). Luminescence, fluorescence, and biomass determination For luminescence measurements, samples were taken from the batch cultivations every hour and 200 ml of a sample (diluted when necessary) was applied on white microtiter well plates in three parallel samples. The luminescent signal was PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 4 / 16 Engineered Synthetic Coculture for Improved Performance tools are widely available for the strain [21, 30, 31]. A. baylyi ADP1 exhibits a relatively wide substrate range and is known to efficiently utilize organic acids such as acetate as a sole carbon and energy source [32], although the detailed mechanism for the uptake and utilization of acetate has not yet been characterized. Moreover, our recent studies suggest that ADP1 neither produces harmful overflow metabolites nor exhibits substrate inhibition (S. Santala and V. Santala, unpublished data). In order to establish a functional symbiotic coculture system with directed carbon flow, and to produce unambiguous data for analyzing the coculture performance, a mutant strain of A. baylyi ADP1 made deficient of utilizing glucose was employed; according to the metabolic model of ADP1, the disruption of a high-affinity gluconate permease (gntT, ACIAD0544) blocks the glucose pathway. Due to the exceptional glucose utilization pathway of ADP1, a glucose molecule is oxidized to gluconate on the outer surface of the inner membrane by an electron carrier associated to glucose dehydrogenase, pyrroloquinoline quinine (PQQ). Subsequently, the high-affinity gluconate permease GntT transports gluconate into cells [32]. Here, a knock-out mutant strain A. baylyi ADP1DgntT::Kanr/tdk [18] was employed. When glucose is sufficiently available in the coculture, E. coli cells uptake glucose into the cells in excess and return a significant amount of the carbon into the medium in the form of acetate. Thus, in a coculture, the glucose negative mutant strain of ADP1 is dependent on the end- metabolites of E. coli, enabling an experimental validation of the carbon flow and fate. A schematic illustration of the proposed carbon flow is presented in Figure 1. In order to establish a functional symbiotic coculture system with directed carbon flow, and to produce unambiguous data for analyzing the coculture performance, a mutant strain of A. baylyi ADP1 made deficient of utilizing glucose was employed; according to the metabolic model of ADP1, the disruption of a high-affinity gluconate permease (gntT, ACIAD0544) blocks the glucose pathway. Due to the exceptional glucose utilization pathway of ADP1, a glucose molecule is oxidized to gluconate on the outer surface of the inner membrane by an electron carrier associated to glucose dehydrogenase, pyrroloquinoline quinine (PQQ). Subsequently, the high-affinity gluconate permease GntT transports gluconate into cells [32]. Here, a knock-out mutant strain A. baylyi ADP1DgntT::Kanr/tdk [18] was employed. When glucose is sufficiently available in the coculture, E. Establishing a synthetic coculture In natural ecosystems, different species interact through diverse forms of cooperation, such as mutualism, parasitism, or symbiosis, creating a complex network of integrated metabolic pathways. From a synthetic biology point of view, cocultures can provide several advantages over monocultures by performing multi-step tasks and being more catabolically versatile [25–27]. In this study, a synthetic coculture was constructed using two strains of different species, E. coli K12 and A. baylyi ADP1, and the effects of connecting the carbon metabolism of these strains were investigated. For the construction of a functional, synthetic coculture, the bacterial species for the community were chosen based on characteristics supporting one another. To promote straight- forward system design and engineering, the availability of readily applicable engineering tools and well-characterized metabolic networks was considered essential here [28, 29]. In microbiological terms, the strains also share similar preferences for growth conditions. A. baylyi ADP1 has been recently introduced as a competitive model organism for genetic studies [18] and metabolic engineering purposes [22, 23], and genetic A. baylyi ADP1 has been recently introduced as a competitive model organism for genetic studies [18] and metabolic engineering purposes [22, 23], and genetic 5 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 coli cells uptake glucose into the cells in excess and return a significant amount of the carbon into the medium in the form of acetate. Thus, in a coculture, the glucose negative mutant strain of ADP1 is dependent on the end- metabolites of E. coli, enabling an experimental validation of the carbon flow and fate. A schematic illustration of the proposed carbon flow is presented in Figure 1. For a functional community, the compatibility of the two strains is evidently essential. In order to monitor the performance and growth of ADP1DgntT::Kanr/ tdk in the coculture, pVKK81-T-lux containing a bacterial luciferase genes luxCDABE (from P. luminescens [33]) were transformed into the strain by natural transformation. A luminescent colony was selected from a plate containing 10 mg/ ml tetracycline. The resulting strain ADP1DgntT::Kanr/tdk[lux_tetr] was desig- nated as ABlux. In order to monitor the growth dynamics of E. coli and ABlux, the strains were cultivated both separately and in cocultures for 12 h in batch cultures. A minimal salts medium MA/9 supplemented with 50 mM glucose was used in the experiment. Biomass and luminescence were determined from the cultivations hourly. Selective plate count was also applied in order to determine the individual cell numbers of E. coli and ABlux. The total biomass of the coculture and the proportion of ABlux of the total biomass are presented in Figure 2. The use of luminescent construct enabled specific real-time monitoring of ABlux performance during the cocultivation (Figure 2, the inlet), and allowed the observation of potential fluctuations and problem situations in the cultures, not readily detectable by other means. According to the luminescence signal data, ABlux starts to grow in the coculture after a short lag phase, which was subsequently confirmed by the 5-hour timepoint plate count. After 11 h of cultivation, the strain reached an approximately 6% proportion of the total biomass. 6 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Engineered Synthetic Coculture for Improved Performance Figure 1. The proposed carbon flow in the wild type Escherichia coli culture and in the coculture of engineered Acinetobacter baylyi ADP1 and E. coli. A) E. coli culture supplied with excess glucose readily shifts to an overflow metabolism (*), producing large amounts of acetate into the culture medium. Acetate inhibits growth and reduces product formation, and carbon flow is directed off the product route. B) In a coculture involving directed carbon flow, the strain A. PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 baylyi ADP1 is made deficient in glucose utilization by a knock-out of gluconate permease gntT and is solely dependent on the end-metabolites (acetate) of E. coli. Carbon is further metabolized and can be directed to biomass and the product of interest. Metabolic pathways in the figure are simplified and only the main products are shown. Figure 1. The proposed carbon flow in the wild type Escherichia coli culture and in the coculture of engineered Acinetobacter baylyi ADP1 and E. coli. A) E. coli culture supplied with excess glucose readily shifts to an overflow metabolism (*), producing large amounts of acetate into the culture medium. Acetate inhibits growth and reduces product formation, and carbon flow is directed off the product route. B) In a coculture involving directed carbon flow, the strain A. baylyi ADP1 is made deficient in glucose utilization by a knock-out of gluconate permease gntT and is solely dependent on the end-metabolites (acetate) of E. coli. Carbon is further metabolized and can be directed to biomass and the product of interest. Metabolic pathways in the figure are simplified and only the main products are shown. doi:10.1371/journal.pone.0113786.g001 Figure 2. The total biomass of the coculture of E. coli and ADP1DgntT::Kanr/tdk[lux_tetr] (ABlux), and the proportion of ABlux in the total biomass. The strain ABlux is deficient in utilizing glucose. During the cultivation, the growth of ABlux was monitored in real-time via a luminescence reporter luxCDABE (the inlet). The cells were cultivated in a minimal salts medium supplied with glucose for 12 h. The mean and standard deviation of the two independent cultures are shown. Note the logarithmic scale in biomass and luminescence y-axes. Total biomass – line with squares; proportion of ABlux cells in total biomass – columns. doi:10.1371/journal.pone.0113786.g002 Figure 2. The total biomass of the coculture of E. coli and ADP1DgntT::Kanr/tdk[lux_tetr] (ABlux), and the proportion of ABlux in the total biomass. The strain ABlux is deficient in utilizing glucose. During the cultivation, the growth of ABlux was monitored in real-time via a luminescence reporter luxCDABE (the inlet). The cells were cultivated in a minimal salts medium supplied with glucose for 12 h. The mean and standard deviation of the two independent cultures are shown. Note the logarithmic scale in biomass and luminescence y-axes. Total biomass – line with squares; proportion of ABlux cells in total biomass – columns. doi:10.1371/journal.pone.0113786.g002 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 7 / 16 Engineered Synthetic Coculture for Improved Performance with the total decrease in glucose concentration being 9 mM. Detectable amounts of acetate could not be observed in the coculture until the 11 h timepoint, indicating efficient acetate utilization by ABlux. The acetate concentration in the end of the cultivation (2 mM) was lower compared to that of the monoculture of E. coli. The strain ABlux did not exhibit growth in the monoculture due to the lack of suitable carbon source, and neither consumption of glucose nor accumulation of end-metabolites was detected (data not shown). We discovered that the coculture was beneficial for E. coli, as the cells grew faster in the cocultivation compared to the monoculture in the studied conditions. As expected, the proportion of ADP1 cells in the coculture increased as a result of E. coli acetate production. According to the E. coli growth curve, the consumption of acetate by ABlux positively affected the E. coli growth, which could be observed as a shorter lag phase and more sufficient growth. Even though the concentration of acetate did not reach the detection limit until the 8–10 h timepoint the cultures already showed differential performance after the 5 h timepoint, indicating that acetate already hinders growth at very low concentra- tions. Eventually, the coculture resulted in more efficient glucose utilization by E. coli and a lower overall acetate concentration in the culture. Thus, it could be concluded that the constructed carbon channeling system diminished the negative effects of acetate and favorably affected the E. coli growth, and a relatively low proportion of the supporting strain ABlux in the community was able to benefit the growth. Production of recombinant protein by E. coli in the synthetic coculture Engineered Synthetic Coculture for Improved Performance Figure 3. E. coli biomasses and substrate/end-metabolite concentrations. The cells were cultivated in a minimal salts medium supplied with glucose for 12 h. A) E. coli cell number in monoculture (empty squares) and in coculture (filled squares). B) Glucose (squares) and acetate (circles) concentrations in monoculture (empty symbols) and in coculture (filled symbols). The mean and standard deviation of two independent culture samples are shown. Note the logarithmic scale in biomass y-axis. Figure 3. E. coli biomasses and substrate/end-metabolite concentrations. The cells were cultivated in a minimal salts medium supplied with glucose for 12 h. A) E. coli cell number in monoculture (empty squares) and in coculture (filled squares). B) Glucose (squares) and acetate (circles) concentrations in monoculture (empty symbols) and in coculture (filled symbols). The mean and standard deviation of two independent culture samples are shown. Note the logarithmic scale in biomass y-axis. doi:10.1371/journal.pone.0113786.g003 The biomass of E. coli in the monoculture and in the coculture was calculated according to optical densities and plate counts: the data is presented in Figure 3A. In the E. coli monoculture, the growth shifted to an exponential phase after 5– 6 hours of lag phase and reached a total cell number of ,6.15?108 ml21 after 12 h of cultivation. In the coculture, the lag phase for E. coli was shorter, 4–5 h, and the culture reached a slightly higher cell number of 6.63?108 ml21 at 12 h timepoint. The consumption of glucose and accumulation of end-metabolites in E. coli monoculture and coculture were analyzed by HPLC (Figure 3B). The consumption of glucose in E. coli monoculture was nearly linear after 7 h of cultivation, with a total decrease of 6 mM in the glucose concentration. Acetate could be reliably detected after an 8 h timepoint, the concentration reaching 4 mM at the end of the cultivation. End-metabolites other than acetate were not detected. In the coculture, the glucose was utilized more rapidly and efficiently, PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 8 / 16 Engineered Synthetic Coculture for Improved Performance Figure 4. Biomasses and fluorescence of E. coli expressing sfGFP/pAK400c (ECsf) and A. baylyi ADP1DgntT::Kanr/tdk expressing empty plasmid pBAV1C-ara (ABc) in mono and cocultures. Fluorescence is produced solely by E. coli expressing GFP. The strains were cultivated in a minimal salts medium supplemented with 50; 100; or 250; mM glucose for 24 h. A) Biomasses (optical density, OD600) of ECsf monocultures and cocultures (ECsfABc). B) Fluorescence signals measured for monocultures and cocultures demonstrating the production of recombinant protein in cultures. The mean and standard deviation of two independent cultures are shown. Figure 4. Biomasses and fluorescence of E. coli expressing sfGFP/pAK400c (ECsf) and A. baylyi ADP1DgntT::Kanr/tdk expressing empty plasmid pBAV1C-ara (ABc) in mono and cocultures. Fluorescence is produced solely by E. coli expressing GFP. The strains were cultivated in a minimal salts medium supplemented with 50; 100; or 250; mM glucose for 24 h. A) Biomasses (optical density, OD600) of ECsf monocultures and cocultures (ECsfABc). B) Fluorescence signals measured for monocultures and cocultures demonstrating the production of recombinant protein in cultures. The mean and standard deviation of two independent cultures are shown. Fluorescence is produced solely by E. coli expressing GFP. The strains were cultivated in a minimal salts medium supplemented with 50; 100; or 250; mM glucose for 24 h. A) Biomasses (optical density, OD600) of ECsf monocultures and cocultures (ECsfABc). B) Fluorescence signals measured for monocultures and cocultures demonstrating the production of recombinant protein in cultures. The mean and standard deviation of two independent cultures are shown. doi:10.1371/journal.pone.0113786.g004 doi:10.1371/journal.pone.0113786.g004 cocultures are presented in Figure 4. The cocultures grew to higher optical densities and produced more fluorescence with all studied glucose concentrations compared to the respective monocultures, and the highest optical density and fluorescence signal were obtained in the culture containing 50 mM glucose. Interestingly, the more glucose is present in the medium, the more significant is the difference between the mono- and cocultures. This indicates that high substrate concentrations may hinder E. coli growth by direct substrate inhibition and by accelerated overflow metabolism. In the cultures containing 250 mM glucose, a fluorescence signal of approximately 2-fold and an optical density of 3- fold higher were obtained for the coculture compared to the monoculture. Thus, the batch experiments further emphasized the coculture relevance for improved culture performance and productivity in variable substrate concentrations. cocultures are presented in Figure 4. Production of recombinant protein by E. coli in the synthetic coculture It was demonstrated that the coculture is beneficial for E. coli growth and biomass production. The next step was to study the effects of the coculture on production of the heterologous product that competes with the cell building blocks, demonstrated here by recombinant protein production. Green fluorescent protein (GFP) was chosen for the proof-of-principle production platform in order to enable real-time monitoring of production dynamics and comparability between the mono and cocultures. In addition, GFP has previously been successfully expressed both in E. coli and A. baylyi ADP1 [21]. A plasmid containing a superfolder variant of GFP (BBa_I746909) under inducible lactose promoter was constructed. The resulting plasmid sfGFP/pAK400c was transformed to E. coli, the clones were selected from chloramphenicol plates, and the strain was designated as ECsf. To enable A. baylyi ADP1 growth in the coculture, plasmid pBAV1C-ara (described elsewhere [22]) without an insert was transformed to the mutant strain ADP1DgntT::Kanr/tdk and selected from the chloramphenicol plates, resulting in a strain designated as ABc. The potential support of ABc for ECsf growth and protein production in a coculture was studied in minimal medium batch cultures supplied with different glucose concentrations: 50 mM, 100 mM, and 250 mM. The amount of GFP produced was estimated by fluorescence determination. The end-point biomasses (OD600) and fluorescence signals for monocultures and 9 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Engineered Synthetic Coculture for Improved Performance Production of recombinant protein by the synthetic coculture in a bioreactor PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Production of recombinant protein by the synthetic coculture in a bioreactor The previous experiments demonstrated that ABc efficiently supports ECsf growth in cocultures, being especially beneficial in cultures containing high substrate concentrations by neglecting the negative effects of acetate. In an ideal case, the secreted acetate could be directed to the product by the supportive strain. For such an approach to be practical, a single expression construct that is functional in both strains should be used. Indeed, an expression vector exploitable both in E. coli and A. baylyi ADP1 have been described [21]. Derived from the original vector, a plasmid pBAV1C-T5-GFP was constructed and transformed to E. coli and A. baylyi ADP1DgntT::Kanr/tdk. The vector contains the gene gfp under a constitutive strong promoter (T5), and a chloramphenicol resistance gene (cat). The resulting strains were designated as ECg and ABg, respectively. To provide sufficient supply of nutrients and building blocks for the strains under strong over-expression, and to study the coculture functionality in simple unoptimized batch conditions without pH buffering or control, cultivations were performed in a rich medium supplied with glucose. The batch cultivations for the ECg and ABg coculture and ECg monoculture were carried out in a rich medium supplied with 100 mM glucose and aeration. The cultivations were conducted in a bioreactor in order to reveal more information about the culture characteristics and dynamics; oxygen partial pressure, pH, biomass production (OD), To provide sufficient supply of nutrients and building blocks for the strains under strong over-expression, and to study the coculture functionality in simple unoptimized batch conditions without pH buffering or control, cultivations were performed in a rich medium supplied with glucose. The batch cultivations for the ECg and ABg coculture and ECg monoculture were carried out in a rich medium supplied with 100 mM glucose and aeration. The cultivations were conducted in a bioreactor in order to reveal more information about the culture characteristics and dynamics; oxygen partial pressure, pH, biomass production (OD), proportions of the coculture strains (%, CFU), substrate and end-metabolite concentrations, and fluorescence signals were monitored throughout the 10-hour cultivations (Figure 5). The coculture started to grow clearly faster after four hours of cultivation, reaching a final optical density of 13, which was ,3-fold higher compared to the biomass of ECg monoculture (OD 4.3). The cocultures grew to higher optical densities and produced more fluorescence with all studied glucose concentrations compared to the respective monocultures, and the highest optical density and fluorescence signal were obtained in the culture containing 50 mM glucose. Interestingly, the more glucose is present in the medium, the more significant is the difference between the mono- and cocultures. This indicates that high substrate concentrations may hinder E. coli growth by direct substrate inhibition and by accelerated overflow metabolism. In the cultures containing 250 mM glucose, a fluorescence signal of approximately 2-fold and an optical density of 3- fold higher were obtained for the coculture compared to the monoculture. Thus, the batch experiments further emphasized the coculture relevance for improved culture performance and productivity in variable substrate concentrations. 10 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Engineered Synthetic Coculture for Improved Performance Figure 5. Monitored bioreactor cultivations of the monoculture ECg and the coculture ECgABg. Both strains produce GFP by expressing the same genetic construct pBAV1C-T5-GFP. The cultivations were performed in a bioreactor in a rich medium supplied with 100 mM glucose and aeration. A) Culture parameters; pH (large circles) and oxygen partial pressure (pO2 %, small circles) for ECg monoculture (empty circles) and ECgABg coculture (filled circles B) Total biomasses (OD600) of the cultures and proportion of ABg cells (%, CFU) in the coculture. C) Fluorescence signals (cts) for the monoculture ECg and coculture ECgABg demonstrating the production of recombinant protein in the cultures. The mean and standard deviation of 2–4 independent culture samples are shown. ECg - E. coli expressing pBAV1C-T5-GFP, ABg - A. baylyi ADP1DgntT::Kanr/tdk expressing pBAV1C-T5-GFP. Figure 5. Monitored bioreactor cultivations of the monoculture ECg and the coculture ECgABg. Both strains produce GFP by expressing the same genetic construct pBAV1C-T5-GFP. The cultivations were performed in a bioreactor in a rich medium supplied with 100 mM glucose and aeration. A) Culture parameters; pH (large circles) and oxygen partial pressure (pO2 %, small circles) for ECg monoculture (empty circles) and ECgABg coculture (filled circles) B) Total biomasses (OD600) of the cultures and proportion of ABg cells (%, CFU) in the coculture. C) Fluorescence signals (cts) for the monoculture ECg and coculture ECgABg demonstrating the production of recombinant protein in the cultures. The mean and standard deviation of 2–4 independent culture samples are shown. ECg - E. coli expressing pBAV1C-T5-GFP, ABg - A. baylyi ADP1DgntT::Kanr/tdk expressing pBAV1C-T5-GFP. doi:10.1371/journal.pone.0113786.g005 the culture. This implies an efficient internal buffering system in the coculture, which retains favorable growth conditions. Acetate concentrations started to differentiate after the 6 h timepoint. At the end of the cultivation, the acetate concentration in the ECg monoculture was 13 mM, which probably affected the growth and cell performance negatively. In the coculture, the acetate concentration was only 3 mM, indicating successful recycle of acetate from the medium to the biomass and the product. The final glucose concentrations were 65 mM in the coculture and 90 mM in the monoculture. For the biomass samples collected at the end of the cultivations, the obtained CDWs were 2.1 g/l and 5.1 g/l for ECg and ECgABg cultures, respectively. Control cultivations without glucose supplementation were carried out in batch bottles. Production of recombinant protein by the synthetic coculture in a bioreactor The initial proportions of the two strains were equal but the proportion of ABg seemed to increase fastest between the 6–8 h timepoints, assumingly in parallel with the exponential growth phase of ECg. It was noted that when cultured in a rich medium, the proportion of ABg cells (20–30%) was much higher compared to the minimal medium coculture where the proportion of ABlux was approximately 6% by the end of the cultivation. The relatively high biomass of ABg did not, however, negatively affect ECg growth, which further supports the hypothesis of ADP1 not producing compounds inhibiting E. coli growth. proportions of the coculture strains (%, CFU), substrate and end-metabolite concentrations, and fluorescence signals were monitored throughout the 10-hour cultivations (Figure 5). The coculture started to grow clearly faster after four hours of cultivation, reaching a final optical density of 13, which was ,3-fold higher compared to the biomass of ECg monoculture (OD 4.3). The initial proportions of the two strains were equal but the proportion of ABg seemed to increase fastest between the 6–8 h timepoints, assumingly in parallel with the exponential growth phase of ECg. It was noted that when cultured in a rich medium, the proportion of ABg cells (20–30%) was much higher compared to the minimal medium coculture where the proportion of ABlux was approximately 6% by the end of the cultivation. The relatively high biomass of ABg did not, however, negatively affect ECg growth, which further supports the hypothesis of ADP1 not producing compounds inhibiting E. coli growth. The fluorescence signals differentiated after 4 hours of cultivation, the final fluorescence signals being approximately 4-fold higher in the coculture compared to the ECg monoculture. The higher biomass and consequently the higher fluorescence signal of the coculture can be explained by the more propitious growth conditions; in both cultures, pH started to drop after 3–4 hours of cultivation, but the decline of pH in ECg monoculture was faster compared to the coculture. In the monoculture, the final pH of 5.4 was reached at 8–9 h timepoint. In the coculture, pH decreased more moderately, but increased rapidly after the 8 h timepoint, reaching a final pH of 6.5, which is very close to the initial pH of PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 11 / 16 doi:10.1371/journal.pone.0113786.g005 Engineered Synthetic Coculture for Improved Performance such conditions (data not shown), suggesting that the relevance of the coculture lies for the most part in the interactive glucose-acetate metabolism. The previous minimal medium cultivations constituted of ECsf and ABc strains demonstrated that both biomass and product titers could be improved, even though ABc was not contributing to the protein production. In a minimal medium, ABc is completely dependent on the carbon provided by ECsf growth, and the proportion of ABc cells is low, whereas rich medium enables more rapid and independent growth of ABc (as for ABg), resulting in significantly higher cell proportions consuming valuable building blocks from protein production. Thus, we were interested to see how the increased number of ABc cells affects the protein production in a rich medium when only ECg contributes to protein production (Figure S1). In short, it was observed that the bioprocess trends, acetate production, glucose consumption, and biomass production were very similar compared to the ECgABg bioreactor coculture. According to the fluorescence signals, the recombinant protein titer was 50% higher in the ECgABc coculture compared to the ECg monoculture, which is expectedly less than improvements gained in ECgABg coculture. It can be concluded that as protein overexpression is highly dependent on the available biocomponents consumed by both the strains, it is not energetically affordable to maintain the system in a rich medium with high proportions of the supportive strain, without it participating in the production. Thus, the availability of genetic tools and the possibility to rationally engineer and commit both the strains to the production are crucial to take full advantage of the carbon redirection, as demonstrated here. Furthermore, the product example (recombinant protein) is constituted of amino acids instead of carbon based molecules, and thus the benefits of carbon rerouting could be potentially better exploited in production of hydrocarbons, such as fatty acid derived compounds for bioenergy [22, 23]. Our findings suggest it is possible to tune the coculture balance, nature, and dynamics by altering the medium, substrate concentration, and genetic constructs. g The accumulation of acetate is a widely recognized problem in bioprocessing of E. coli. This study demonstrates that by exploiting a coculture with optimally chosen strains, the growth, carbon utilization, and product formation can be improved through symbiotic interactions, and the negative effects of acetate can be diminished. It was found that the coculture does not grow sufficiently in PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 12 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 Conclusions Synthetic biology broadens the possibilities to tune sophisticated production platforms, and coculturing is seen as a promising new frontier for taking the bioproduction to the next level [35, 36]. This study demonstrates that rationally engineered synthetic cocultures can improve biomass production, culture viability, and product formation in simple unoptimized batch conditions. The study extends applications of novel type of bioprocess optimization, and provides clues for the development of functional, readily engineered, and dynamic cocultures for synthetic biology applications. Engineered Synthetic Coculture for Improved Performance bioprocesses have demanded decades of optimization work to build up a cost- effective bioproduction platform [34]. Thus, improvement of the utility, profitability, and viability of cocultures requires significant research efforts. It is impossible to exceed the production rates and yields of highly tuned and optimized systems at a single cell level, but the power of cocultures most probably lie in processes that cannot be readily optimized (e.g. processes exploiting diverse industrial streams) or in processes involving variable conditions. Supporting Information Figure S1. Bioreactor data of ECg and ABc coculture. doi:10.1371/journal.pone.0113786.s001 (PDF) Author Contributions Conceived and designed the experiments: SS VS. Performed the experiments: SS VS. Analyzed the data: SS VS. Contributed reagents/materials/analysis tools: SS. Wrote the paper: SS. Supervised and coordinated the study: MK VS. Acknowledgments We want to thank Dr. Anniina Kivisto¨ and Rahul Mangayil for their assistance in HPLC analyses, as well as Antti Larjo for the computational metabolic analysis. 4. Rabinovitch-Deere CA, Oliver JW, Rodriguez GM, Atsumi S (2013) Synthetic Biology and Metabolic Engineering Approaches To Produce Biofuels. Chemical reviews. PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 3. Purnick PE, Weiss R (2009) The second wave of synthetic biology: from modules to systems. Nature reviews Molecular cell biology 10: 410–422. 2. Lee SK, Chou H, Ham TS, Lee TS, Keasling JD (2008) Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 19: 556–563. Employing the coculture enabled the use of a high substrate concentration in a simple batch culture, and the over-flow metabolism of E. coli was successfully exploited in producing the protein of interest. Fluctuations in sugar concentrations in industrial feedstocks can impose restrictions for single strain cultures (6), whereas high substrate concentration is not an issue for the coculture system presented here. Thus, with regards to sustainable and economical carbon sources, exploiting a well-designed coculture may broaden the possibilities to exploit challenging liquors with high concentrations of carbohydrates. Nevertheless, cocultures are complex systems involving numerous interactions. Despite the possibility to reduce these interactions, it is very difficult to identify all the mechanisms that are potentially beneficial for the culture. E. coli based 13 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786 December 3, 2014 References 1. Keasling JD (2008) Synthetic biology for synthetic chemistry. ACS chemical biology 3: 64–76. 1. Keasling JD (2008) Synthetic biology for synthetic chemistry. ACS chemical biology 3: 64–76. 1. Keasling JD (2008) Synthetic biology for synthetic chemistry. ACS chemical biology 3: 64–76. 2. Lee SK, Chou H, Ham TS, Lee TS, Keasling JD (2008) Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. 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Separable States on Density Matrices and Emergent Systems
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Yasmín Navarrete 1,*,† and Sergio Davis 2,3,† Yasmín Navarrete 1,*,† and Sergio Davis 2,3,† 1 Instituto de Filosofía y Ciencias de la Complejidad, Los Alerces 3024 Ñuñoa, Santiago 7780192, Chile Research Center in the Intersection of Plasma Physics, Matter and Complexity (P mc), Comisión Chilena de Energía Nuclear, Casilla 188-D, Santiago 8320000, Chile g g 3 Departamento de Física, Facultad de Ciencias Exactas, Universidad Andres Bello, Sazié 2212 piso 7, Santiago 8370136, Chile 3 Departamento de Física, Facultad de Ciencias Exactas, Universidad Andres Bello, Sazié 2212 piso 7, S i 8370136 Chil 3 Departamento de Física, Facultad de Ciencias Exactas, Universidad Andres Bello, Sazié 2212 piso 7, Santiago 8370136, Chile g * Correspondence: yasmin.navarrete@gmail.com † These authors contributed equally to this work. Abstract: In this paper, we present an analytical description of emergence from the density matrix framework as a state of knowledge of the system, and its generalized probability formulation. This description is based on the idea of fragile systems, wherein the observer modifies the system by the measurement (i.e., the observer effect) in order to detect possible emergent behavior. We propose the use of a descriptor, based on quantum mutual information, to calculate if subsystems of systems have inner correlations. This may contribute to a definition of emergent systems in terms of emergent information. Keywords: emergence; quantum theory; density matrix; subadditivity 1. Introduction From quantum mechanics, it is well known that any separable state belongs to a composite space that can be factored into individual states from separate subspaces. One state is said to be correlated if it is not separable. As a matter of fact, to determine if a state is separable or not is not trivial and the problem is classed as NP-hard from the theory of complex systems [1]. Citation: Navarrete, Y.; Davis, S. Quantum Mutual Information, Fragile Systems and Emergence. Entropy 2022, 24, 1676. https:// doi.org/10.3390/e24111676 Academic Editor: Masahito Hayashi Received: 12 September 2022 Accepted: 11 November 2022 Published: 17 November 2022 Citation: Navarrete, Y.; Davis, S. Quantum Mutual Information, Fragile Systems and Emergence. Entropy 2022, 24, 1676. https:// doi.org/10.3390/e24111676 Academic Editor: Masahito Hayashi Received: 12 September 2022 Accepted: 11 November 2022 Published: 17 November 2022 entropy entropy entropy 2. Emergent Systems Several definitions of emergence exist, taking into account different aspects of their origin or behavior. For instance, Peter Checkland [7] defines emergence as “the principle that entities exhibit properties which are meaningful only when attributed to the whole, not to its parts”. Emergent systems are structured in such a way that their components interact, allowing for the structure of global patterns, depicted as a consequence of interre- lations/correlations between subsystem elements, them being the result of complex and self-organizing processes. This process may be triggered by an external stimulus. There are basically three types of emergence: simple, weak and strong ones, described as follows: Simple emergence is composed by the combination of certain properties and relation- ships between elements in a non-linear behavior. For instance, in order to achieve the flight of an airplane, we cannot consider the motors, the propulsion system and their wings separately; all of these properties must be considered together because they are interconnected and they have interrelations through which flight emerges. This type of emergence can be predicted from the functioning of its parts and it is referred to as the concept of synergy, which means interacting or working together [8]. Weak emergence describes the emergence of properties of systems that may be pre- dictable (not completely) and also reducible. They can be reduced to basic rules at an initial time. After a while, the behavior can be unpredictable, as is mentioned in chaos theory [9]; nevertheless, it is possible to make computational simulations about such systems because of the knowledge of the basic rules. Weak emergence is the product of complex system dynamics (i.e., non-linear behavior, spontaneous order and adaptation); an example of the latter is cellular automaton, known as Conway’s Game of Life [10–12]. Strong emergence is a case of non-expected emergence, as well as weak emergence. The difference lies in its non-reducible behavior, which appears just when the system is running. As it is systematically determined by low-level attributes, it is not possible to deduce it from the components at lower levels. The consciousness phenomenon is one example of this type of emergence, and appears as a construction process. There is likely no algorithm from the bottom up because it is a dynamical process evolving along time at the highest level, with non-linear relations at the lower ones. Citation: Navarrete, Y.; Davis, S. Quantum Mutual Information, Fragile Systems and Emergence. Entropy 2022, 24, 1676. https:// doi.org/10.3390/e24111676 A density operator, in quantum mechanics, is used to describe the statistical state of a quantum system. The usual meaning of it is that the eigenvalues are the probabilities of finding a system in one state corresponding to the eigenvectors. In a physical sense, we can see its elements as relative frequencies corresponding to an appropriate ensemble of N identical copies of the system that are in several possible states under a certain setup or preparation protocol. Thus, we have a superposition of quantum states |vi⟩with probabilities pi (real numbers) satisfying ∑pi = 1. However, it can be shown that the density operator formalism can be recovered in a Bayesian formalism for noncommutative expectations, wherein the system depends on the order of the measurements. This is not restricted just to a quantum mechanical system, and can be understood under the framework of what we have called a fragile system [2]. This operator provides a convenient mean for describing quantum systems whose state is not completely known, it being mathematically equivalent to a state vector approach [3,4]. Academic Editor: Masahito Hayashi Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. By applying the entropy to the density matrix, we can obtain the degree of disinfor- mation of the state of the system. The systems can be composed of subsystems and, using the subadditivity property (the probability of the whole is less than that of its parts) [5], it is possible to quantify if the entropy of the whole is less than that of its parts. Holzer and De Meer [6] make a comparison between the information at the system level with the information at a lower level. As they state, “this measure gives a high value of emergence for systems with many dependencies (interdependent components) and a low value of emergence for systems with few dependencies (independent components)”; therefore, the Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/entropy Entropy 2022, 24, 1676. https://doi.org/10.3390/e24111676 Entropy 2022, 24, 1676 2 of 10 information of the whole is more than the information of the parts. In that sense, the entropy can be a good parameter to measure a type of emergence in systems. This paper is organized as follows. In Section 2, we talk about emergent systems and its current definitions. In Section 3, we define fragile systems as ones that are modified by the act of measurement because of the change in internal variables. In Section 4, we introduce the density matrix formalism. In Section 5, we depict a mathematical formulation of emergent systems within the density matrix formalism. In Section 6, we show a concrete example of a subadditive system. Finally, we provide some concluding remarks in Section 7. 2. Emergent Systems g Another way of conceptualizing emergence is the separation of levels of complexity of the system at different spatial or temporal scales [13]. y p p Some main characteristics of strong emergence to consider are the following: Non-reducible phenomenon: the global state of the emergent system cannot be explain- able, and neither is it reducible to its sub-system components. Downward causation: emergent high-level properties appear from a non-obvious con- sequence of low-level properties, but, at the same time, all processes at the lower level of hierarchy are constrained by and act in coherence with the laws of the higher level [14]. Wholeness: a phenomenon wherein a complex, interesting high-level function appears as a result of combining low-level mechanisms in straightforward ways. Entropy 2022, 24, 1676 3 of 10 3 of 10 Radical novelty emergence: a phenomenon wherein a system is designed according to certain principles. Interesting unexpected properties arise from the behavior of sub-system elements [15]. Radical novelty emergence: a phenomenon wherein a system is designed according to certain principles. Interesting unexpected properties arise from the behavior of sub-system elements [15]. In general, emergent systems are common in nature and technology. One example of the latter is the speed of a vehicle affected by the center of gravity, the driver skills, weather and friction, among other attributes (in this case, we can predict the emergent property of the speed from the relation between components, it being the case of weak emergence). It is possible to find strong emergent properties in the consciousness phenomenon [16], human body and social phenomena, among others. One important example is ’self-awareness’, which is a result of the interconnection of neurons in the brain [17]. In the case of biological systems (as well as in the case of social phenomena), emergent models are appropriate for describing those situations. We can observe the characteristics mentioned regarding strong emergence: non-reducibility, radical novelty, wholeness and downward causation. The main aspect of biological emergent systems is that they may be observed from inside them by the system (“from internal control of a system which might be fully con- trolled by an observer/controller architecture that is part of the system” [18]); this is in concordance with the autopoiesis theory proposed by the Chilean biologists H. Maturana and F. 3. Fragile Systems In simple terms, a fragile system is one that is affected by the measurement [21]. This distinguishes it from a non-fragile (classical) system, which is not modified upon observation. Because any system (being fragile or not) possesses information, we will think of a system as a “black box” that can be found in different internal states, which is to be denoted by λ. In general, λ contains many degrees of freedom, but we will not make use of that inner structure here. The internal state λ contains all of the information necessary to describe any aspect of the system. The crucial difference between a fragile system and a non-fragile one is that, in a fragile system, access to the internal state λ is impossible, because it is precisely this internal state that is modified by the measurement. As the modification of the state λ depends on the details of the environment when carrying out the measurement (which we do not know or control with accuracy), the outcome of a measurement is unavoidably stochastic, and a mathematical formulation requires probability theory [22]. 2. Emergent Systems Varela [19] to define the self-maintaining chemistry of any living cell, which is under the perspective of chemical organization theory used to formalize autopoietic structures, “providing a basis to operationalize goals as an emergent process” [20]. When the measure- ment is affected by the observer, it is the case of what we will call a fragile system. In the next section, we will go deeper into the concept. 4. The Density Operator In this section, we formulate the density matrix operator, as a previous concept, in order to define emergent systems using the calculation of entropies. The density operator is a positive semi-definite Hermitian operator of trace one. If A is the matrix representation of an arbitrary observable ˆA, we can write A =      a1 a2 ... aN      a1a2 · · · aN  . (1) (1) Entropy 2022, 24, 1676 4 of 10 Hence, we have Hence, we have Hence, we have A =      a1a1 · · · a1aN a2a1 · · · a2aN ... ... ... aNa1 · · · aNaN      where ai is the conjugate of the element ai, and, in the case of real numbers, the same element. We can take the average of different measurements represented by different matrices A of the same observable, and then normalize and diagonalize it; finally, we obtain the density matrix of a mixture as below: ˆρ = N ∑ i pi|v⟩⟨v| (2) (2) where pi = P(λi|I) and λi is obtained from the eigen-value problem λi|vi⟩= λi ˆA [21]. where pi = P(λi|I) and λi is obtained from the eigen-value problem λi|vi⟩= λi ˆA [21]. We can formulate the density matrix operator by the use of a complex Hilbert space just as in Von Neumann’s formulation of quantum theory [23]. For this, we consider an arbitrary orthonormal basis set |n⟩(n = 1, . . . , N) with ⟨i|j⟩= δ(i, j), and define the density operator [24] ˆρ as ˆρ := N ∑ i=1 N ∑ j=1 ρij|i⟩⟨j|, (3) (3) with ρij complex numbers. Imposing that ˆρ is Hermitian, we see that the diagonal elements ρii must be real and ρij = ρji. It is always possible to make a choice of such complex matrix elements ρij so that they are proportional to the elements pij; these are given by ρij = ⟨ϵi| ˆρ ϵj = 1 2η  [pij + pji] + i[pij −pji]  , (4) (4) where η = ∑i pii is a normalization factor that imposes Tr( ˆρ) = 1. Pure and Mixed States Consider an ensemble of measurements {|vi⟩}N i=1. If the state vector is known [25], the ensemble represents a pure state. Assuming that the system is in the state |v⟩, we can expand it with respect to the eigenvector of a Hermitian operator ˆA as follows: |v⟩ = ∑ n λn|vn⟩, (5) ˆA|v⟩ = λn|v⟩. (6) (5) (6) Finally, we can define a pure state by the following term [25]: Finally, we can define a pure state by the following term [25]: ˆρ = |v⟩⟨v|. (7) (7) Because ˆρ2 = (|v⟩⟨v|)(|v⟩⟨v|) = |v⟩(⟨v|v⟩)⟨v| = ˆρ, we can distinguish a density operator of a pure state by tracing; then, we have Because ˆρ2 = (|v⟩⟨v|)(|v⟩⟨v|) = |v⟩(⟨v|v⟩)⟨v| = ˆρ, we can distinguish a density operator of a pure state by tracing; then, we have Tr( ˆρ) = Tr  ˆρ2 = 1. (8) (8) When we cannot repeat exactly the same initial condition, because of the noise of the system, we represent this situation in a mathematical formulation in terms of an operator called a density matrix for mixed states. This is a superposition of pure states [26]: ˆρ = ∑ i pi|v⟩⟨v|, (9) (9) Entropy 2022, 24, 1676 5 of 10 where the weights of each measurement satisfy the normalization condition ∑i pi = 1 . Each pi is the probability of finding a system in a given pure state. where the weights of each measurement satisfy the normalization condition ∑i pi = 1 . Each pi is the probability of finding a system in a given pure state. p p y g y g p In contrast to a pure state, when we have a mixed-density matrix, the trace of the square density matrix is given by the inequality Tr  ˆρ2 < 1. (10) (10) Hence, Tr ˆρ2 , known as the degree of purity [25], can be used to distinguish between pure and mixed states in a basis-independent manner. 5. Density Operator Formalism and Emergent Systems Earlier work by Prokopenko et al. [13] has set the basis for a discussion of complexity, self-organization and the emergence of classical systems in information-theoretical terms, particularly in terms of Shannon entropy and mutual information. Taking all of this into account, we will rephrase the earlier arguments in terms of the (von Neumann) entropy of density matrices. However, first, let us review the behavior of the information entropy (or Shannon entropy) for classical, correlated systems. We adopt the Bayesian view by Jaynes [27] and others of the Shannon entropy related to the information content of a model based on, in principle, subjective probabilities, but consistent with known facts. Shannon entropy is then a measure of missing information in a probabilistic model about some aspect of reality, and is therefore dependent of the state of knowledge used to construct said model. For a state of knowledge I, where we ask an arbitrary question with N possible answers, denoted by the proposition A1, A2, . . . , AN, the Shannon entropy is defined as S(I) := − N ∑ i=1 pi ln pi (11) (11) where pi is the probability P(Ai|I) of the answer Ai being true under I. Please note that, for two ‘observers’ with different states of knowledge I1 and I2, the Shannon entropies S(I1) and S(I2) that they assign to an unknown question will, in general, be different. For instance, if the first observer knows that A1 is true, whereas the second only knows that either A1 or A2 is true, then S(I1) = 0 because P(Ak|I1) = δ1k, but S(I2) > 0 because 0 < P(A1|I2) < 1 and P(A2|I2) = 1 −P(A1|I2) > 0. In the case where the question involves the unknown value of one or more variable, the information entropy directly translates in terms of the probability distribution. For in- stance, for the joint probability distribution P(X, Y|I) of the variables X and Y under the state of knowledge I, we have SXY(I) := −∑ x ∑ y P(x, y|I) ln P(x, y|I). (12) (12) Using the product rule of probability, Using the product rule of probability, Using the product rule of probability, P(x, y|I) = P(y|x, I)P(x|I), (13) (13) this entropy can always be separated into two terms [28], SXY(I) = SX(I) + D SY|X E I, (14) (14) where the first term is the entropy of the variable X, and the second term, D SY|X E I := −∑ x P(x|I)∑ y P(y|x, I) ln P(y|x, I) (15) (15) Entropy 2022, 24, 1676 6 of 10 is the expected value of the conditional entropy of Y given X. This conditional entropy cannot be negative, it being the expected value of a non-negative quantity. is the expected value of the conditional entropy of Y given X. This conditional entropy cannot be negative, it being the expected value of a non-negative quantity. It is possible to extend the Bayesian idea of probabilities as degrees of belief constrained on the available information to quantum systems [29,30]. Let ρ12 = ρ1 ⊗ρ2. We can obtain the eigenvalues of ρ1 ai = {ρ1}ii and ρ2 bj = {ρ2}ii. Hence, the diagonal elements of ρ12 are given by all products of the form aibj, where i = 1, ..., n1 and j = 1, ..., n2. Here, n1 and n2 are the dimensions of ρ1 and ρ2, respectively; then: S(ρ12) = −Tr(ρ12 ln ρ12) (16) = − n1 ∑ i=1 n2 ∑ j=1 (aibj) ln aibj  . (16) On the other hand, the sum of the entropy of each system is given by On the other hand, the sum of the entropy of each system is given by S(ρ1) + S(ρ2) = − n1 ∑ i=1 ai ln(ai) − n2 ∑ j=1 bj ln bj  . (17) (17) Thus, the entropy for an ensemble (ρ12) for which the subsystems are uncorrelated is just equal to the sum of the entropies of the reduced ensembles for the subsystems. When there are correlations, we should expect an inequality instead (called the subadditivity property of entropy [5,31,32]), since, in this case, ρ12 contains additional information concerning the correlations, which is not present in ρ1 and in ρ2 (those are the partial traces of ρ12, respectively), as in (18) S(ρ12) ≤S(ρ1) + S(ρ2). (18) Given this inequality, we can use the mutual information formulation Given this inequality, we can use the mutual information formulation I := S(ρ1) + S(ρ2) −S(ρ12), (19) (19) such that I > 0 whenever there is subadditive behavior and I = 0 for additivity. Using the product rule of probability, From Equation (19), we obtain a descriptor of weak emergency in systems that are correlated. In these types of systems, new information emerges from the relation of its parts in terms of correlations. 6. An Example of a Subadditive System (25) Γ1 : a = 1, b = 1, Γ2 : a = 1, b = 3, Γ3 : a = 3, b = 1, Γ4 : a = 3, b = 3, Γ5 : a = 2, b = 2, Γ6 : a = 2, b = 4, Γ7 : a = 4, b = 2, Γ8 : a = 4, b = 4. (25) (25) Let us now define the measurement M(a, b) by M(a, b) :=          1 if a = b, 2, if a > b, 3 if a < b. (26) (26) such that the system undergoes the following transitions: Γ1 ↔Γ4, Γ5 ↔Γ8, Γ2 ↔Γ6 and Γ3 ↔Γ7. In this way, if the system has a = 3 and b = 1, that is, it is in the state Γ3, and we perform the measurement M, we will obtain a value M = 2 and the state will change to a′ = 4 and b′ = 2, that is, to Γ7. This makes the system fragile regarding the measurement M, with the integers a and b being the hidden variables of the system. Lastly, we will define the parity of each integer as 0 if the number is odd, and 1 if it is even. In this way, there are only four observable states, namely s1 = (0, 0), s2 = (0, 1), s3 = (1, 0), s4 = (1, 1), (27) (27) where the first and second position of the tuple represent the parity bit of a and b, respec- tively. Of these states, only s1 and s4 are consistent with the constraint I. Considering the allowed transitions, we have that where the first and second position of the tuple represent the parity bit of a and b, respec- tively. Of these states, only s1 and s4 are consistent with the constraint I. Considering the allowed transitions, we have that P(s, s′|I) =    1 4 if s ∈{s1, s4} and s′ ∈{s1, s4}, 0 otherwise. (28) (28) The matrix elements pij = P(s = si, s′ = sj|I) are given in Table 1, together with the density matrix elements ρij. The latter are simply ρij = 1 2η pij + pji + i[pij −pji]  = pij η = 2pij, (29) (29) because η = ∑4 i=1 pii = 1 2. 6. An Example of a Subadditive System As is depicted in Equation (4), we can write the density matrix as follows: ρij = ⟨ϵi| ˆρ ϵj = 1 2η  [pij + pji] + i[pij −pji]  . (20) (20) but where we now interpret pij as pij = P(ϵi, ϵ′ j|I), (21) η = ∑ i pii (22) (21) (22) (22) where primed states are states after a measurement. f h l l h ld h where primed states are states after a measurement. B f th i li ti l it t h ld th t where primed states are states after a measurement. Because of the marginalization rule, it must hold that ∑ i pij = P(ϵ′ j|I) for all j = 1, . . . , N, ∑ i pij = P(ϵ′ j|I) for all j = 1, . . . , N, (23) (23) while, simultaneously, while, simultaneously, ∑ j pij = P(ϵi|I) for all i = 1, . . . , N (24) ∑ j pij = P(ϵi|I) for all i = 1, . . . , N (24) Entropy 2022, 24, 1676 7 of 10 7 of 10 must be true. As an example, consider an abstract system composed of two integers, a and b, such that a ∈{1, 2, 3, 4} and b ∈{1, 2, 3, 4}. Let us set the constraint I that a and b are either both even or both odd; then, there are eight allowed states, namely must be true. As an example, consider an abstract system composed of two integers, a and b, such that a ∈{1, 2, 3, 4} and b ∈{1, 2, 3, 4}. Let us set the constraint I that a and b are either both even or both odd; then, there are eight allowed states, namely Γ1 : a = 1, b = 1, Γ2 : a = 1, b = 3, Γ3 : a = 3, b = 1, Γ4 : a = 3, b = 3, Γ5 : a = 2, b = 2, Γ6 : a = 2, b = 4, Γ7 : a = 4, b = 2, Γ8 : a = 4, b = 4. 6. An Example of a Subadditive System The states given by Equation (27) are equivalent to the ket notation (|00⟩, |11⟩, |01⟩, |10⟩). The density matrix elements ρij correspond to the density operator of a pure, but entangled state, ˆρAB = 1 2  |00⟩⟨00| + |00⟩⟨11| + |11⟩⟨00| + |11⟩⟨11|  = Φ+ Φ+ (30) (30) Entropy 2022, 24, 1676 8 of 10 with |Φ+⟩as one of the Bell states, with |Φ+⟩as one of the Bell states, Φ+ := 1 √ 2  |00⟩+ ⟨11|  . (31) (31) Table 1. The set of joint probabilities pij = P(s = si, s′ = sj|I) (third column) for the fragile system arising from the two-integer example described in Section 6. The fourth column shows the values of the elements of the corresponding density matrix. i j pij ρij 1 1 1⁄4 1⁄2 1 2 0 0 1 3 0 0 1 4 1⁄4 1⁄2 2 1 0 0 2 2 0 0 2 3 0 0 2 4 0 0 3 1 0 0 3 2 0 0 3 3 0 0 3 4 0 0 4 1 1⁄4 1⁄2 4 2 0 0 4 3 0 0 4 4 1⁄4 1⁄2 The reduced density matrices are The reduced density matrices are ρA = 1 2 1 0 0 1  , ρB = 1 2 1 0 0 1  . Hence, ˆρAB ̸= ˆρA ⊗ˆρB, and the operator ˆρAB is not separable. The values of the von Neumann entropy are SA = SB = ln 2 and SAB = 0, the latter being a pure state; therefore, the system is subadditive. With this example, we have found a density operator that is not only correlated but could describe an emergent system, since the entropy of the whole is less than that of its parts. 7. Concluding Remarks In this work, we reviewed the idea of emergence in the context of quantum mechanics and fragile systems in order to detect emergent behavior in systems under the framework of the density matrix theory. We proposed the mutual information as a descriptor of emergence in fragile systems [21]. When the parameter I > 0, there is more information in the whole than its parts; therefore, new information emerges as a result of the interaction of the subsystems as correlations. We have presented a concrete example of an operator that could describe a subadditive system. We believe that this formulation may give some insights about adaptative, self- sustainable and interconnected models. In particular, we aim to describe how, in an entangled system, the observer is part of the observation by being an inseparable element of it; for instance, by considering the side effect that produces the act of measurement (i.e., the observer effect [33–35]). Entropy 2022, 24, 1676 9 of 10 9 of 10 Author Contributions: Conceptualization, Y.N.; methodology, Y.N. and S.D.; software, S.D.; vali- dation, Y.N. and S.D.; formal analysis, Y.N. and S.D.; writing—original draft preparation, Y.N. and S.D.; writing—review and editing, Y.N. and S.D. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by ANID FONDECYT 1220651 grant. Funding: This research was funded by ANID FONDECYT 1220651 grant. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References g g y pp mers, D.J. The Conscious Mind: In Search of a Fundamental Theory; Oxford Paperbacks: Oxford, UK, 1996. 16. Varela, F.J. 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Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum
EvoDevo
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Developmental coupling of larval and adult stages in a complex life cycle insights from limb Developmental coupling of larval and adult stages in a complex life cycle insights from limb © 2013 Lee 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. * Correspondence: ysuzuki@wellesley.edu †Equal contributors Department of Biological Sciences, Wellesley College, 106 Central St., Wellesley, MA 02481, USA Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum Alison K Lee†, Christie C Sze†, Elaine R Kim and Yuichiro Suzuki* Alison K Lee†, Christie C Sze†, Elaine R Kim and Yuichiro Suzuki* Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum L l Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum Lee et al. Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 RESEARCH Open Access Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 Background side-effects caused by gene silencing, it is difficult to know how alterations of larval limb morphologies during embryogenesis affect the adult morphology. Thus, in order to assess the continuity between larval leg and adult leg patterning, the larval appendage morphology was altered by molecular perturbations during regeneration, to assess the potential impact on the regenerated adult morphology. Tribolium is well suited for genetically perturbing regener- ation, given its short life cycle, fully sequenced genome [12], and amenability to functional analyses using RNA interference (RNAi) [13]. Importantly, it develops external larval appendages, such as legs and antennae, which are capable of complete regeneration in a relatively short amount of time [14]. The larvae are capable of regenerating their appendages, and within two molts, much of the larval appendage morphology is restored. Once they become a pupa or an adult, Tribolium do not regenerate their appendages. By disrupting larval leg re-patterning, we can begin to address how larval leg patterning and adult leg patterning might be linked. Complex life cycles, which contain two or more morpho- logically discrete postembryonic phases, are found ubiqui- tously among many metazoan taxa [1]. Such complex life cycles can serve as adaptive strategies that allow different developmental stages to evolve independently to occupy larger ecological niches [1]. For example, holometabolous insects, or insects that undergo complete metamorphosis, produce distinct larval and adult morphologies that are capable of freely adapting to different ecological habitats. The evolution of two morphologically distinct stages is thought to be a key innovation that greatly aided the diversification of these insects [1,2]. However, whether or not the larval morphology is completely free to evolve without influencing the adult phenotype remains unclear. The molecular mechanisms underlying the transition from larval to adult stages are well-understood, yet the cellular origins of the adult body remain an enigma. In higher dipterans and a number of other insect species, clusters of cells form a distinct tissue called an imaginal disc, which develops inside the larva. In fruit flies, much of the adult body forms from these imaginal discs, and the larval cells make limited contributions to the adult body [3]. However, in most other insects, adult structures do not arise from free-floating imaginal discs, but rather develop from cells associated with larval structures. In lepidopterans, for example, imaginal cells embedded within the larval tissues replace much of the larval-specific cells to generate the adult structures [4,5]. Background In other insects, such as beetles, polymorphic larval cells are thought to make direct contribution to the developing adult body [5,6], although more studies are needed before definitive conclusions can be drawn. In this study, we knocked down the expression of four transcription factors that have been previously identified as key regulators of normal appendage patterning: dachshund (dac), Distal-less (Dll), abrupt (ab) and spineless (ss). dac codes for a transcription factor required for development of the mid-limb segment along the proximal-distal (PD) axis in Drosophila and Tribolium [15,16]. In Manduca, its embryonic expression likewise suggests a conserved role in mid-limb segment patterning [15-17]. In Tribolium, Dac has been determined to play a critical role in both adult mid-leg segment patterning [9,18] and adult antennal segmentation [8]. dac expression in Tribolium has been analyzed in the embryonic stage [15], but whether it has a major function in patterning larval legs or antennae during this stage is not known. Given that most insects do not make their entire adult body from set-aside imaginal discs, the larval morphology likely imposes some constraint on the development of the adult morphology. To determine the extent to which various stages of a complex life cycle can independently evolve, an understanding of the developmental relation- ships among the different stages is essential. In this study, we examined the consequence of genetic perturbations during larval appendage regeneration, to examine the degree of developmental continuity that might exist between larval and adult morphologies. Dll is a limb-patterning gene encoding a homeodomain transcription factor required for distal limb development in insects [8,9,18-20]. In Tribolium, Dll is required for proper leg patterning along the PD axis during embryonic development [20] and metamorphosis [9,18]. Dll has also been established to be important for antenna segmentation in the developing Tribolium pupa [8] and embryo [20]. In addition to Dac and Dll, several additional transcrip- tion factors have also been identified to be important for patterning limbs during metamorphosis [8,9]. We chose to investigate the role of two of these transcription factors during regeneration: the Bric-a-brac Tramtrack Broad complex (BTB) protein, Ab, and the basic-helix-loop- helix-PAS transcription factor, Ss. Ab is a BTB-zinc finger transcription factor necessary for antennal and tarsal segmentation in Tribolium. Knockdown of ab in Tribolium larvae results in a reduction of the number of adult anten- nal segments [8] and fusion of tarsomeres of the adult legs [9]. Abstract Background: A complex life cycle, such as complete metamorphosis, is a key innovation that can promote diversification of species. The evolution of a morphologically distinct larval stage is thought to have enabled insects to occupy broader ecological niches and become the most diverse metazoan taxon, yet the extent to which larval and adult morphologies can evolve independently remains unknown. Perturbation of larval limb regeneration allows us to generate larval legs and antennae with altered limb morphologies, which may be used to explore the developmental continuity that might exist between larval and adult appendages. In this study, we determined the roles of several appendage patterning transcription factors, abrupt (ab), dachshund (dac), Distal-less (Dll), and spineless (ss), in the red flour beetle, Tribolium castaneum, during larval appendage regeneration. The functions of these genes in regenerating and non-regenerating limbs were compared using RNA interference. Results: During limb regeneration, dac and ss were necessary to re-pattern the same larval structures as those patterned during embryogenesis. Removal of these two genes led to larval appendage patterning defects that were carried over to the adult legs. Surprisingly, even though maternal knockdown of ab had minimal effects on limb allocation and patterning in the embryo, it was necessary for blastema growth, an earlier phase of regeneration. Finally, knockdown of Dll prevented the blastema-like bumps from re-differentiating into appendages. Conclusions: Our results suggest that, similar to vertebrates, the re-patterning phase of Tribolium larval limb regeneration relies on the same genes that are used during embryonic limb patterning. Thus, the re-patterning phase of regeneration is likely to be regulated by taxon-specific patterning mechanisms. Furthermore, Ab and Dll appear to play important roles during blastema proliferation and re-differentiation, respectively. Finally, our results show that continuity exists between larval and adult limb patterning, and that larval and adult leg morphologies may be developmentally coupled. Thus, the evolution of imaginal discs may have been a key step towards completely removing any developmental constraints that existed between larval and adult phenotypes. Keywords: Limb regeneration, Distal-less, Dachshund, Spineless, Abrupt, Tribolium castaneum, Patte * Correspondence: ysuzuki@wellesley.edu †Equal contributors Department of Biological Sciences, Wellesley College, 106 Central St., Wellesley, MA 02481, USA Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Page 2 of 16 Background During postembryonic development of Drosophila, We chose to work with a model holometabolous insect, Tribolium castaneum, whose legs and antennae undergo two major morphogenetic transitions during the life cycle: 1) embryonic to larval appendages during embryogenesis, and 2) larval to adult appendages during metamorphosis [7]. Knockdown studies in Tribolium have shown that the embryonic patterning of larval legs and the metamorphic patterning of adult legs rely on overlapping but distinct sets of genes [8-11]. Because embryonic gene knockdown often leads to lethality of the animal due to pleiotropic Page 3 of 16 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 mRNA isolation and cDNA synthesis Larvae were dissected in 1X-PBS (0.02 M phosphate, 0.15 M NaCl, 0.0038 M NaH2PO4, 0.0162 M Na2HPO4; pH 7.4) to remove the gut and the fat body. The remaining tissue was homogenized in Trizol (Invitrogen, Carlsbad, CA, USA), and RNA was subsequently extracted using chloroform, treated with DNase (Promega, Fitchburg, WI, USA) and precipitated in isopropyl alcohol. cDNA was synthesized from 1 μg of total RNA using the cDNA syn- thesis kit (Thermo Fisher Scientific, Pittsburgh PA, USA) following the manufacturer’s instructions. mRNA isolation and cDNA synthesis Larvae were dissected in 1X-PBS (0.02 M phosphate, 0.15 M NaCl, 0.0038 M NaH2PO4, 0.0162 M Na2HPO4; pH 7.4) to remove the gut and the fat body. The remaining tissue was homogenized in Trizol (Invitrogen, Carlsbad, CA, USA), and RNA was subsequently extracted using chloroform, treated with DNase (Promega, Fitchburg, WI, USA) and precipitated in isopropyl alcohol. cDNA was synthesized from 1 μg of total RNA using the cDNA syn- thesis kit (Thermo Fisher Scientific, Pittsburgh PA, USA) following the manufacturer’s instructions. Ab is involved in establishing and maintaining muscle attachments and morphogenesis of adult appendages: viable ab mutants have deformed antennae and bristles, gnarled legs and wing venation defects [21-23]. Its role during embryonic limb development is not known although it has been identified as a regulator of neuromuscular junction development in Drosophila, where it is expressed in muscle cells but not neurons [21,24]. Ss is also required for leg segmentation in Tribolium and Drosophila during metamorphosis [9,25]. Knockdown of ss in Tribolium results in the development of adult legs with fused tarsomeres. Background In addition, homeotic transforma- tions of the distal portions of the adult antennae into a tibia-like segment with a terminal claw result from ss knockdown during the larval stage [8,10]. Similarly, in Drosophila, Ss is required for specifying antennal identity, and ss mutants exhibit antenna-to-leg transformations [25-28]. During Tribolium embryonic antennal development, Ss plays a critical role in establishing larval antennal identity [10,29]. Silencing of ss during embryogenesis results in the formation of newly hatched larvae with legs instead of antennae [10,29]. However, its removal does not appear to influence embryonic leg development; thus, embryos develop larval legs normally even when ss is silenced, while ss knockdown during metamorphosis results in adults with fused tarsomeres. Cloning and double-stranded RNA (dsRNA) synthesis Sequences of Dll [AF317551], dac [XM_964678], ab [XM_969854] and ss [EU912437] were obtained from confirmed sequences deposited in GenBank [8,10,18], and primers were designed outside of highly conserved regions to avoid off-target knockdown of gene expression. The list of primers used for cDNA amplification is given in Table 1. The amplified cDNA product was isolated and subsequently cloned into a TOPO TA vector (Invitrogen, Carlsbad, CA, USA). Following plasmid identity confirm- ation by sequencing, plasmid DNA was linearized via restriction digestion. Each of the strands of the dsRNA was synthesized with the T3 and T7 MEGAscript Kits (Ambion) following the manufacturer’s instructions. Single-stranded RNA (ssRNA) was combined and annealed to form dsRNA [30]. The annealed product was analyzed by gel electrophoresis to confirm annealing, and stored at −80°C until use. The final concentrations of the dsRNA were 2 μg/μL for ab, dac, Dll and ampr (plasmid gift from Dr Takashi Koyama, Gulbenkian Institute, Lisbon), and 2 μg/μL or 4 μg/μL for ss. We hypothesized that the removal of patterning genes re- quired for larval limb development would also be required for larval limb regeneration. We also hypothesized that disrupting the larval limb patterning during regeneration would influence the adult limb patterning and illustrate the continuity that exists between larval and adult limb patterning. Thus, we determined the phenotypes of em- bryonic appendages resulting from silencing Dac and Ab, and the effects of dac, Dll, ab and ss knockdowns during larval limb regeneration in Tribolium. We found that both Ab and Dll are necessary for limb regeneration prior to patterning, suggesting regeneration-specific functions of these genes. Background We also found that perturbed patterning of larval limbs leads to corresponding alterations in the adult limb morphology, indicating the continuity of patterning across larval and adult structures. DsRNA injection A l Approximately 0.5 μg (0.25 μL) of dsRNA was injected into the dorsal side of the day-0 fifth or sixth instar larvae (the final instar in our colony is typically the seventh or eighth instar), between the first and second abdominal segments, with a pulled 10-μL glass capillary needle connected to a syringe. Control larvae were injected with the same amount of bacterial ampr dsRNA. To visualize the effects of dsRNA on developing embryos, parental RNAi was conducted on female Tribolium adults [20]. Approximately 0.5 μL of dsRNA was injected into the abdominal body cavity of the adult female insects. The eggs from the dsRNA-injected female adults were separated out from the flour every 4 to 5 days, starting one week after dsRNA injection. The eggs were maintained at 29°C for another 4 to 5 days to allow the eggs to develop. The developing unhatched embryos or early hatched larvae (first instar) were then processed with lactic acid to clear the egg shell and the hard cuticle for fluorescent Results Embryonic functions of dac and ab We first report the effects of maternal dac and ab dsRNA injection on embryonic appendage development, since knockdown embryonic phenotypes for these genes have not yet been reported. The results of maternal RNAi are summarized in Table 2. Dll and ss knockdown produced phenotypes similar to those reported previously (see Additional file 1 and Tables 2 and 3) [10,20,29,33]. Leg and antennal ablations All of the dac knockdown embryos (n = 47) (Table 3) developed normal antennal morphology (Figure 1E), similar to antennae of the control ampr dsRNA-treated embryos (Figure 1B). In contrast, 49% of the dac dsRNA-injected embryos (Table 3) had partially fused femur and tibiotarsus (Figure 1D and 1F), which were not seen in the ampr dsRNA-injected control embryos (Figure 1A and 1C). The coxa and trochanter, however, were unaffected in size or segmentation. Thus, dac knock- down caused a reduction in size of the femur-tibiotarsal leg segments, whereas the antennae remained unaffected (Figure 1E and 1F). Two days after dsRNA injections, the larval mid- and hindlegs were ablated at the coxa on one side [14]. The larvae were placed on double-sided tape after anesthesia, and the legs were ablated using a pair of microscissors. The forelegs and the contralateral mid- and hindlegs served as internal controls for analyzing the effect of each dsRNA on regeneration of larval and adult legs. The larval antenna from the left side of the head was ablated using a razor blade two days after dsRNA injection. Anesthetized larvae were placed ventral side uppermost on double-sided tape, and the antennae were sliced off using the razor blade. The contralateral antenna served as the internal control. Larvae were stored in a 70% ethanol: 15% glycerol solution until imaged. The legs and heads were mounted in 80% glycerol and imaged using light and fluorescence microscopy, respectively. The majority of ab knockdown embryos produced through maternal RNAi exhibited an abnormal scrunched phenotype (Table 3; Figure 1G-I), where the body and appendages all appeared broader and compressed. The antenna (n = 57) and legs (n = 58) of the ab-knockdown embryos both exhibited this characteristic scrunched phenotype, where the cuticle failed to be fully extended. However, these appendages had the typical number of segments in the right proportions. This suggests that Ab does not affect the structural formation or patterning of these appendages in the embryo. Beetle husbandry Tribolium casteneum strain GA-1 was obtained from Dr Richard Beeman (USDA ARS Biological Research Unit, Grain Marketing & Production Research Center, Manhattan, KS, USA). Beetles were raised on organic wheat flour containing 5% nutritional yeast, and stocks were maintained in plastic containers in a 29°C incubator with approximately 50% relative humidity. No ethical approval was necessary to work with Tribolium as they are common household insect pests. Page 4 of 16 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Table 1 Primers used for ab, dac, Dll and ss dsRNA synthesis and knockdown verification Gene Application Forward primer (5′->3′) Reverse primer (5′->3′) ab RNAi ACTCACAAAGGAGAAGGGAAA CGTTGGTATTGAAAGGATGG Knockdown verification GCCTGTGATGGATGTTCGT GTCTTGGGTCTGTCGCTCT ss RNAi ATTACTCAAAACTGGCGCTTC TGTTGTGTTAGTGGGAGGAGTT Knockdown verification GGAGATACCGCAGAAGGAAG TTGTAGTCAGGGGCGATGT dac RNAi CAGCATCGCATCTTCAAC CTCCTCCCTCAGCCTTTCT Knockdown verification ACTGCACTACGGCCAGTTC TCGTCCATGTCTTGATCCTT Dll RNAi GGATAACAAACCCTTCACGAC GCCTCTCCAACGATAAACAC Knockdown verification GGTGTGTTCGTAGTGCTTCC CGCCTTCATCATCTTCTTGT RNAi, RNA interference; ab, abrupt; ss, spineless; dac, dachshund; Dll, Distal-less. microscopy as previously described [31,32]: unhatched embryos were incubated in 75% lactic acid, while hatched larvae were incubated overnight at 60°C in 10% ethanol: 90% lactic acid solution. These were then stored in 70% ethanol: 15% glycerol solution, or mounted in 80% glycerol. Embryos were imaged and scored using fluorescence microscopy. microscopy as previously described [31,32]: unhatched embryos were incubated in 75% lactic acid, while hatched larvae were incubated overnight at 60°C in 10% ethanol: 90% lactic acid solution. These were then stored in 70% ethanol: 15% glycerol solution, or mounted in 80% glycerol. Embryos were imaged and scored using fluorescence microscopy. Knockdown verification d h h To determine whether gene expression was knocked down in response to dsRNA injection, gene expression was analyzed using semiquantitative PCR. Day-0 sixth-instar larvae were injected with dac, Dll, ab, ss or control ampr dsRNA. For each treatment, two days after molting into the seventh instar, three larvae were pooled in Trizol and homogenized to isolate their RNA. After DNAse treat- ment, 1 μg of RNA was converted to cDNA and amplified using semiquantitative PCR. The PCR was repeated until the optimum cycle numbers were obtained, such that the bands were unsaturated. The following cycle numbers were used: dac = 42; Dll = 39; ab = 38; ss = 40; rp49 = 34. Besides the scrunched phenotype, ab knockdown dramatically decreased the number of bristles that were present on the surface of the embryonic cuticle. Compared to the ampr dsRNA-injected embryos (Figure 1J), there were fewer bristles present on the dorsal tergites of ab knockdown embryos (Figure 1K). This suggests that Ab is important for embryonic bristle formation and cuticular expansion, but not for appendage segmentation or patterning. Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Page 5 of 16 Page 5 of 16 Table 2 Summary of the knockdown effects observed in this study Appendage Gene No ablation Ablation Maternal RNAi Larval RNAi Larval RNAi Larva Larva Pupa/adult Larva Leg Dll Loss of distal leg segments Loss of tissue identity and regression Loss of tissue identity and regression Bump formation and no outgrowths dac Partial fusion of femur and tibiotarsus No effect Loss of distal femur and tibia and fusion of proximal tarsomeres Partial fusion of femur and tibiotarsus ab Slightly compressed but segment number is unaffected No effect Partial loss of tarsomeres Small bump formation and no outgrowths ss No effect No effect Fusion of tarsomeres No effect Antenna Dll Loss of distal antennal segments Loss of tissue identity Partial transformation into a leg Bump formation and no outgrowths dac No effect No effect Partial loss of antennal segments No effect ab Slightly compressed, but segment number is unaffected No effect Partial loss of antennal segments No outgrowths in some larvae; others no effect ss Partial transformation into a leg No effect Partial transformation into a leg Partial transformation into a leg The leg and antennal phenotypes observed after maternal RNA interference (RNAi) or larval RNAi of ab, dac, Dll and ss expression with and without ablation are summarized. Knockdown of dac expression prevents regeneration of the medial segments of legs When smaller larvae were injected, the larvae molted three times, allowing us to see the effect of dac knockdown on the regenerating larval leg. When ampr dsRNA-injected larvae molted three times, the morphology of all segments was restored (Figure 3A). In contrast, when dac expression was knocked down, the femur and tibiotarsus of the regenerated legs were partially fused (Figure 3B). This leg phenotype was similar to the embryonic legs of dac-knockdown larvae (compare Figures 1F and 3B). We next examined the effects of larval RNAi during regeneration. All of the phenotypic effects observed in this study are summarized in Table 2. To examine the role of Dac during Tribolium leg regeneration, dac dsRNA was injected into day-0 fifth- or sixth-instar larvae. Two days later, the mid- and hindlegs on one side of these larvae were ablated, and the regeneration of the ablated append- age after every molt was recorded (Table 4). After one molt, the site of ablation was completely healed in all larvae (n = 18) (Table 4; Figure 2A and C). After two molts, all larvae (n = 16) began to regenerate truncated legs (Table 4). The regenerating mid- and hindlegs of the dac dsRNA-injected animals had regenerated the coxa, trochanter and distal claw, but the femur and tibiotarsal segment appeared to be shortened (Figure 2D) compared to the ampr dsRNA-injected regenerating limbs (Figure 2B). As reported previously, adults that developed after dac dsRNA-injection in the larval phase lacked the distal femur and tibia and had fused proximal tarsomeres [9,18]. When legs were ablated in these dac dsRNA-injected animals, the regenerated legs in adults that underwent two molts prior to metamorphosis had smaller femurs and fused tarsomeres, unlike those of the uncut contralat- eral legs (n = 12) (Table 4; Figure 4C and D). Knockdown verification d h h Dll, Distal-less; dac, dachshund; ab, abrupt; ss, spineless. Table 2 Summary of the knockdown effects observed in this study Table 2 Summary of the knockdown effects observed in this study Appendage Gene No ablation Knockdown of dac expression prevents regeneration of the medial segments of legs Eggs and newly hatched larvae from female beetles injected with of 2 μg/μL ampicillin-resistance (ampr) (A-C), dachshund (dac) (D-F) and abrupt (ab) (G-I) double-stranded (ds)RNA were collected and processed to compare the phenotypes of resulting antennae and legs. Newly hatched ampr dsRNA-treated larvae were used as a control. The antennae (B,E,H) and legs (C,F,I) were visualized under fluorescence microscopy and are marked with arrowheads. Right panel shows a color-level inversion image with the leg segments highlighted in yellow. (J) Lateral view of the abdomen of a newly hatched larva obtained from female adults injected with ampr dsRNA. (K) Lateral view of the abdomen of the late embryo obtained from female adults injected with ab dsRNA. Scale bars represent 0.1 mm. Figure 1 Effects of patterning gene knockdown on embryonic appendage development. Eggs and newly hatched larvae from female beetles injected with of 2 μg/μL ampicillin-resistance (ampr) (A-C), dachshund (dac) (D-F) and abrupt (ab) (G-I) double-stranded (ds)RNA were Figure 1 Effects of patterning gene knockdown on embryonic appendage development. Eggs and newly hatched larvae from female beetles injected with of 2 μg/μL ampicillin-resistance (ampr) (A-C), dachshund (dac) (D-F) and abrupt (ab) (G-I) double-stranded (ds)RNA were collected and processed to compare the phenotypes of resulting antennae and legs. Newly hatched ampr dsRNA-treated larvae were used as a control. The antennae (B,E,H) and legs (C,F,I) were visualized under fluorescence microscopy and are marked with arrowheads. Right panel shows a color-level inversion image with the leg segments highlighted in yellow. (J) Lateral view of the abdomen of a newly hatched larva obtained from female adults injected with ampr dsRNA. (K) Lateral view of the abdomen of the late embryo obtained from female adults injected with ab dsRNA. Scale bars represent 0.1 mm. Knockdown of dac expression prevents regeneration of the medial segments of legs The femur and the segment corresponding to fused tarsomeres were Table 3 Summary of the effects of ab, dac, Dll and ss dsRNA injection on embryonic appendage development Treatment (dsRNA) Total number of embryos Antenna Leg Unaffected F/Sc/T* Short Unaffected F/Sc/T* Short ampr 49 49 (100%) 0 (0%) 0 (0%) 49 (100%) 0 (0%) 0 (0%) dac 47 47 (100%) 0 (0%) 0 (0%) 23 (49%) 24 (F)(51%) 0 (0%) Dll 53 4 (8%) 0 (0%) 49 (92%) 4 (8%) 0 (0%) 49 (92%) ss 58 0 (0%) 58 (T)(100%) 0 (0%) 58 (100%) 0 (0%) 0 (0%) ab 65 7 (11%) 57 (Sc)(88%) 1 (1%) 7 (11%) 58 (Sc)(89%) 0 (0%) *F/Sc/T denotes embryos that are fused (F), scrunched (Sc) or transformed (T). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. Eggs collected from female beetles injected with ab, dac, Dll and ss dsRNA (2 μg/μL) of were collected and processed to determine the phenotypes of the developing embryonic appendages, the antennae and legs. The ampr-treated embryos were used as the control. The antenna and legs were scored according to the following categories: unaffected, F/Sc/T (fused, scrunched, or transformed compared to the control), or short (lengths of segments are different from the control). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. mary of the effects of ab, dac, Dll and ss dsRNA injection on embryonic appendage development Table 3 Summary of the effects of ab, dac, Dll and ss dsRNA injection on embryonic appendage d *F/Sc/T denotes embryos that are fused (F), scrunched (Sc) or transformed (T). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. Eggs collected from female beetles injected with ab, dac, Dll and ss dsRNA (2 μg/μL) of were collected and processed to determine the phenotypes of the developing embryonic appendages, the antennae and legs. The ampr-treated embryos were used as the control. The antenna and legs were scored according to the following categories: unaffected, F/Sc/T (fused, scrunched, or transformed compared to the control), or short (lengths of segments are different from the control). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. Page 6 of 16 Lee et al. EvoDevo 2013, 4:20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Figure 1 Effects of patterning gene knockdown on embryonic appendage development. The fifth- or sixth-instar larvae were injected with ampr, dac, Dll, ab and ss dsRNA, and their legs were ablated. ampr dsRNA injections served as controls. *Regeneration in the dac dsRNA-injected insects was different from that in the control ampr dsRNA-injected insects. ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ab, abrupt; ss, spineless. Knockdown of dac expression prevents regeneration of the medial segments of legs Table 4 Summary of effects of patterning gene knockdowns on larval leg regeneration in Tribolium Larvae after 2 molts Pupae after 1 molt Pupae after 2 molts dsRNA injected Concentration Total number Number with regenerated legs Number with no regenerated legs Died before pupation after 2 larval molts Number with leg regeneration Number with no leg regeneration Number with leg regeneration Number with no leg regeneration ampr 2 μg/μL 23 10 0 3 13 0 7 0 dac 2μg/μL 18 16* 0 4 2 0 12* 0 Dll 2μg/μL 18 0 18 16 0 0 0 2 ab 2 μg/μL 28 0 19 13 0 9 0 6 ss 4 μg/μL 18 11 0 3 7 0 8 0 2 μg/μL 10 7 0 1 3 0 6 0 The fifth- or sixth-instar larvae were injected with ampr, dac, Dll, ab and ss dsRNA, and their legs were ablated. ampr dsRNA injections served as controls. *Regeneration in the dac dsRNA-injected insects was different from that in the control ampr dsRNA-injected insects. ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ab, abrupt; ss, spineless. ummary of effects of patterning gene knockdowns on larval leg regeneration in Tribolium Table 4 Summary of effects of patterning gene knockdowns on larval leg regene The fifth- or sixth-instar larvae were injected with ampr, dac, Dll, ab and ss dsRNA, and their legs were ablated. ampr dsRNA injections served as controls. *Regeneration in the dac dsRNA-injected insects was different from that in the control ampr dsRNA-injected insects. ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ab, abrupt; ss, spineless. Page 7 of 16 Page 7 of 16 Lee et al. EvoDevo 2013, 4:20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 http://www.evodevojournal.com/content/4/1/20 Figure 2 The effect of patterning gene knockdown on Tribolium larval leg regeneration one or two molts after leg ablation. (A,C,E,G,I,K) Regenerating larval legs after one molt in larvae injected with ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA (C), Distal-less (Dll) dsRNA (E), abrupt (ab) dsRNA (G), spineless (ss) dsRNA (2 μg/μl) (I) and ss dsRNA (4 μg/μl) (K). (B,D,F,H,J,L) Regenerating larval legs after two molts in larvae injected with ampr dsRNA (B), dac dsRNA (D), Dll dsRNA (F), ab dsRNA (H), ss dsRNA (2 μg/μl) (J) and ss dsRNA (4 μg/μl) (L). Arrowheads indicate the bases of the ablated legs. Middle panel shows the close-up image of the hindleg. Knockdown of dac expression prevents regeneration of the medial segments of legs Right panel shows a color-level inversion image with the regenerating leg segments highlighted in yellow. Cx, coxa; tr, trochanter; f, femur; tt, tibiotarsus. Scale bars correspond to 0.1 mm. Figure 2 The effect of patterning gene knockdown on Tribolium larval leg regeneration one or two molts after leg ablation. (A,C,E,G,I,K) Regenerating larval legs after one molt in larvae injected with ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA (C), Distal-less (Dll) dsRNA (E), abrupt (ab) dsRNA (G), spineless (ss) dsRNA (2 μg/μl) (I) and ss dsRNA (4 μg/μl) (K). (B,D,F,H,J,L) Regenerating larval legs after two molts in larvae injected with ampr dsRNA (B), dac dsRNA (D), Dll dsRNA (F), ab dsRNA (H), ss dsRNA (2 μg/μl) (J) and ss dsRNA (4 μg/μl) (L). Arrowheads indicate the bases of the ablated legs. Middle panel shows the close-up image of the hindleg. Right panel shows a color-level inversion image with the regenerating leg segments highlighted in yellow. Cx, coxa; tr, trochanter; f, femur; tt, tibiotarsus. Scale bars correspond to 0.1 mm. (Table 5; Figure 5B). After two molts, all of the dac dsRNA-injected larvae (n = 14) regenerated their cut antennae. They either partially developed a new segment distal to the site of ablation, or fully regenerated their larval antennae, as observed in the ampr dsRNA-injected animals (Figure 5B). All regenerated antennae were indis- tinguishable from the unablated contralateral antennae reduced in length, and in some cases, were missing. These data indicate that Dac plays a role in re-patterning the medial segments during leg regeneration and that the alteration in larval leg patterning is carried over to the adult leg patterning. The silencing of dac expression during the larval stage did not appear to affect the regeneration of antennae Page 8 of 16 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Figure 3 Regenerated legs of larvae after three molts post gene knockdown and leg ablation. (A-C) Regenerating larval legs injected with ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA (B) and spineless (ss) dsRNA (4 μg/μl) (C). The mid- and hindlegs (marked with arrowheads) were removed. A close-up of the hindleg is shown in the center, and right panels show a color-level inversion image with the regenerated leg segments highlighted in yellow. Fourth-instar larvae were injected on day 0, and legs were removed on day 2. Dll is essential for regeneration of appendages Dll is essential for regeneration of appendages Injection of Dll dsRNA into day-0 sixth-instar larvae followed by leg ablation resulted in no regeneration of limbs. After one molt, all of the larvae (n = 18) were able to heal the wound around the area of leg ablation (Figure 2E; Table 4), similar to the ampr dsRNA-treated animals. After two or more molts (Table 4), however, none of the Dll dsRNA-injected larvae were able to regenerate their ablated limbs (n = 18), even though the ampr dsRNA-treated animals regenerated their limbs. All of the ablated leg segments, such as the femur, tibiotarsus and claw, were missing (Figure 2F). In the absence of Dll, only a round blastema-like structure was present on the ablated legs after two molts (Figure 2F), whereas in the control ampr dsRNA-injected animals, all of the leg segments were re-established after two molts (Figure 2B). In addition, the morphology of the contralateral, unablated legs gradually became distorted with increasing number of Similarly, day-0 sixth-instar larvae (n = 10) injected with 2 μg/μL Dll dsRNA followed by antennal ablation did not regenerate any structures after two molts (Table 5; Figure 5C). They formed rounded large blastema-like bumps that failed to grow back any of the antennal seg- ments even after multiple supernumerary molts. Thus, Dll appears to be necessary for re-differentiation during larval appendage regeneration. Knockdown of dac expression prevents regeneration of the medial segments of legs Figure 3 Regenerated legs of larvae after three molts post gene knockdown and leg ablation. (A-C) Regenerating larval legs injected with ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA (B) and spineless (ss) dsRNA (4 μg/μl) (C). The mid- and hindlegs (marked with arrowheads) were removed. A close-up of the hindleg is shown in the center, and right panels show a color-level inversion image with the regenerated leg segments highlighted in yellow. Fourth-instar larvae were injected on day 0, and legs were removed on day 2. molts, indicating that Dll is required to maintain leg identity, as previously reported [18]. Most larvae continued to molt as larvae and never metamorphosed. This inability to metamorphose was also observed previously in unablated larvae, so it was likely independent of wounding. The two adults that metamorphosized after more than two molts post Dll dsRNA injection did not regenerate any structures on their ablated side (Table 4; Figure 4I). Given that the blastema-like bump in these Dll dsRNA-injected larvae were prominent, Dll appears to be required for re-differentiation of the blastema-like bump into a functional leg, but not for the formation of the bump. (Figure 6B) in all adults that pupated after two molts (n = 12), and both ablated and unablated antennae had reduced numbers of segments as reported previously [8,18]. This indicates that Dac does not play a major role during antennal regeneration, whereas it is necessary for proper antennal development during metamorphosis. Ab is required for blastema development The identity of the observed structure and the claw-like structures could not be determined, but the abnormal structure showed that Ab strongly influences growth/patterning during regener- ation, in addition to regulating the growth of blastemas. This was in contrast to the unablated legs of the adults obtained from ab dsRNA-injected larvae, which were affected such that the tarsomeres were reduced in number (Figure 4F), but the femur and the claws at the tip of the tarsus remained unaffected, as reported previously [8,9]. Thus, although silencing of ab only affects tarsal segmentation during metamorphosis, it prevents legs from re-forming during regeneration. The effect of removing larval Ab expression on the regeneration of antennae was variable. As with the ampr dsRNA-injected control animals, sixth-instar larvae injected with 2 μg/μL ab dsRNA after one molt were able to heal their wounds and sometimes form blastema-like structures. After two molts, a few larvae (n = 4) were able to regener- ate a new segment on the site of antennal ablation, whereas the rest (n = 7) did not regenerate any new struc- tures (Figure 5D; Table 5). However, the majority of the animals that were injected with ab dsRNA and underwent antennal ablation died as larvae, as they got trapped in the old cuticle during the larval-larval molts. The head often became trapped in the old cuticle, and the larvae eventually died, presumably from starvation. Ab is required for blastema development Similarly, the failed to show any signs of leg regeneratio structures were visible at the ablated area mass of unidentifiable tissue was observed w femur nor tibia regenerated (Figure 4E, arro particular adult that had developed a large the second larval molt post injection, a appendage developed (Figure 4F). The str sclerotized and exhibited normal pigmentatio development. However, this appendage had o with several abnormal protrusions, as well claw-like protrusions (Figure 4F) at the bound the coxa and the segment. The identity of t structure and the claw-like structures co determined, but the abnormal structure s Ab strongly influences growth/patterning dur ation, in addition to regulating the growth o Table 5 Summary of the effects following kno Larvae after 2 dsRNA injected Concentration Total number Number with regenerated antennae Nu no r a ampr 2 μg/μL 14 10 dac 2 μg/μL 27 14 Dll 2 μg/μL 10 0 ab 2 μg/μL 14 4 0.5 μg/μL 9 2 ss 2 μg/μL 22 21* The left antennae of fifth- and sixth-instar larvae treated with concentrations were ablated. The numbers of Tribolium treated regeneration was different from the control ampr dsRNA-inject all cases, after one molt, the wound healed and a smooth, rounded covering formed (Figure 2G). After the second molt, the legs had still not regenerated in the ab dsRNA-injected larvae (n = 19) (Table 4), and a small bump was observed (Figure 2H), indicating that little cell proliferation had taken place. Beyond the coxa, none of the larval leg segments were distinguishable. Although many larvae died prior to metamorphosis (n = 13), none of the animals that pupated after two larval molts regenerated pupal legs (n = 6). Similarly, the adults also failed to show any signs of leg regeneration: often no structures were visible at the ablated areas, or a tiny mass of unidentifiable tissue was observed with neither femur nor tibia regenerated (Figure 4E, arrows). In one particular adult that had developed a large bump after the second larval molt post injection, a malformed appendage developed (Figure 4F). The structure was sclerotized and exhibited normal pigmentation and bristle development. However, this appendage had one segment with several abnormal protrusions, as well as a pair of claw-like protrusions (Figure 4F) at the boundary between the coxa and the segment. Ab is required for blastema development We next examined the effect of silencing ab during the larval stage on the regeneration of ablated larval legs. In Page 9 of 16 Lee et al. EvoDevo 2013, 4:20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 http://www.evodevojournal.com/content/4/1/20 Figure 4 (See legend on next page.) Page 10 of 16 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 (See figure on previous page.) Figure 4 The effects of appendage patterning gene knockdowns on adult leg regeneration in Tribolium. (A,B) Adult leg phenotype of larvae injected with 2 μg/μL ampicillin-resistance (ampr) double-stranded (ds)RNA. Adults formed from pupae that experienced one molt (A) or two molts (B) after dsRNA injection. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. (C,D) Adults that formed after larvae were injected with 2 μg/μL dachshund (dac) dsRNA and underwent one molt (C) and two molts (D) before pupation. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. Arrowheads indicate the segment on the unablated leg that is missing in the ablated leg. (E,F) Adults that formed after larvae were injected with 2 μg/μL abrupt (ab) dsRNA and underwent two molts before pupation. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. (E) Arrows indicate the site where the legs should have regenerated. (F) Arrow indicates the claw-like structure that developed at the base of the leg. (G,H) Adults that formed after larvae were injected with 4 μg/μL ss dsRNA and underwent one molt (G) and two molts (H) before pupation. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. (I) Adults that formed after larvae were injected with 2 μg/μL Distal-less (Dll) dsRNA and underwent three molts before pupation. Arrows indicate the sites where the legs should have regenerated. all cases, after one molt, the wound he smooth, rounded covering formed (Figure 2G second molt, the legs had still not regenerat dsRNA-injected larvae (n = 19) (Table 4), bump was observed (Figure 2H), indicating th proliferation had taken place. Beyond the co the larval leg segments were distinguishabl many larvae died prior to metamorphosis (n of the animals that pupated after two l regenerated pupal legs (n = 6). Ab is required for blastema development Only two animals survived to metamorphose after one molt: one of them regenerated an antenna that resembled the unablated contralateral antenna, and the other became a pharate adult that did not exhibit any signs of antennal regeneration Table 5 Summary of the effects following knockdown of patterning genes on larval antennal regeneration in Tribolium Larvae after 2 molts Pupae after 1 molt Pupae after 2 molts dsRNA injected Concentration Total number Number with regenerated antennae Number with no regenerated antennae Died before pupation after 2 larval molts Number with antennal regeneration Number with no antennal regeneration Number with antennal regeneration Number with no antennal regeneration ampr 2 μg/μL 14 10 0 0 4 0 10 0 dac 2 μg/μL 27 14 0 2 10 3 12 0 Dll 2 μg/μL 10 0 7 7 0 3 0 0 ab 2 μg/μL 14 4 7 11 2 1 0 0 0.5 μg/μL 9 2 7 8 0 0 1 0 ss 2 μg/μL 22 21* 0 9 0 1 12* 0 The left antennae of fifth- and sixth-instar larvae treated with abrupt (ab), dachshund (dac), Distal-less (Dll) and spineless (ss) double-stranded (ds)RNA at various concentrations were ablated. The numbers of Tribolium treated with the ampicillin-resistance (ampr) dsRNA are also included as a control. *For ss, the pattern of regeneration was different from the control ampr dsRNA-injected regeneration; the regenerating antennae transformed into a leg. Table 5 Summary of the effects following knockdown of patterning genes on larval a 5 Summary of the effects following knockdown of patterning genes on larval antennal regenerati cts following knockdown of patterning genes on larval antennal regeneration in Tribolium The left antennae of fifth- and sixth-instar larvae treated with abrupt (ab), dachshund (dac), Distal-less (Dll) and spineless (ss) double-stranded (ds)RNA at various concentrations were ablated. The numbers of Tribolium treated with the ampicillin-resistance (ampr) dsRNA are also included as a control. *For ss, the pattern of regeneration was different from the control ampr dsRNA-injected regeneration; the regenerating antennae transformed into a leg. Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Page 11 of 16 Figure 5 Effects of patterning gene knockdown on larval antennal regeneration. Larvae were treated with 2 μg/μL ampicillin-resistance (ampr) (A), dachshund (dac) (B), Distal-less (Dll) (C), abrupt (ab) (D) and spineless (ss) (E) double-stranded (dsRNA). Left panels, the head of the treated larvae; middle panels, unablated antennae; right panels, ablated antennae. Ab is required for blastema development (E) Arrow indicates the claw-like structure in the regenerated antenna of ss dsRNA-injected larva. The arrowhead indicates the ectopic bristle on the transformed antenna. Figure 5 Effects of patterning gene knockdown on larval antennal regeneration. Larvae were treated with 2 μg/μL ampicillin-resistance (ampr) (A), dachshund (dac) (B), Distal-less (Dll) (C), abrupt (ab) (D) and spineless (ss) (E) double-stranded (dsRNA). Left panels, the head of the treated larvae; middle panels, unablated antennae; right panels, ablated antennae. (E) Arrow indicates the claw-like structure in the regenerated antenna of ss dsRNA-injected larva. The arrowhead indicates the ectopic bristle on the transformed antenna. dsRNA (Table 5). There were two larvae with regenerating antennae (developing new structures on the site of ablation) after two molts; one of these larvae molted successfully into an adult with a perfectly regenerated antenna that resembled the uncut control antenna. However, the remainder of the larvae (n = 7) did not show signs of antennal regeneration after two molts. In addition, decreasing the concentration to 0.5 μg/μL did not decrease the rate of larval mortality (8 larvae died after two molts). Thus, at either concentration, (not shown). The remaining larvae that survived past two molts (n = 11) were unable to properly eclose or meta- morphose, and ultimately died as larvae. The observed deaths were not confined to larvae with ablated limbs: many unablated Ab-knockdown larvae also died prior to metamorphosis (not shown). To see if we could obtain higher survival rates, we injected fifth-instar larvae (n = 9) with 0.5 μg/μL ab dsRNA. The degree of antennal regeneration was variable, as with the larvae injected with a higher concentration of Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Page 12 of 16 Lee et al. EvoDevo 2013, 4:20 Figure 6 Effects of patterning gene knockdown on adult antennal regeneration. Adults that formed after larvae were treated with 2 μg/μL ampicillin-resistance (ampr) (A), dachshund (dac) (B) and spineless (ss) (C) double-stranded (ds)RNA. All injected animals underwent two molts prior to the prepupal stage. Comparable pictures were taken under the same magnification. Figure 6 Effects of patterning gene knockdown on adult antennal regeneration. Adults that formed after larvae were treated with 2 μg/μL ampicillin-resistance (ampr) (A), dachshund (dac) (B) and spineless (ss) (C) double-stranded (ds)RNA. All injected animals underwent two molts prior to the prepupal stage. Comparable pictures were taken under the same magnification. Ss is not required for leg regeneration When ss was silenced prior to leg ablation during the larval stage, both ablated legs began to regenerate by the second molt (n = 11 for 4 μg/μL and n = 7 for 2 μg/μL; Table 4; Figure 2J). These legs were similar to the regen- erating legs of ampr dsRNA-injected larvae and regenerated all of the larval leg segments. When smaller larvae were injected, larvae that molted three times had restored the morphology of leg segments, similar to ampr dsRNA-injected larvae (compare Figure 3A and C). ss-knockdown without leg ablations resulted in the formation of adults with phenotypes similar to those obtained by Shippy et al. (2009) and Angelini et al. (2012) (Figure 4G and H) [9,10]. As described previously, the tarsomeres were fused and were shorter than those of the control ampr dsRNA-injected adults. In the adults obtained from ss dsRNA-injected larvae followed by leg ablations, the regenerated legs were morphologically indistinguishable from the contralateral unablated legs (Table 4; Figure 4G and H). From these results, we infer that Ss is not necessary for larval leg regeneration. In the unablated antenna of ss-knockdown larvae, the antennae transformed partially into a leg-like structure during metamorphosis (n = 14). These animals have one large structure that appears to comprise a fusion of the femur, tibia and tarsomeres (Figure 6C; [10]). The adult structure on the ablated side had distinct leg-like struc- tures with clearly distinguishable tibia and tarsomeres, in contrast to the contralateral unablated antennae, where the tibia and tarsomeres appeared to be fused. Presumably, the earlier transformation of the regenerating antenna into leg-like phenotype accounts for the more enhanced leg- like morphology observed in the ss-knockdown adult. Thus, similar to the effect of dac knockdown in regenerating legs, the antennal patterning defects in ss dsRNA-injected larvae were reflected in the adult antennal morphology. Taken together, our results indicate that Ss plays an im- portant role in maintaining the identity of the antennae during larval antennal regeneration and metamorphosis. When ss was knocked down in day-0 fifth- or sixth- instar larvae, all of the ablated antennae (n = 22) (Table 5) healed and formed rounded structures as seen in the control ampr-knockdown animals after the first molt (Figure 5A). After the second molt, all of the 2 μg/μL ss Ab is required for blastema development most ab dsRNA-injected larvae were unable to regen- erate their ablated antennae after two molts (n = 7/11 for 2 μg/μL and n = 7/9 for 0.5 μg/μL; Table 5). Combined, these results indicate that Ab is required for blastema development during regeneration of both legs and antennae. dsRNA-injected larvae (n = 21) developed leg-like struc- tures in the place of a regenerated antenna (Figure 5E; Table 5). The regenerated antennae developed a claw and a bristle pattern resembling those seen in larval legs (Figure 5E, arrow and arrowhead). The unablated antennae remained unaffected and did not transform, indicating that Ss is only required when the larval antenna is regenerating. Such heteromorphic transformation of regenerating appendages was never seen with the knockdowns of any of the other transcription factors investigated here. Discussion I hi d In this study, we examined the roles of limb-patterning genes during embryonic development and regeneration, and the degree of developmental coupling between larval and adult limb-patterning (Table 2). We found that dac- knockdown embryos developed legs with abnormally shortened medial segments, while the antennae developed normally. In addition, silencing of dac resulted in loss of the medial segments of the regenerating legs; this altered phenotype was carried over to the adult leg morphology. However, no major effects were detected in the regener- ating antennae. Knockdown of Dll completely inhibited regeneration of the ablated appendages, although a promin- ent blastema-like structure formed. ab-knockdown embryos exhibited a unique scrunched phenotype throughout their entire body, including their antennae and legs; however, each of the segments of these appendages was distin- guishable. These embryos also displayed abnormal bris- tle patterning on the dorsal tergites. Ab was found to be essential for blastema growth/development. Ss-knock- down larvae were able to regenerate their limbs, but a heteromorphic transformation of the larval antenna to a leg was observed when the antennae were removed. This larval heteromorphic effect was also carried over to the adult stage. Legs regenerated normally when the expression of ss was knocked down. However, knockdown of ss in the fifth or sixth instars affected the regeneration of the antennae. The ablated antenna underwent a heteromorphic transformation and developed into a structure that resem- bled a leg. A similar transformation of an antenna to a leg was also observed in embryos [10,29]. The embryonic phenotypes resulting from dsRNA treatment suggest that Ss is not involved in the embryonic patterning of legs but plays a key role in specifying antennal identity. In contrast, during metamorphosis, Ss is required for patterning the distal segments of the developing adult legs [8-10,25]. Taken together, the functions of Ss and Dac during regen- eration are similar to those seen during embryonic, rather than metamorphic, development (Table 2). Dac and Ss function similarly during regeneration and embryonic development Although dac dsRNA-injected Tribolium were able to regenerate both the larval legs and antennae after two molts (Tables 4 and 5), knockdown of Dac expression Knockdown verification We performed semiquantitative PCR to determine the gene expressions of dac, Dll, ab and ss in response to dsRNA injections. We confirmed that gene expression Page 13 of 16 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Page 13 of 16 disrupted the patterning of the medial segments in the re- generating legs (Table 2; Figure 3B). Our results indicate that Dac acts specifically in the legs to pattern the medial segments during regeneration, which is consistent with past studies of Dac in cricket leg regeneration [34]. In the antennae, Dac seems to have no apparent role during larval regeneration, and the regenerated adult antennae were indistinguishable from the unablated antennae (Figures 5B and 6B). Similar effects of patterning were observed in the dac-knockdown embryos: in embryos affected by dac dsRNA, the antennae developed normally, but the legs had a shortened femur-tibiotarsus segment, akin to the regenerated larval legs of dac-knockdown larvae. These findings are consistent with previous studies, which have determined that dac is most strongly expressed in the legs and less so in the antennae [15,35], suggesting that the role of Dac in patterning the medial segments of the antenna is largely confined to metamorphosis [8], and not regeneration or embryonic development. Thus, Dac func- tions similarly during larval appendage regeneration and embryonic development (Table 2). was knocked down through dsRNA injection by comparing the expression in ampr dsRNA-injected animals with that in the dsRNA-injected animals (Figure 7). Compared to the control larvae, the target gene expression was reduced in larvae injected with dsRNA of the respective genes (Figure 7). Thus, we established that the phenotypes shown in this study were the effects resulting from the knockdown of each gene. Developmental uncoupling between larval and adult morphologies An unresolved issue in holometabolous insect development and evolution is the developmental continuity between larval legs and adult legs. At the cellular level, there appears to be a smooth transition between the larval appendages and adult appendages in most insects except in the case of lepidopterans, where the adult appendages actually arise from set-aside imaginal cells that reside within the larval legs and proliferate during the final instar [4]. What is less clear is how patterning of larval append- ages affects adult appendages. Our study shows that larval appendage patterning is inherited by the adult appendages. For example, when Dac is silenced in the regenerating larval legs, the medial segments are deleted, and this patterning alteration is also observed in the adult stage. While unablated adult legs are also affected, the regenerated leg is more severely affected. This implies that either the fates of the presumptive adult cells are already specified by the time they pupate or the number of cells present in the larval appendage constrains the adult leg morphology. Regardless of the mechanism, our study indicates that larval appendage patterning might pose a developmental constraint on the adult appendages. Thus, while the evolu- tion of metamorphosis allowed the generation of two distinct morphs, the larva and the adult, which are each adapted to a different habitat, the two life-history stages may constrain each other, at least in some holometabolous insects, such as beetles. Unlike the effect of silencing ab on regeneration, Ab was not required for the initial allocation and subsequent for- mation of either antennae or legs during embryonic devel- opment (Table 2). During metamorphosis, the tarsomeres became fused in ab-knockdown animals; however, the rest of the leg was unaffected. Thus, while Ab is involved in the distal patterning of adult legs during normal development, Ab appears to play a distinct role during regeneration by affecting the development and growth of the blastema (Table 2). Together, these data indicate that the role of Ab during regeneration is separate from its embryonic and metamorphic function in normal development. The ab-knockdown phenotype is similar to that obtained when Wnt-1 expression is silenced. When Wnt-1 is silenced, the blastema fails to grow, and the legs are never able to regenerate, whereas the amount of antennal regen- eration is variable between individuals [14]. Thus, Ab and the Wnt signaling pathway may influence the same process during regeneration and may potentially interact. Developmental uncoupling between larval and adult morphologies It is interesting that both Wnt-1 and Ab knockdown animals form rudimentary wings during metamorphosis (unpub- lished observations and [14]). The heteromorphic transformations seen in ss knock- down animals have interesting implications for under- standing the developmental basis for naturally occurring heteromorphic transformations. The theory of homeotic transformation was first developed by Bateson (1894) to describe the replacement of one structure, such as the antennae, with a completely different one, such as the tarsus of a leg [53]. Though at first believed to be isolated examples of developmental anomalies, various studies have demonstrated that heteromorphic transformations could be experimentally induced by physical mutilation during late development [54,55]. For example, injuring the antennae of a sawfly, Cimbex axillaris, can cause the heteromorphic transformation of the adult antenna into a leg [55]. However, the molecular basis of postembryonic heteromorphic transformation of an appendage has not been well-characterized. Our study demonstrates that given the continuity of larval and adult stages, alteration of a single transcription factor, such as Ss, during a spe- cific time period of larval life can induce the homeotic Ab is required for initiation of regeneration specific embryonic patterning genes are used to re-pattern the appendages. Whether or not the first steps of limb re- generation, such as wound healing and blastema formation, are evolutionarily conserved processes remains to be seen. Our study shows that Ab plays an essential role in limb regeneration. Beyond the formation of a small bump, appendage regeneration appeared to be stunted in ab- knockdown animals. This indicates that Ab may be involved in cell proliferation of blastema cells during leg regeneration. The effects of ab knockdown on antenna regeneration, though less definitive given that the antennae of a few larvae regenerated after two molts (Table 5), also indicated that Ab is required for the initiation of antennal regeneration. Developmental uncoupling between larval and adult morphologies Dll is required for initiation of leg outgrowth during regeneration None of the animals injected with Dll dsRNA were able to regenerate any structure distal to the site of ablation in either the legs (Tables 2 and 4; Figure 2E, 2F and 4I) or the antennae (Tables 2 and 5; Figure 5C). For all of the Dll-knockdown animals, a large bump formed on the ablated sites and remained throughout the molts, indi- cating that the bump is able to grow. However, no append- ages ever differentiated from this bump, and the legs were absent from the adult (Figure 4I). At this point, we do not have any blastema markers so we are unable to determine the nature of this bump. However, given the previous finding that Dll is required to maintain the appendage identity [18], Dll may be required for re- differentiation of cells inside this bump. Figure 7 Knockdown verification of double-stranded RNA (dsRNA)-injected larvae using semiquantitative polymerase chain reaction. Day-0 sixth-instar larvae were injected with 2 μg/μL ampicillin-resistance (ampr), dachshund (dac), Distal-less (Dll), abrupt (ab) and spineless (ss) dsRNA, and total RNA was isolated from day-2 seventh-instar larvae. This RNA was converted to cDNA, and gene-specific primers were used to amplify the gene products. ampr dsRNA-injected larvae served as controls. The cycle numbers were as follows: ab = 38; dac = 42; Dll = 39; ss = 40; rp49 = 34. Figure 7 Knockdown verification of double-stranded RNA (dsRNA)-injected larvae using semiquantitative polymerase chain reaction. Day-0 sixth-instar larvae were injected with 2 μg/ ll ( r) d h h d (d ) l l ( ll) b Figure 7 Knockdown verification of double-stranded RNA (dsRNA)-injected larvae using semiquantitative polymerase chain reaction. Day-0 sixth-instar larvae were injected with 2 μg/μL ampicillin-resistance (ampr), dachshund (dac), Distal-less (Dll), abrupt (ab) and spineless (ss) dsRNA, and total RNA was isolated from day-2 seventh-instar larvae. This RNA was converted to cDNA, and gene-specific primers were used to amplify the gene products. ampr dsRNA-injected larvae served as controls. The cycle numbers were as follows: ab = 38; dac = 42; Dll = 39; ss = 40; rp49 = 34. Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Page 14 of 16 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Conclusions 8. Angelini DR, Kikuchi M, Jockusch EL: Genetic patterning in the adult capitate antenna of the beetle Tribolium castaneum. Dev Biol 2009, 327:240–251. 8. Angelini DR, Kikuchi M, Jockusch EL: Genetic patterning in the adult capitate antenna of the beetle Tribolium castaneum. Dev Biol 2009, 327:240–251. Our study indicates that larval limb morphology is linked to the adult limb patterning in Tribolium. We propose that the evolution of free-floating imaginal discs may have been a key step towards to completely removing the developmental constraint that existed between the larval and adult phenotypes. Thus, the evolution of insects is characterized by a gradual loss of developmental coupling between the juvenile and adult stages. 9. Angelini DR, Smith FW, Jockusch EL: Extent with modification: leg patterning in the beetle Tribolium castaneum and the evolution of seri homologs. G3 (Bethesda) 2012, 2:235–248. 9. Angelini DR, Smith FW, Jockusch EL: Extent with modification: leg patterning in the beetle Tribolium castaneum and the evolution of serial homologs. G3 (Bethesda) 2012, 2:235–248. g 10. Shippy TD, Yeager SJ, Denell RE: The Tribolium spineless ortholog specifies both larval and adult antennal identity. Dev Genes Evol 2009, 219:45–51. 10. Shippy TD, Yeager SJ, Denell RE: The Tribolium spineless ortholog specifies both larval and adult antennal identity. Dev Genes Evol 2009, 219:45–51. 10. Shippy TD, Yeager SJ, Denell RE: The Tribolium spineless ortholog specifies both larval and adult antennal identity. Dev Genes Evol 2009, 219:45–51. 11. Namigai EKO, Suzuki Y: Functional conservation and divergence of BMP ligands in limb development and lipid homeostasis of holometabolous insects. Evol Dev 2012, 14:296–310. y 11. Namigai EKO, Suzuki Y: Functional conservation and divergence of BMP ligands in limb development and lipid homeostasis of holometabolous insects. Evol Dev 2012, 14:296–310. 11. Namigai EKO, Suzuki Y: Functional conservation and divergence of BMP ligands in limb development and lipid homeostasis of holometabolous insects. Evol Dev 2012, 14:296–310. 12. Richards S, Gibbs RA, Weinstock GM, Brown SJ, Denell R, Beeman RW, Gibbs R, Bucher G, Friedrich M, Grimmelikhuijzen CJ, Klingler M, Lorenzen M, Richards S, Roth S, Schröder R, Tautz D, Zdobnov EM, Muzny D, Gibbs RA, Weinstock GM, Attaway T, Bell S, Buhay CJ, Chandrabose MN, Chavez D, Clerk-Blankenburg KP, Cree A, Dao M, Davis C, Chacko J: The genome of the model beetle and pest Tribolium castaneum. Nature 2008, 452:949–955. References 1. Moran NA: Adaptation and constraint in the complex life-cycles of animals. Annu Rev Ecol Syst 1994, 25:573–600. 2. Truman JW, Riddiford LM: The origins of insect metamorphosis. Nature 1999, 401:447–452. 2. Truman JW, Riddiford LM: The origins of insect metamorphosis. Nature 1999, 401:447–452. 3. Siegel JG, Fristrom JW: The biochemistry of imaginal disc development. In The genetics and biology of Drosophila vol. 2A. Edited by Ashburner M, Wright TRF. New York: Academic Press; 1978. 3. Siegel JG, Fristrom JW: The biochemistry of imaginal disc development. In The genetics and biology of Drosophila vol. 2A. Edited by Ashburner M, Wright TRF. New York: Academic Press; 1978. 4. Tanaka K, Truman JW: Development of the adult leg epidermis in Manduca sexta: contribution of different larval cell populations. Dev Genes Evol 2005, 215:78–89. 4. Tanaka K, Truman JW: Development of the adult leg epidermis in Manduca sexta: contribution of different larval cell populations. Dev Genes Evol 2005, 215:78–89. 5. Truman JW, Riddiford LM: Endocrine insights into the evolution of metamorphosis in insects. Annu Rev Entomol 2002, 47:467–500. 5. Truman JW, Riddiford LM: Endocrine insights into the evolution of metamorphosis in insects. Annu Rev Entomol 2002, 47:467–500. 6 Nagel RH: Metathetely in larvae of the confused flour beetle 5. Truman JW, Riddiford LM: Endocrine insights into the evolution of metamorphosis in insects. Annu Rev Entomol 2002, 47:467–500. 6. Nagel RH: Metathetely in larvae of the confused flour beetle (Tribolium confusum Duval). Ann Entomol Soc Am 1934, 27:425–428. p 6. Nagel RH: Metathetely in larvae of the confused flour beetle (Tribolium confusum Duval). Ann Entomol Soc Am 1934, 27:425–428. 6. Nagel RH: Metathetely in larvae of the confused flour beetle (Tribolium confusum Duval). Ann Entomol Soc Am 1934, 27:425–428. 7. Truman J, Riddiford L: The morphostatic actions of juvenile hormone. Insect Biochem Mol Biol 2007, 37:761–831. 7. Truman J, Riddiford L: The morphostatic actions of juvenile hormone. Insect Biochem Mol Biol 2007, 37:761–831. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 19. Cohen SM, Bronner G, Kuttner F, Jurgens G, Jackle H: Distal-less encodes a homoeodomain protein required for limb development in Drosophila. Nature 1989, 338:432–434. Additional file Additional file 1: Effects of Dll and ss knockdown on embryonic development. (A,D) Whole-body image of the embryo injected with Dll (A) and ss (D) dsRNA. (B) Head of a Dll knockdown embryo. Arrowheads point to the antennae that lacked the distal flagellum. (C) Legs of Dll-knockdown embryo that lacked the distal portions (arrowheads). Close-up image is shown to the right. (E) Head of a ss-knockdown embryo. Arrowheads indicate the antennae that had transformed into legs. (F) Legs were unaffected by ss knockdown. Dll, Distal-less; ss, Spineless; dsRNA, double-stranded RNA. 13. Tomoyasu Y, Denell RE: Larval RNAi in Tribolium (Coleoptera) for analyzing adult development. Dev Genes Evol 2004, 214:575–578. 14. Shah M, Namigai E, Suzuki Y: The role of canonical Wnt signaling in leg regeneration and metamorphosis in the red flour beetle Tribolium castaneum. Mech Dev 2011, 128:342–400. 15. Prpic NM, Wigand B, Damen WG, Klingler M: Expression of dachshund in wild-type and Distal-less mutant Tribolium corroborates serial homologies in insect appendages. Dev Genes Evol 2001, 211:467–477. Authors’ contributions AKL participated in the design of the study, carried out the molecular genetic studies and drafted the manuscript. CCS participated in the design of the study, carried out the molecular genetic studies and drafted the manuscript. ERK carried out the molecular genetic studies and helped to draft the manuscript. YS participated in the design and coordination of the study, carried out the molecular genetic studies and helped to draft the manuscript. All authors read and approved the final manuscript. 20. Bucher G, Scholten J, Klingler M: Parental RNAi in Tribolium (Coleoptera). Curr Biol 2002, 12:R85–R86. 21. Hu S, Fambrough D, Atashi JR, Goodman CS, Crews ST: The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections. Genes Dev 1995, 9:2936–2948. 22. Cook O, Biehs B, Bier E: Brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila. Development 2004, 131:2113–2124. Abbreviations 16. Mardon G, Solomon NM: Rubin GM: dachshund encodes a nuclear protein required for normal eye and leg development in Drosophila. Development 1994, 120:3473–3486. Ab: Abrupt; ampr: Ampicillin-resistance gene; BTB: Bric-a-brac tramtrack broad complex; dac: Dachshund; Dll: Distal-less; dsRNA: Double-stranded RNA; FGF: Fibroblast growth factor; PBS: Phosphate-buffered saline; Ab: Abrupt; ampr: Ampicillin-resistance gene; BTB: Bric-a-brac tramtrack broad complex; dac: Dachshund; Dll: Distal-less; dsRNA: Double-stranded RNA; FGF: Fibroblast growth factor; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; PD: Proximal-distal; RNAi: RNA interference; ss: Spineless; ssRNA: Single-stranded RNA. 17. Tanaka K, Truman JW: Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta. Dev Biol 2007, 305:539–550. g p PCR: Polymerase chain reaction; PD: Proximal-distal; RNAi: RNA interference; ss: Spineless; ssRNA: Single-stranded RNA. 18. Suzuki Y, Squires DC, Riddiford LM: Larval leg integrity is maintained by Distal-less and is required for proper timing of metamorphosis in the flour beetle, Tribolium castaneum. Dev Biol 2009, 326:60–67. Received: 20 March 2013 Accepted: 4 June 2013 Published: 4 July 2013 transformation of the regenerating larval antennae and subsequently influence the adult antennal identity. Received: 20 March 2013 Accepted: 4 June 2013 Published: 4 July 2013 Compartmentalization of developmental processes has been proposed to be a major source of evolvability for different stages of complex life cycles [1]. In insects with larval limbs that transform into adult limbs, developmental coupling between larval and adult stages might constrain evolutionary change. Many derived insect species have independently evolved imaginal discs that have enabled complete separation of larval and adult life stages [1,2,56]. Thus, in addition to promoting rapid growth in an organ- ism with a short life cycle, the evolution of imaginal discs may serve as a final step towards complete dissociation of larval and adult structures. Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Page 15 of 16 Page 15 of 16 Re-patterning phase of limb regeneration relies on taxon- specific regulation Limb regeneration in most metazoans, including Tribolium, can be classified into four general stages: wound healing, blastema formation, blastema cell proliferation, and re- patterning [14,36-38]. Although we have found that both Dac and Ss are necessary for the re-patterning of certain appendages in Tribolium, these genes most likely are not conserved throughout the Metazoa, because they are spe- cific to the patterning of arthropod appendage segments. Similarly, genes that are necessary for normal limb patterning and re-patterning in vertebrates, like fibroblast growth factor (FGF) [39-46], appear not to play significant roles, at least during the earliest stages of Tribolium limb re-patterning [47]. Thus, while certain factors appear to play roles in regeneration across all regenerating metazoans [14,47-52], genes involved in re-patterning are unlikely to be a part of any conserved gene regulatory networks underlying limb regeneration. Rather, it appears that once the regeneration pathway is activated, taxon- Acknowledgements Okada TS: A brief history of regeneration research - For admiring Professor Niazi’s discovery of the effect of vitamin A on regeneration. J Biosci 1996, 21:261–271. 30. Hughes C, Kaufman TC: RNAi analysis of Deformed, proboscipedia and sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the hemipteran head. Development 2000, 127:3683–3694. 55. Edwards JS, Reddy GR, Rani MU: Central projections of a homoeotic regenerate, antennapedia, in a stick insect, Carausius-morosus (Phasmida). J Neurobiol 1989, 20:101–114. 31. Bolognesi R, Farzana L, Fischer TD, Brown SJ: Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum. Curr Biol 2008, 18:1624–1629. 56. Svacha P: What are and what are not imaginal discs: reevaluation of some basic concepts (Insecta, Holometabola). Dev Biol 1992, 154:101–117 32. Shippy TD, Guo JH, Brown SJ, Beeman RW, Denell RE: Analysis of maxillopedia expression pattern and larval cuticular phenotype in wild- type and mutant Tribolium. Genetics 2000, 155:721–731. doi:10.1186/2041-9139-4-20 Cite this article as: Lee et al.: Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum. EvoDevo 2013 4:20. 33. Beermann A, Jay DG, Beeman RW, Hulskamp M, Tautz D, Jurgens G: The short antennae gene of Tribolium is required for limb development and encodes the orthologue of the Drosophila Distal-less protein. Development 2001, 128:287–297. 34. Nakamura T, Mito T, Bando T, Ohuchi H, Noji S: Dissecting insect leg regeneration through RNA interference. Cell Mol Life Sci 2008, 65:64–72. 35. Dong PDS, Chu J, Panganiban G: Proximodistal domain specification and interactions in developing Drosophila appendages. Development 2001, 128:2365–2372. 36. Bergantinos C, Corominas M, Serras F: Cell death-induced regeneration in wing imaginal discs requires JNK signalling. Development 2010, 137:1169–1179. 37. Bosch M, Serras F, Martin-Blanco E, Baguna J: JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs. Dev Biol 2005, 280:73–86. 38. Sustar A, Schubiger G: A transient cell cycle shift in Drosophila imaginal disc cells precedes multipotency. Cell 2005, 120:383–393. 39. D’Jamoos CA, McMahon G, Tsonis PA: Fibroblast growth factor receptors regulate the ability for hindlimb regeneration in Xenopus laevis. Wound Repair Regen 1998, 6:388–397. 40. Mullen LM, Bryant SV, Torok MA, Blumberg B, Gardiner DM: Nerve dependency of regeneration: the role of distal-less and FGF signaling in amphibian limb regeneration. Development 1996, 122:3487–3497. 41. Acknowledgements 23. Biehs B, Sturtevant MA, Bier E: Boundaries in the Drosophila wing imaginal disc organize vein-specific genetic programs. Development 1998, 125:4245–4257. We thank Drs John Cameron, Jennifer Hood-DeGrenier, Kaye Peterman, Michelle LaBonte and Kimberly O’Donnell, and the members of the Suzuki lab, for their support and critical reading of the manuscript, and Dr Elizabeth Jockusch for helpful discussions. We thank Dr Richard Beeman and Dr Yoshinori Tomoyasu for the Tribolium beetles. This study was supported in part by Wellesley College and by the National Science Foundation Grant number IOS-1027453 to YS. 24. Caygill EE, Johnston LA: Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs. Curr Biol 2008, 18:943–950. Page 16 of 16 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 25. Duncan DM, Burgess EA, Duncan I: Control of distal antennal identity and tarsal development in Drosophila by spineless-aristapedia, a homolog of the mammalian dioxin receptor. Genes Dev 1998, 12:1290–1303. 49. Yokoyama H, Ogino H, Stoick-Cooper CL, Grainger RM, Moon RT: Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration. Dev Biol 2007, 306:170–178. 50. Yokoyama H: Initiation of limb regeneration: the critical steps for regenerative capacity. Develop Growth Differ 2008, 50:13–22. 26. Emerald BS, Curtiss J, Mlodzik M, Cohen SM: Distal antenna and distal antenna related encode nuclear proteins containing pipsqueak motifs involved in antenna development in Drosophila. Development 2003, 130:1171–1180. 51. Vinarsky V, Atkinson DL, Stevenson TJ, Keating MT, Odelberg SJ: Normal newt limb regeneration requires matrix metalloproteinase function. Dev Biol 2005, 279:86–98. 27. Struhl G: Spineless-aristapedia: a homeotic gene that does not control the development of specific compartments in Drosophila. Genetics 1982, 102:737–749. 52. Shimizu H, Zhang XM, Zhang JS, Leontovich A, Fei KY, Yan L, Sarras MP: Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases. Development 2002, 129:1521–1532. 28. Emmons RB, Duncan D, Duncan I: Regulation of the Drosophila distal antennal determinant spineless. Dev Biol 2007, 302:412–426. 53. Bateson W: Materials for the Study of Variation Treated with Especial Regard to Discontinuity in the Origin of Species. London: Macmillan; 1894. 29. Toegel JP, Wimmer EA, Prpic NM: Loss of spineless function transforms the Tribolium antenna into a thoracic leg with pretarsal, tibiotarsal, and femoral identity. Dev Genes Evol 2009, 219:53–58. y 54. Acknowledgements Lee Y, Grill S, Sanchez A, Murphy-Ryan M, Poss KD: Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration. Development 2005, 132:5173–5183. 42. Poss FD, Shen JX, Nechiporuk A, McMahon G, Thisse B, Thisse C, Keating MT: Roles for Fgf signaling during zebrafish fin regeneration. Dev Biol 2000, 222:347–358. 43. Whitehead GG, Makino S, Lien CL, Keating MT: fgf20 is essential for initiating zebrafish fin regeneration. Science 2005, 310:1957–1960. 44. Giampaoli S, Bucci S, Ragghianti M, Mancino G, Zhang F, Ferretti P: Expression of FGF2 in the limb blastema of two Salamandridae correlates with their regenerative capability. Proc R Soc Lond B Biol Sci 2003, 270(1530):2197–2205. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 45. Poulin ML, Patrie KM, Botelho MJ, Tassava RA, Chiu IM: Heterogeneity in the expression of fibroblast growth-factor receptors during limb regeneration in newts (Notophthalmus-viridescens). Development 1993, 119:353–361. • Convenient online submission • Thorough peer review 46. Han MJ, An JY, Kim WS: Expression patterns of Fgf-8 during development and limb regeneration of the axolotl. Dev Dyn 2001, 220:40–48. 47. Mitten EK, Jing D, Suzuki Y: Matrix metalloproteinases (MMPs) are required for wound closure and healing during larval leg regeneration in the flour beetle, Tribolium castaneum. Insect Biochem Mol Biol 2012, 42:854–864. 48. Acknowledgements Nakamura T, Mito T, Tanaka Y, Bando T, Ohuchi H, Noji S: Involvement of canonical Wnt/Wingless signaling in the determination of the positional values within the leg segment of the cricket Gryllus bimaculatus. Develop Growth Differ 2007, 49:79–88. 48. Nakamura T, Mito T, Tanaka Y, Bando T, Ohuchi H, Noji S: Involvement of canonical Wnt/Wingless signaling in the determination of the positional values within the leg segment of the cricket Gryllus bimaculatus. Develop Growth Differ 2007, 49:79–88.
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The effect of the ionosphere on remote sensing of sea surface salinity from space: absorption and emission at L band
IEEE transactions on geoscience and remote sensing
2,002
public-domain
7,022
Manuscript received June 1, 2001; revised December 13, 2001. D. M. Le Vine is with the Microwave Sensors Branch/Code 975, Labora- tory for Hydrospheric Processes, Goddard Space Flight Center, Greenbelt, MD 20771 USA (e-mail: dmlevine@priam.gsfc.nasa.gov). S. Abraham is with the Science System and Applications, Inc., Microwave Sensors Branch/Code 975, Goddard Space Flight Center, Greenbelt, MD 20771 USA (e-mail: sabraham@synth.gsfc.nasa.gov). Publisher Item Identifier S 0196-2892(02)04604-1. 771 771 IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 I. INTRODUCTION T T HE SALINITY of the open ocean is important for under- standing ocean circulation and for modeling energy ex- change with the atmosphere. For example, salinity gradients affect mixed layer processes, which influence fluxes of heat near the surface [1]. Salinity and temperature determine water density and are important factors in large-scale ocean circu- lation [2]. Also, changes in salinity are primarily caused by changes in freshwater (evaporation, precipitation, melting ice, or river input). These changes are manifestations of elements of the water cycle, which are poorly known over the ocean [3]. The Effect of the Ionosphere on Remote Sensing of Sea Surface Salinity From Space: Absorption and Emission at L Band David M. Le Vine, Fellow, IEEE, and Saji Abraham, Member, IEEE However, measurement of sea surface salinity (SSS) in the open ocean presents a special challenge. This is so because the dynamic range of SSS in the open ocean is relatively small (about 5 K) and the requirements for a scientifically useful mea- surement (about 0.05 K) put a severe constraint on radiometric performance. A change of 0.05 K at L-band corresponds to a change of about 0.1 psu [7] where “psu” denotes changes mea- sured on the practical salinity scale [11]. To put the challenge of this measurement in perspective, the dynamic range associated with changes in soil moisture is on the order of 100 K [12] and measurement requirements are 1–4 K [13]. Abstract—The purpose of this work is to examine the effects of Faraday rotation and attenuation/emission in the ionosphere in the context of a future remote sensing system in space to mea- sure salinity. Sea surface salinity is important for understanding ocean circulation and for modeling energy exchange with the at- mosphere. A passive microwave sensor in space operating near 1.4 GHz (L-band) could provide global coverage and complement in situ arrays being planned to provide subsurface profiles. However, the salinity signal is relatively small and changes along the prop- agation path can be important sources of error. It is shown that errors due to the ionosphere can be as large as several psu. The dominant source of error is Faraday rotation but emission can be important. Among the important potential sources of error at L-band is the ionosphere. The ionosphere causes a change in the direction of polarization (Faraday rotation) and because the ionosphere is lossy at L-band, there is both attenuation and emission along the signal path. As will be shown here, both phenomena can cause errors that are important for remote sensing of salinity at the 0.1-psu level of accuracy. A brief background is given in Ap- pendices A and B to define Faraday rotation and attenuation in the ionosphere. In Section II, the magnitude of Faraday rotation and its effect on observed brightness temperature are presented. In Sections III–IV, attenuation and its effect on brightness tem- perature (emission) are discussed. Index Terms—Ionospheric electromagnetic propagation, mi- crowave radiometry, ocean salinity, remote sensing. U.S. Government work not protected by U.S. copyright. Manuscript received June 1, 2001; revised December 13, 2001. A. Magnitude Top: variation of vertical total electron content (VTEC) with solar activity. Bottom: Faraday rotation at the location 30 N, 220 E during high solar activity (June 1989). The data are for a sensor altitude of 675 km and looking perpendicular to the satellite heading to the right. The solid and dashed lines are for local time noon and 6 am, respectively. Rz. (Rz is a 12-month, running mean centered on the middle of the month of interest.) The data are for June and the solid line is for a local time of noon and the dashed line for 6 am. The lower panel shows Faraday rotation at this location for several incidence angles, . This example illustrates some general characteristics: VTEC increases with solar zenith angle and solar activity, and for a fixed sensor orientation Faraday rotation increases with VTEC and incidence angle. Faraday rotation varies as VTEC ; hence, orientation of the sensor with respect to the magnetic field affects the rate of change with incidence angle and VTEC. These approximations are reasonable because is slowly varying with altitude and because there is little curvature of the ray path. In (2), is in tesla, is in degrees, and VTEC dz is the vertical total electron con- tent at the sub-satellite point in total electron content units (10 electrons/m ). Rz. (Rz is a 12-month, running mean centered on the middle of the month of interest.) The data are for June and the solid line is for a local time of noon and the dashed line for 6 am. The lower panel shows Faraday rotation at this location for several incidence angles, . This example illustrates some general characteristics: VTEC increases with solar zenith angle and solar activity, and for a fixed sensor orientation Faraday rotation increases with VTEC and incidence angle. Faraday rotation varies as VTEC ; hence, orientation of the sensor with respect to the magnetic field affects the rate of change with incidence angle and VTEC. ( ) It is clear from (1) and (2) that Faraday rotation depends on electron density and magnetic field and also on the orientation of the sensor with respect to the local magnetic field (i.e., on ). Examples of the effect of sensor orientation (scan pattern and look angle) can be found in [16]. A. Magnitude Microwave remote sensing from space could provide the necessary temporal and spatial sampling needed to understand the role of salinity in these ocean processes [2], [4]. Changes in salinity modulate the emissivity of the surface and cause changes in emission that are sufficiently strong in the low frequency portion of the microwave spectrum to be detected with passive sensors [5], [6]. Measurements from space have been proposed [7] and salinity differences were observed from space with the L- band radiometer on SKYLAB 25 [8]. Recently, experiments with L-band radiometers on aircraft have demonstrated that salinity can be retrieved with accuracy useful for studying processes in coastal regions [9], [10]. The rotation of the polarization vector in the ionosphere is due to the change along the propagation path from surface to sensor of the phase, , in (B3). Substituting (A6) into (B4) and integrating along the propagation path, , one obtains ds (1) (1) where is the Faraday rotation in radians and is the angle between the direction of propagation and the Earth magnetic field [14], [15]. To simplify the many calculations needed to make a global map of , (1) has been approximated by making the change of variables ds dz where is the normal to the surface at the subsatellite point nadir and is the polar angle between nadir and the line of sight to the surface (inci- dence angle). Substituting for the plasma frequency, and elec- tron gyro frequency, , from (A2)–(A3) and replacing by its value at an altitude of 400 km [16], one obtains VTEC (2) VTEC IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 772 Top: variation of vertical total electron content (VTEC) with solar activity. Bottom: Faraday rotation at the location 30 N, 220 E during high solar activity 89). The data are for a sensor altitude of 675 km and looking perpendicular to the satellite heading to the right. The solid and dashed lines are for local n and 6 am, respectively. Fig. 1. Top: variation of vertical total electron content (VTEC) with solar activity. Bottom: Faraday rotation at the location 30 N, 220 E during high solar activity (June 1989) The data are for a sensor altitude of 675 km and looking perpendicular to the satellite heading to the right The solid and dashed lines are for local Fig. 1. A. Magnitude For purposes of this paper, it will be assumed that the sensor looks to the right (i.e., across track in the plane perpendicular to the satellite heading). The sensor will be assumed to be in a sun-synchronous orbit with an altitude of 675 km, which is representative of orbits proposed for microwave remote sensing at L-band [4]. Faraday rotation will be computed using the International Reference Ionosphere (IRI-95) [17] to generate the necessary electron density profiles and the International Geomagnetic Reference Field (IGRF) [18] for the magnetic field. Fig. 2 shows the global distribution of Faraday rotation for a sensor with the orientation and altitude given above. The data are for a local time of 6 am (left) and noon (right) for incidence an- glesof20,30,40,and50degrees.ThesedataareforJune,1989,a very active Sun near solar maximum with sunspot number, Rz . Notice the north–south bias in these maps, reflecting that the computation is for summer in the northern hemisphere. Also notice the east–west symmetry, which reflects that the computa- tion is for constant local time (6 am or noon). However, there are significant deviations from these simple generalizations. For ex- ample, notice at noon (right) the peaks in Faraday rotation south of Australia and also over northern Asia. These patterns reflect the complex variations of VTEC with latitude and longitude and Fig. 1 shows the Faraday rotation at a fixed location (30 N, 220 E) over the Pacific Ocean just off the coast of North America. The top panel shows VTEC at this location as a function of solar activity given in terms of the sunspot number LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 773 obal distribution of Faraday rotation for local time of 6 am (left) and noon (right). The data are for high solar activity (June 1989), an altitu di l h lli h di h i h i id l i di d (20 30 40 d 50 d ) distribution of Faraday rotation for local time of 6 am (left) and noon (right). The data are for high solar activity (June 1989), an altitude of 675 km, pendicular to the satellite heading to the right at incidence angles as indicated (20, 30, 40, and 50 degrees). ce of Faraday rotation on orientation with respect to gnetic field (i.e., ). trating the dependence on solar activity, local time and sensor orientation can be found in [16] and [19]. A. Magnitude Additional examples illus- trating the dependence on solar activity, local time and sensor orientation can be found in [16] and [19]. Fig. 2. Global distribution of Faraday rotation for local time of 6 am (left) and noon (right). The data are for high solar activity (June 1989), an altitude of 675 km, and looking perpendicular to the satellite heading to the right at incidence angles as indicated (20, 30, 40, and 50 degrees). the dependence of Faraday rotation on orientation with respect to the local magnetic field (i.e., ). Additional examples illus- the dependence of Faraday rotation on orientation with respect to the local magnetic field (i.e., ). Additional examples illus- the dependence of Faraday rotation on orientation with respect to the local magnetic field (i.e., ). Additional examples illus- trating the dependence on solar activity, local time and sensor orientation can be found in [16] and [19]. trating the dependence on solar activity, local time and sensor orientation can be found in [16] and [19]. trating the dependence on solar activity, local time and sensor orientation can be found in [16] and [19]. IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 774 Fig. 3. Error in brightness temperature due to neglecting Faraday rotation at noon (top) and 6 am (bottom) at 30 N, 220 E for high solar activity (June 1989). The data are for a sensor at altitude of 675 km and looking perpendicular to the satellite heading to the right. The surface is ocean with S = 35 psu and T = 20 C. Fig. 3. Error in brightness temperature due to neglecting Faraday rotation at noon (top) and 6 am (bottom) at 30 N, 220 E for high solar activity (June 1989). The data are for a sensor at altitude of 675 km and looking perpendicular to the satellite heading to the right. The surface is ocean with S = 35 psu and T = 20 C. B. Effect on Brightness Temperature where the “ ” and “ ”signs are for H- and V-polarization, re- spectively. To compute , it has been assumed that the surface is flat and without roughness (waves or foam). In this case, the emissivity is Faraday rotation is a potential source of error for remote sensing because radiation of given polarization at the surface will have mixed polarization when it arrives at the sensor. In particular, given the physical temperature of the surface and the emissivity for horizontal and vertical polarization, respectively, one obtains the following expression for the brightness temperatures, , at the sensor: (5) (5) where are the Fresnel reflection coefficients (6a) (6b) (6a) (3a) (3b) (6b) Inverting (3) without correcting for Faraday rotation can re- sult in error because and are different functions of salinity [6]. and is the relative dielectric constant of sea water and is a function of salinity and temperature. The relationship for L-band reported by [6] has been used here. For purposes of deciding when the error might be significant, it is convenient to examine the change in brightness temperature, , due to Faraday rotation. From (3), one obtains Fig. 3 shows as a function of solar activity at the fixed location (30 N, 220 E). The top panel is for local time of noon and the bottom panel is for 6 am. The data are for the month of June, a sensor at 675 km altitude, and looking to the right with incidence angles of 10, 30, and 50 degrees. The Faraday rotation and VTEC are given in Fig. 1. The calculations were made for (4) LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 775 global distribution of the error in brightness temperature as a function of incidence angle due to neglecting Faraday rotation at 6 am (le ata are for high solar activity (June 1989; Rz = 158) and for a sensor at altitude of 675 km and looking perpendicular to the satellite h face is ocean with S = 35 psu and T = 20 C. urface with psu and C. Notice that incidence angle and solar activity as one would expec Fig. 4. The global distribution of the error in brightness temperature as a function of incidence angle due to neglecting Faraday rotation at 6 am (left) and noon (right). B. Effect on Brightness Temperature The data are for high solar activity (June 1989; Rz = 158) and for a sensor at altitude of 675 km and looking perpendicular to the satellite heading to the right. The surface is ocean with S = 35 psu and T = 20 C. an ocean surface with psu and C. Notice that the error incurred by ignoring Faraday rotation increases with incidence angle and solar activity as one would expect (because Faraday rotation increases with these parameters) and the error IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 776 Fig. 5. Profiles of ionosphere parameters (electron density, collision frequency, temperature, and attenuation) at 30 N, 330 E for local times of noon (solid) and 6 am (dashed) and high solar activity (June 1989). Fig. 5. Profiles of ionosphere parameters (electron density, collision frequency, temperature, and attenuation) at 30 N, 330 E for local times of noon (solid) and 6 am (dashed) and high solar activity (June 1989). be neglected. Neglecting terms of the order compared to unity in (A6) and (B4), one obtains be neglected. Neglecting terms of the order compared to unity in (A6) and (B4), one obtains is much worse at noon than at 6 am. For incidence angles less than 40 degrees, the error at 6 am is less than 0.3 K even at max- imum solar activity. At noon, the error is much larger (Fig. 3, top panel). (7) (8) (7) Fig. 4 shows the global distribution of brightness temperature error, , over the oceans. These calculations were made for an ocean surface with psu, C and for a period of solar maximum (June 1989). The panel on the left is for a local time of 6 am and on the right for noon. Notice that, at 6 am, mid-latitudes and moderate incidence angles the error in brightness temperature is less than 0.3 K. However, much larger values are possible at local noon, even at moderate incidence angles. (To approximate the error in the estimate of salinity, the ratio 0.5 K/psu is useful.) (8) (8) In order to compute , both the electron density and the collision frequency are needed. To estimate the collision fre- quency, we use the “effective” collision frequency defined by [20] (9) (9) where where (10) LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 777 TABLE I INTEGRATED PARAMETERS (NADIR AND LOCATION: 30 N, 330 E) TABLE I INTEGRATED PARAMETERS (NADIR AND LOCATION: 30 N, 330 E) TABLE I the signal that would arrive in the absence of attenuation (and Faraday rotation). Calling this difference , one obtains the signal that would arrive in the absence of attenuation (and Faraday rotation). Calling this difference , one obtains is the temperature of all constituents. The concentrations are in units of cm and the collision frequencies are in Hz. Computations of have been made here using the IRI-95 [17] to obtain electron density profiles and using the Mass- Spectrometer-Incoherent-Scatter (MSIS) model [21] to gen- erate the profiles of temperature and neutral particle density ( and ) needed in (10). Values of were computed as a function of altitude in steps of 1 km and then integrated along the ray path to obtain an optical depth: dz. The optical depth gives the attenuation of power (i.e., brightness temperature). (11a) Since is small, this is approximately Since is small, this is approximately (11b) (11b) Values of at (30 N, 330 E) are given in Table I. These values are for nadir (vertical and horizontal polarization are equal) and it is obvious from (11) that the attenuation increases with incidence angle approximately as . The values given in Table I assume a water surface with a salinity of 35 psu and temperature of 20 C. Values at selected locations around the globe (shown in Fig. 6) are given in Table II for a local time of 6 am and in Table III for a local time of noon. The same values of salinity and temperature are used at each location (35 psu and 20 C). The only variations are with parameters of the iono- sphere. Notice that, at nadir, in the case of high solar activity and noon, it is possible to have a loss of 0.01 K. However, the loss is much less at 6 am, even at high solar activity. Fig. 5, shows vertical profiles at (30 N, 330 E). Profiles are shown for electron density , collision frequency , tem- perature , and attenuation coefficient for local times of 6 am (dashed) and noon (solid). The calculations are for a period of high solar activity (June 1989). Notice the peak in the profile of attenuation coefficient at low altitude (near 100 km). IV. IONOSPHERE EMISSION Loss in the ionosphere also means that there will be emission (additional signal which is not related to surface properties). There are two components of this emission, a signal emitted up- ward toward the radiometer from the column of ionosphere be- tween the sensor and surface and signal emitted downward that is reflected at the surface into the sensor’s field-of-view. Using a conventional radiative transfer model for a uniform layer, one can write these terms as follows [7], [22]: LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE This occurs because the collision frequency increases rapidly in low altitudes. Hence, even though the electron density has decreased substantially from its peak near 300 km (F-region), attenuation is not negligibly small in the lower ionosphere. Integrated values of these parameters are shown in Table I. The table shows values for high and also low solar activity. Values for Faraday rotation and the ellipticity factor are included for reference. Notice how much smaller is than . All of the integrated values are for an altitude of 675 km and a vertical path. The values scale roughly as for incidence angles less than 50 degrees. A. Magnitude of Absorption (10) As shown in Appendix B, the dominant loss due to absorption in the ionosphere is determined by the coefficient [cf. (B3) and (B4)]. As will be shown below, the term (attenuation due to ellipticity) in (B3) is very much smaller at L-band and can In these expressions, the subscripts , , and denote elec- trons, ions, and neutral particles, respectively. is the molec- ular weight of ions and neutral particles (assumed equal) and B. Loss of Brightness Temperature The signal emitted from the surface is and it is attenuated as it propagates through the ionosphere to the sensor. For purposes of estimating the effect of this attenuation, define the loss in brightness temperature to be the difference between the temperature that actually arrives at the sensor and (12) (12) (12) IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 778 Fig. 6. Location where the calculation of ionospheric absorption and emission were made. Fig. 6. Location where the calculation of ionospheric absorption and emission were made. where term is polarization-dependent because reflection at the surface depends on polarization. (13a) dx dz (13b) dx dz (13c) (13a) Examples of the upwelling and downwelling terms in (12) are given at a fixed location (30 N, 330 E) in Fig. 7. The radi- ation is shown for nadir and an incidence angle of 50 degrees as a function of local time. The data are for a period of high solar activity (June 1989) and assume a surface with salinity of 35 psu and temperature of 20 C and a sensor at 675 km. The upwelling radiation (top) is unpolarized. The downwelling ra- diation is the net term, , in (12). This term is different for vertical polarization (middle) and horizontal polarization (bottom) because is different for the two polarizations. (13a) (13a) (13a) dx dz (13b) (13b) (13b) (13b) dx dz (13c) Tables II and III list examples for the several locations around the globe shown in Fig. 6. The data are for the month of June and for high and low solar activity (Rz and 8.5) corresponding to solar maximum and minimum, respectively. The data in Table II are for local time of 6 am and the data in Table III are for noon. Values are presented for optical depth , for the loss in brightness temperature due to attenuation , and for the upwelling radiation and downwelling radiation . The results are for nadir (vertical and horizontal polarization are the same). Notice that the total radiation at the sensor (upwelling plus downwelling) ranges from 0.001 to 0.055 K at 6 am depending on location and solar activity. To put this in perspective, the salinity signal is approximately 0.5 K/psu. Thus, a change of 0.05 K is an error of about 0.1 psu. LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 779 TABLE II IONOSPHERIC ATTENUATION AND EMISSION (6 AM) TABLE II IONOSPHERIC ATTENUATION AND EMISSION (6 AM) TABLE III IONOSPHERIC ATTENUATION AND EMISSION (NOON) V. COMMENTS B. Loss of Brightness Temperature This is important for monitoring salinity in the open ocean but probably not important for applications in the coastal ocean [23]. (13c) (13c) In these expressions, (13a) is the nominal emission from the surface, is the altitude of the sensor (675 km in the numer- ical examples), is the attenuation coefficient for power at altitude as given by (7), is the emissivity of the surface and is the Fresnel reflection coefficient of the surface as defined by (6), and is the temperature of the ionosphere and is the temperature of the surface. is the signal emitted by the ionosphere along the line-of-sight between the surface and sensor. This term is due to thermal emission and is the same for both polarizations. is the radiation emitted by the entire column of ionosphere downward to the surface. This radiation is reflected at the surface and then attenuated as it propagates from the surface toward the sensor. The net contribu- tion is as given in (12). This In these expressions, (13a) is the nominal emission from the surface, is the altitude of the sensor (675 km in the numer- ical examples), is the attenuation coefficient for power at altitude as given by (7), is the emissivity of the surface and is the Fresnel reflection coefficient of the surface as defined by (6), and is the temperature of the ionosphere and is the temperature of the surface. is the signal emitted by the ionosphere along the line-of-sight between the surface and sensor. This term is due to thermal emission and is the same for both polarizations. is the radiation emitted by the entire column of ionosphere downward to the surface. This radiation is reflected at the surface and then attenuated as it propagates from the surface toward the sensor. The net contribu- tion is as given in (12). This V. COMMENTS 3 (error due to Faraday rotation) and Tables II and III (error due to attenuation and emission) that corrections for these phenomena will have to be made to achieve salinity retrievals accurate to 0.1–0.2 psu. Calculations have been presented here illustrating the effect of Faraday rotation and attenuation/emission from the iono- sphere on passive microwave remote sensing at L-band (1.4 GHz). The motivation for this work is current interest in remote sensing of ocean salinity from space. To put the effects of the ionosphere into context, the change of brightness temperature with salinity is about 0.5 K/psu. It is clear from Figs. 2 and The data also indicate some obvious choices for remote sensing. Clearly an optimum choice as far as minimizing errors is 6 am local time and as close to a minimum of solar activity as possible. Local time of 6 am is near the minimum in the daily cycle of the ionosphere, (Fig. 7 is an example of the IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 780 Fig. 7. Diurnal variation of upwelling (top) and downwelling emissions (vertical polarization, middle; horizontal polarization, bottom). The calculations are at 30 N, 330 E during high solar activity (June 1989). The surface is ocean with S = 35 psu and T = 20 C. IRI-95 model [17] is a good representation of mean characteris- tics of the ionosphere but is not a particularly good predictor of current (instantaneous) behavior [24], [25]. It may be possible to improve its ability to predict current behavior given input of local, measured parameters [26], [27]. However, without such input corrections for Faraday rotation and emission will likely have to be based on other models or techniques. (One possibility is to measure the third Stokes parameter [28].) Attenuation is proportional to as in (7) which follows from the Appleton–Hartree equation and simplifications for L-band. This is a “cold” plasma approximation. In the present study, the collision frequency is taken as (see (9)–(10)). It has been suggested that, when , as is the case at L-band, a better approximation is to use [29]. Hence, it is likely that the values for emission and loss given here are underestimated. Also, the results presented here employ approximations that are reasonable at low and middle latitudes. Near the poles, additional care must be taken in the calculation of attenuation [30]. APPENDIX A REFRACTIVE INDEX AT L BAND The (A4) Sin Sin Cos (A1) (A1) LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 781 LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 781 tionalizing the denominator and only keeping terms of lowest der in and and in the numerator, one may ite this as Cos (A5) nally, using the definitions where (B4) nalizing the denominator and only keeping terms of lowest in and and in the numerator, one may this as Cos (A5) ly, using the definitions where (B4) alizing the denominator and only keeping terms of lowest n and and in the numerator, one may his as Cos (A5) , using the definitions where (B4) Rationalizing the denominator and only keeping terms of lowest order in and and in the numerator, one may write this as where Cos (A5) Finally, using the definitions Finally, using the definitions (B4) Cos (A6) Because the two modes propagate with different phase velocity, the polarization vector changes and because the index of refrac- tion is complex, there is attenuation. For example, in the spe- cial case , both modes in (B3) are attenuated by the factor and the polarization vector rotates through an angle . The latter is Faraday rotation. The wave emerges linearly polarized but with reduced amplitude and with the direction of the polarization vector changed. When the two modes do not entirely add in phase and the wave has a slight ellipti- cally polarized component. At L-band is very small (Table I) and will be neglected in this paper. The two dominant effects at L-band are Faraday rotation (determined by ) and attenuation (determined by ). (A6) (A6) and taking the square root of (A5), one obtains (A7) (A7) (A7) APPENDIX B PROPAGATION OF A PLANE WAVE IN THE IONOSPHERE To obtain an expression for the complete path, one needs to sum over all the layers. Assuming that the changes are suffi- ciently gradual that we can neglect reflections as the boundaries and that the layers are sufficiently thin and numerous that sums are approximated by integrals, one obtains Assume a plane (flat) layered ionosphere divided into many layers sufficiently thin that the index of refraction is constant within each layer. That is, neglect curvature; assume that the only variations are vertical and that the vertical variations are slowly varying. We approximate the solution for propagation in this model ionosphere by solving for plane-wave propagation within each layer assuming constant index of refraction and later summing the solutions over all the layers. (B5) To begin, consider a linearly polarized wave propagating in the -direction within the th layer. It has the following form: (B1) (B1) where where dz (B6a) dz (B6b) It is possible to write this linearly polarized wave as the sum of the two circularly polarized waves. Doing so and recognizing that in the ionosphere the two modes propagate with different velocities (i.e., index of refraction, , in (A7)), one obtains (B6a) (B2) ACKNOWLEDGMENT The authors wish to thank the Dr. D. Bilitza (National Space Science Data Center, Greenbelt) for providing atmosphere and ionosphere models. where and denote unit vectors along the and axes, respec- tively, and the subscript “ ” on has been dropped to make the notation easier. Substituting for from (A7), one obtains APPENDIX A REFRACTIVE INDEX AT L BAND The effective refractive index for electromagnetic wave propagation in the ionosphere is given by the Appleton–Hartree equation [31], [32] (A1), shown at the bottom of the page, where is frequency and is the angle between the direction of propagation and the geomagnetic field and where In these expressions, is the plasma frequency (Hz) In these expressions, is the plasma frequency (Hz) (A2) (A2) (A2) where is the number of electrons per m and is the electron gyro frequency (Hz) Fig. 7. Diurnal variation of upwelling (top) and downwelling emissions (vertical polarization, middle; horizontal polarization, bottom). The calculations are at 30 N, 330 E during high solar activity (June 1989). The surface is ocean with S = 35 psu and T = 20 C. (A3) (A3) At L-band (1.4 GHz) the frequency is sufficiently larger than the maximum plasma frequency and the gyro frequency that simpli- fying assumptions can be made to the classical magneto-ionic theory. In particular, the terms in (A1) proportional to Sin can be neglected as long as . This is called the quasi- longitudinal approximations, and one obtains [14] diurnal variation). Remote sensing from a sun-synchronous orbit with an equatorial crossing time of 6am/6pm would provide observations near this minimum. Fig. 1 and Tables I–III illustrate the dependence on solar activity. For a fixed local time and location, Faraday rotation and emission/absorption increase roughly linearly with the solar activity (IG) index, Rz. Cos (A4) The studies presented here use the IRI-95 and are therefore representative of climatalogical data for the ionosphere. 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Le Vine (M’70–SM’79–F’95) received the B.S. and M.S. degrees in electrical engineering, the M.S. degree in physics, and the Ph.D. degree in elec- trical engineering from the University of Michigan, Ann Arbor. [11] E. L. Lewis, “The practical salinity scale 1978 and its antecedents,” IEEE J. Oceanic Eng., vol. OE-5, pp. 3–8, 1980. [12] P. E. O’Neill, A. Hsu, T. Jackson, and C. Swift, “Ground-based microwave radiometer measurements during the Southern Great Plains ’97 Experiment,” in Proc. IGARSS-98, Seattle, WA, July 1998, pp. 1843–1845. IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 Space Res., vol. 27, no. 1, pp. 153–156, 2001. [20] H. Rishbeth and O. K. Garriott, Introduction to Ionospheric Physics. New York: Academic, 1969. [21] A. E. Hedin, “Extension of the MSIS thermospheric model into the middle and lower atmosphere,” J. Geophys. Res., vol. 96, p. 1159, 1991. [22] H.-J. C. Blume and B. M. Kendall, “Passive microwave measurements of temperature and salinity in coastal zones,” IEEE Trans. Geosci. Remote Sensing, vol. GE-20, pp. 394–404, 1982. [23] G. S. E. Lagerloef, “Final Report of the First Workshop Salinity Sea Ice Working Group (SSIWG),”, http://www.esr.org, 1998. Saji Abraham (M’01) received the B.Sc. degree in physics from Kerala University, India, and the M.Sc. degree in space physics from Andhra University, India, and the Ph.D. degree in space physics from the University of Delhi, India. He has been supporting NASA projects since 1997 and is currently employed with Science System and Applications, Inc. (SSAI), Greenbelt, MD. His current research includes developing forward models to simulate brightness temperature at microwaves for space-born sensors and sea surface salinity retrieval algorithms. He has worked in research areas related to ionosphere radio wave propagation techniques and has published scientific articles on lower ionosphere dynamics and its coupling with the lower atmosphere through planetary waves and Quasi-biennial Oscillations (QBO). Dr. Abraham is a member of the American Geophyical Union and the IEEE Geoscience and Remote Sensing Society. Saji Abraham (M’01) received the B.Sc. degree in physics from Kerala University, India, and the M.Sc. degree in space physics from Andhra University, India, and the Ph.D. degree in space physics from the University of Delhi, India. [24] A. J. Preble, D. N. Anderson, B. G. Fejer, and P. H. Doherty, “Compar- ison between calculated and observed F region electron density profiles at Jicamarca, Peru,” Radio Sci., vol. 4, pp. 857–866, 1994. [25] W. S. Schreiner, R. E. Markin, and G. H. Born, “Correction of single frequency altimeter measurements for ionosphere delay,” IEEE Trans. Geosci. Remote Sensing, vol. 35, pp. 271–277, Feb. 1997. [26] A. Komjathy, K. Gold, and G. Born, “A comparison of updating PRISM and IRI-95 using JPL-derived GIM’s for single frequency satellite altimetry,” in Ionospheric Determination and Specification for Ocean Altimetry and GPS Surface Reflection Workshop. Pasadena, CA: Jet Propulsion Lab., 1997. [27] A. Komjathy, R. B. Langley, and D. IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 He has been employed as an Electronics Engineer at the Harry Diamond Laboratory, Department of the Army, and as a Research Engineer at the Radiation Laboratory, University of Michigan. After leaving the University of Michigan, he became Assistant Professor in the Department of Electrical Engineering at the University of Maryland, College Park. In 1973, he joined NASA’s Goddard Space Flight Center, Greenbelt, MD, where he is currently a member of the Microwave Sensors Branch. His current research has focused on the development of techniques for microwave remote sensing of the environment from space. [13] T. J. Schmugge, P. E. O’Neill, and J. Wang, “Passive microwave soil moisture research,” IEEE Trans. Geosci. Remote Sensing, vol. GE-24, p. 12, 1986. [14] A. R. Thompson, J. M. Moran, and G. W. Swenson, Interferometry and Synthesis in Radio Astronomy. New York: Wiley, 1986. [15] G. H. Millman, “The geometry of earth’s magnetic field at ionospheric heights,” J. Geophys. Res., vol. 64, no. 7, p. 717, 1959. [16] D. M. Le Vine and S. Abraham, “Faraday rotation and passive microwave remote sensing of soil moisture from space,” in Microw. Radiomet. Remote Sens. Earth’s Surf. Atmosphere, P. Pampaloni and S. Paloscia, Eds: VSP, 2000, pp. 89–96. Dr. Le Vine is a member of the International Union of Radio Science (URSI, Commisions B, E and F) and the American Geophysical Union. Within the IEEE he is affiliated with the Antennas and Propagation Society and the Geoscience and Remote Sensing Society. He was president of the local (Washington, DC) section of the Antennas and Propagation Society and has been active in en- gineering accreditation. He is currently a member of the IEEE Committee on Engineering Accreditation Activities (CEAA) and is one of the IEEE represen- tatives on the Engineering Accreditation Commission (EAC) of ABET. He is a member of Eta Kappa Nu, Tau Beta Pi, and Phi Kappa Phi. He was a Univer- sity of Michigan Fellow and NASA/ASEE Faculty Fellow and is the recipient of several service awards from the Goddard Space Flight Center. pp [17] D. Bilitza, “International Reference Ionosphere—Status 1995/1996,” Adv. Space Res., vol. 20, no. 9, pp. 1751–1754, 1997. [18] C. E. Barton, “International geomagnetic reference field: The seventh generation,” J. Geomagn. Geoelectr., vol. 49, pp. 123–148, 1997. [19] S. Abraham and D. M. Le Vine, “Evaluation of IRI-95 to correct errors caused by Faraday rotation in passive microwave remote sensing from space,” Adv. REFERENCES [1] R. Murtugudde and A. Busalacchi, “Simulation of the tropical oceans with an ocean GCM coupled to an atmospheric mixed layer model,” J. Climate, vol. 9, pp. 1795–1815, 1998. [1] R. Murtugudde and A. Busalacchi, “Simulation of the tropical oceans with an ocean GCM coupled to an atmospheric mixed layer model,” J. Climate, vol. 9, pp. 1795–1815, 1998. pp [2] G. Lagerloef, C. Swift, and D. M. Le Vine, “Sea surface salinity: The next remote sensing challenge,” Oceanography, vol. 8, pp. 44–50, 1995. pp [2] G. Lagerloef, C. Swift, and D. M. Le Vine, “Sea surface salinity: The next remote sensing challenge,” Oceanography, vol. 8, pp. 44–50, 1995. [3] R. W. Schmitt, “The ocean freshwater cycle,” in JSC Ocean Observing Development Panel. College Station, TX: Texas A&M Univ., 1994, p. 40. [3] R. W. Schmitt, “The ocean freshwater cycle,” in JSC Ocean Observing Development Panel. College Station, TX: Texas A&M Univ., 1994, p. 40. [4] D. M. Le Vine, J. B. Zaitzeff, E. J. D’Sa, J. L. Miller, C. T. Swift, and M. Goodberlet, “Sea surface salinity: Toward and operational remote sensing system,” in Satellites, Oceanography and Society, D. Halpern, Ed. Amsterdam, The Netherlands: Elsevier, 2000, pp. 321–335. sensing system,” in Satellites, Oceanography and Society, D. Halpern, Ed. Amsterdam, The Netherlands: Elsevier, 2000, pp. 321–335. (B3) Ed. Amsterdam, The Netherlands: Elsevier, 2000, pp. 321–3 IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 Bilitza, “Ingesting GPS-derived TEC data into the International Reference Ionosphere for single fre- quency radar altimeter ionosphere delay corrections,” Adv. Space Res., vol. 22, no. 6, pp. 793–801, 1998.
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Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection
BMC plant biology
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RESEARCH ARTICLE Open Access © 2011 Lang 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: Among the various biochemical markers, fatty acids or lipid profiles represent a chemically relatively inert class of compounds that is easy to isolate from biological material. Fatty acid (FA) profiles are considered as chemotaxonomic markers to define groups of various taxonomic ranks in flowering plants, trees and other embryophytes. Results: The fatty acid profiles of 2076 microalgal strains from the culture collection of algae of Göttingen University (SAG) were determined in the stationary phase. Overall 76 different fatty acids and 10 other lipophilic substances were identified and quantified. The obtained FA profiles were added into a database providing information about fatty acid composition. Using this database we tested whether FA profiles are suitable as chemotaxonomic markers. FA distribution patterns were found to reflect phylogenetic relationships at the level of phyla and classes. In contrast, at lower taxonomic levels, e.g. between closely related species and even among multiple isolates of the same species, FA contents may be rather variable. Conclusion: FA distribution patterns are suitable chemotaxonomic markers to define taxa of higher rank in algae. However, due to their extensive variation at the species level it is difficult to make predictions about the FA profile in a novel isolate. Lang et al. BMC Plant Biology 2011, 11:124 Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection Imke Lang1,2, Ladislav Hodac3, Thomas Friedl3 and Ivo Feussner1* Imke Lang1,2, Ladislav Hodac3, Thomas Friedl3 and Ivo Feussner1* * Correspondence: ifeussn@gwdg.de 1Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Biochemistry, Göttingen, Germany Full list of author information is available at the end of the article Background saturated and mono unsaturated FAs as well as trace amounts of PUFAs, mostly linoleic acid (LA, 18:2(9Z, 12Z): where x:y(z) is a fatty acid containing X carbons and y double bonds in position z counting from the car- boxyl end)). On the other hand Chromalveolate algae contain significant amounts of PUFAs [6]. The analysis of the overall fatty acid profiles as well as the occurrence of fatty acids (FAs) in different lipid classes in microalgae is an emerging field which is expected to reveal the identification of novel FAs with a variety of new func- tional groups [1]. Despite a number of reports has been carried out and published, describing the contents as well as the composition of polyunsaturated fatty acids (PUFAs) in mostly marine microalgae [2-4], systematic approaches that include different or even many genera of microalgae and particularly those from freshwaters or terrestrial habi- tats are still missing [5]. Among the various biochemical markers, FA or lipid profiles represent a chemically relatively inert class of compounds that is easy to isolate from biological material and FA profiles are considered as chemotaxonomic mar- kers to define groups of various taxonomic ranks in flow- ering plants, trees and other Embryophytes [7,8]. Beside the identification of novel FAs, some recent stu- dies report on the use of FAs and lipid profiles of algae as biomarkers [1,9-11]. Viso et al. determined profiles of FAs of nine different marine algal groups and they were able to define even species-specific lipid compositions [4]. More- over they found a roughly taxon specific profile when the cells were cultured under identical growth conditions. Various strains and species of the cyanobacterium Nostoc Based on current knowledge, FA composition divides microalgae roughly into two groups, i.e. on one hand the cyanobacteria and green algae (Chlorophyta and Strepto- phyta) which contain low amounts of FAs, predominantly * Correspondence: ifeussn@gwdg.de 1Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Biochemistry, Göttingen, Germany Full list of author information is available at the end of the article Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 2 of 16 identified by comparison to mass spectra with highest similarity to the proposed substance in the MS-library (Nist02 or Wiley98). By this some methyl esters of branched FAs were detected, for example 12-methyl-14:0 or 3, 7, 11, 15-tetramethyl-16:0. Background Whereas for most of the FAMEs, authentic standards or MS references were avail- able, for some other substances only “best hit” identifica- tion was possible. The DMOX derivatives enabled the identification of the remaining 12 FAMEs. Unidentified substances have yet to be verified with authentic stan- dards, which are not available at this time point. The com- plete database is shown as additional file 1. were screened for their FA content and the application of a FA-based cluster analysis has been described for their identification [12]. FA and lipid composition have also been used as bio- markers to distinguish closely related microalgae at the species and the generic levels [11,13]. Hitherto no sys- tematic analysis has been carried out on a large scale basis on either the profiles of lipids or FAs in microalgae. Therefore, we determined the FA profiles of all available microalgal strains of the SAG culture collection of micro- algae http://www.epsag.uni-goettingen.de which is one of the most diverse and comprehensive resources of microal- gae. At present (March 2011) 2291 strains of mainly microscopic algae including a considerable variety of cya- nobacteria is available. They comprise almost all phyla and classes of eukaryotic algae, but an emphasis is put on algae from freshwaters and terrestrial habitats. Bacteria in algal cultures (as contaminations or some- times even through symbiosis) are well known and can be found in culture strains of almost any algal culture collec- tion. Only a small fraction (about 20%) of the studied SAG strains may be in axenic state. Therefore, also the FA con- tent of the contaminating bacteria may have contributed to the obtained FA profile. To test this, we measured methyl-15:0 and methyl 17:0 that are regarded as markers for bacterial contaminations [4]. Only 34 strains out of the 2076 analyzed strains contained small amounts methyl- 15:0. This observed low rate of contaminating bacteria was supported by microscopic controls which are routine in the perpetual maintenance of algal strains (data not shown). In summary, we conclude that only 1-2% of the strains may have been contaminated and that there is only a minor influence of bacterial contaminations on the observed algal culture FA profiles. Distribution patterns of FAs may be valuable also as a proxy to identify certain groups, species and strains of microalgae of particular interest for applied research, i.e. Background due to the presence of certain FAs and/or high percen- tages of total FA content. We also tested whether the detected FA distribution patterns are meaningful in a phy- logenetic context at various taxonomic levels, i.e. to define taxonomic groups of microalgae by their FA patterns. It would assist predicting FA content and/or presence of other valuable compounds if the phylogenetic relation- ships of algae were reflected in their FA distribution patterns. In addition we compared the measured major FA pro- files of 10 randomly chosen strains from different classes with published data (Table 2), and it should be noted that only one out of the 10 strains that were chosen from the published data originated from the SAG collection. For 6 strains the FA profiles were very similar. In case of the 4 remaining strains major differences were observed in the degree of desaturation of the FAs with different chain lengths, which may be explained by the different cultiva- tion conditions used in the different studies. Here the focus was set on esterified long chain FAs (C- 14 - C-24), which were analysed via Gas chromatography (GC) with or without mass spectrometry (MS). The large number of data obtained, were added into a database to document the FA profiles of the studied microalgal strains. 1. A database of FA profiles from diverse microalgae 1. A database of FA profiles from diverse microalgae The characterisation of FA profiles of the SAG microalgal strains was performed by screening long chain FAs (C-14 - C-24) esterified within lipids. A total of 2076 culture strains from the SAG (equal 91% of the SAG’s holding) were screened. A database was established which con- tained all identified FAs and some other hydrophobic metabolites. An overview of all substances identified in the algal strains screened is shown in Table 1. A total of 86 different substances were identified by mass spectrometry, 76 of which represent methyl esters of FAs. Out of the 76 fatty acids, 36 substances were identified by their mass spectrum and by retention time according to a standard substance, and the other 40 fatty acids were identified by their mass spectra only. The remaining 10 substances were identified by their mass spectra only as well. In com- parisons with a standard substance, the compound was Results and Discussion 1. A database of FA profiles from diverse microalgae 2. Patterns of fatty acid composition 2. Patterns of fatty acid composition FAME profiles were rather different among strains. As an example, FAME profiles from four different genera, i.e. Chroococcus (Cyanobacteria), Closteriopsis (Chlorophyta, Trebouxiophyceae), Pseudochantransia (Rhodophyta) and Prymnesium (Chromalveolates, Haptophyta) are pre- sented in Figure 1. Therefore it was anticipated to recover certain different FA distribution patterns between phyla, classes and genera of microalgae. In addi- tion, it was tested whether differences in FA patterns can also be found for groups at lower taxonomic rank, i.e. between species of the same genus or even among multi- ples isolates of the same species. Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 3 of 16 Table 1 Overview of the FAMEs identified and other substances found in the analysed SAG microalgal strains 86 substances, 76 methyl esters of FAs methyl esters of saturated straight-chain FAs methyl esters of branched chain FAs methyl esters of monoenoic FAs 14:0 12-methyl-14:0 14:1 (7Z) 16:0 13-methyl-14:0 14:1 (9Z) 17:0 14-methyl-15:0 15:1 (10Z) 18:0 14-methyl-16:0 16:1 (5Z) 19:0 methyl-3, 7, 11, 15-tetramethyl-16:0 16:1 (7Z) 20:0 16- o. 2. Patterns of fatty acid composition 15-methyl-17:0 16:1 (9Z) 21:0 17-methyl-18:0 16:1 (11Z) 22:0 6, 10, 14 trimethyl-2-pentadecanone 17:1 (8Z) 23:0 17:1 (9Z) 24:0 17:1 (10Z) 18:1 (9E) methyl esters of dienoic FAs methyl esters of trienoic FAs 18:1 (9Z) 15:2 16:3 (4Z, 7Z, 10Z) 18:1 (11Z) 16:2 (7Z, 10Z) 16:3 (6Z, 9Z, 12Z) 19:1 (11Z) 16:2 (9Z, 12Z) 16:3 (7Z, 10Z, 13Z) 20:1 (11Z) 17:2 (7Z, 10Z) 17:3 22:1 (13Z) 17:2 (9Z, 12Z) 18:3 (5Z, 9Z, 12Z) 24:1 (15Z) 18:2 (6Z, 9Z) 18:3 (6Z, 9Z, 12Z) 18:2 (8Z, xZ)* 18:3 (8Z, 11Z, 14Z) 18:2 (9E, 12E) 18:3 (9Z, 12Z, 15Z) 18:2 (9Z, 12Z) 19:3 18:2 (9Z, 14Z) 19:3 18:2 (11Z, 14Z) 20:3 (7Z, 10Z, 13Z) 19:2 (9Z, 12Z) 20:3 (8Z, 11Z, 14Z) 20:2 (11Z, 14Z) 20:3 (11Z, 14Z, 17Z) 22:2 (13Z, 16Z) 22:3 methyl esters of tetra-, penta-, and hexaenoic FAs other substances 16:4 (4Z, 7Z, 10Z, 13Z) (8Z, 11Z)-heptadeca-8, 11-dienal 16:4 (6Z, 9Z, 12Z, 15Z) 3-(3, 5-ditertbutyl-4-hydroxyphenyl) propionate 18:4 (5Z, 9Z, 12Z, 15Z) 3, 7, 11, 15-tetramethyl-2-hexadecen-1-ol 18:4 (6Z, 9Z, 12Z, 15Z) 8-(2-octylcyclopropyl) octadecanoate 19:4 2, 3, 4, 5- tetramethyl-3-hexen 20:4 (5Z, 8Z, 11Z, 14Z) (5Z, 8Z, 11Z)-15, 16 epoxy 5, 8, 11-octadecadienoate 20:4 (8Z, 11Z, 14Z, 17Z) Tetradecanamide 22:4 (7Z, 10Z, 13Z, 16Z) Hexadecanamide 18:5 (3Z, 6Z, 9Z, 12Z, 15Z) (9Z)-Octadecenamide 20:5 (5Z, 8Z, 11Z, 14Z, 17Z) 9, 10-methylene tetradecanoate 22:5 (4Z, 7Z, 10Z, 13Z, 16Z) 22:5 (7Z, 10Z, 13Z, 16Z, 19Z) 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) h k d (*) h d bl b d l l d Table 1 Overview of the FAMEs identified and other substances found in the analysed SAG microalgal strains Table 1 Overview of the FAMEs identified and other substances found in the analysed SAG microalgal strains 86 substances, 76 methyl esters of FAs For the marked (*) FAMEs the double bond positions were only tentatively assigned. occurrence of these four PUFAs in a certain group of microalgae is given as the percentage of strains with a certain FA from all examined strains in Table 3. Because the SAG culture collection focuses on micro- scopic algae from terrestrial habitats, the Haptophyta, Dinophyta and Phaeophyceae were just poorly repre- sented. Therefore, the recovered distribution patterns in 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection occurrence of these four PUFAs in a certain group of microalgae is given as the percentage of strains with a certain FA from all examined strains in Table 3. Because the SAG culture collection focuses on micro- scopic algae from terrestrial habitats, the Haptophyta, Dinophyta and Phaeophyceae were just poorly repre- sented. Therefore, the recovered distribution patterns in occurrence of these four PUFAs in a certain group of microalgae is given as the percentage of strains with a certain FA from all examined strains in Table 3. The distribution patterns of FAs among and within the 17 groups (phyla or classes) of microalgae and the cya- nobacteria comprised by the examined strains was investigated in more detail for four PUFAs which are of high nutritional interest (Table 3). The frequency of Because the SAG culture collection focuses on micro- scopic algae from terrestrial habitats, the Haptophyta, Dinophyta and Phaeophyceae were just poorly repre- sented. Therefore, the recovered distribution patterns in Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 4 of 16 Table 2 Comparison of the major FA composition of algae observed in this study against data published previously Species FA (% of total) Ref 14:0 16:0 16:1 16:2 16:3 16:4 18:0 18:1 18:2 18:3 18:4 20:4 20:5 22:6 Bacillariophyceae Phaeodactylum 9.2 26.8 45.4 - - - 0.7 4.6 - - - - 12.3 1.1 a tricornutum 9.4 23.7 35.8 - - - 6.0 3.3 4.4 3.2 0.2 - 13.3 0.9 b 6.7 14.7 43.6 2.0 - - - 15.8 0.5 0.4 1.1 - 14.4 0.7 e Thalassiosira weissflogii 25.9 28.8 28.7 - - 7.4 1.5 3.3 - 0.3 - - 4.0 0.1 b 8.8 36.6 40.5 - - - - 14.0 - - - - - - e Chlorophyceae Dunaliella primolecta 0.4 21.8 4.5 0.9 2.5 12.3 0.8 6.4 6.2 41.1 4.1 - - - b 0.6 26.0 0.9 - - - 1.6 16.3 7.0 38.7 0.6 - - - e Nannochloris sp. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection 1.8 15.1 16.6 - 0.2 - 1.0 57.7 0.6 0.8 0.3 5.9 - - b 13.3 17.8 - - - - - 23.9 10.8 28.2 6.1 - - - e Parietochloris incisa - 10.0 2.0 1.0 1.0 - 3.0 16.0 17.0 3.0 - 46.0 1.0 - c 0 19.8 - 5.2 - - 18.2 10.2 14.3 14.3 - 14.0 4.3 - e Cyanophyceae Nostoc commune 0.3 43.5 11.3 0.4 - - 1.5 6.9 19.3 16.3 - - - - d - 25.3 24.1 - - - - - 12.5 38.1 - - - - e Synechocystis sp. 13.4 26.5 43.6 - - - 3.5 8.0 0.2 4.7 - - - - b 42.5 18.8 30.1 - - - - - - 14.2 - - - - e Haptophyceae Pavlova lutheri 11.8 23.6 28.3 - - - 2.0 12.4 - - - - 12.1 9.7 a 10.1 11.1 26.3 - - - - 5.2 0.6 0.5 9.1 0.3 18.0 9.7 e Prymnesiophyceae Emiliana huxleyi 41.7 17.7 5.5 - - - 2.1 21.7 0.9 5.5 5.0 - - - b 18.8 10.3 - - - - 10.8 42.2 - - 8.7 - - 9.2 e Raphidophyceae Heterosigma akashiwo 6.2 46.3 21.3 - - 0.4 0.5 2.7 1.6 4.2 7.3 - 8.7 0.7 b 6.6 40.0 12.7 4.0 - - - - 4.5 6.7 5.2 3.5 14.8 - e a [3] most frequent FA, present in 15 out of 20 examined groups (Table 3). In the Dinophyta, Haptophyta and Euglenoids DHA-containing strains were particularly fre- quent and DHA was found there in relatively high per- centages of total FA content, i.e. in 60% or more of these strains the DHA proportion was higher than 5%. In the single studied dinophyte strain of Ceratium horridum the DHA proportion was even 29.3%. In the other groups DHA was found in rather low frequencies and also mostly in rather small proportions, i.e. less than 1% of total FA content. Although DHA was found in the Cryp- tophyta and Bacillariophyceae in about every fifth strain, its percentage of total FA content was less than 5% there, except in Cryptomonas baltica SAG 18.80 (Cryptophyta) where it is was 13.7%. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection Despite DHA was found in rather low frequencies in the green algae (Chlorophyta), the sec- ond highest DHA content of all SAG strains, 18.9% of total FA, was found in the chlorophyte Chlorococcum these and other poorly represented groups may not be representative for the whole group. For instance, for Phaeophyceae mainly microscopic forms (e.g., Ectocar- pus and the freshwater genus Bodanella) were available and the examined Rhodophyta strains covered mostly freshwater forms or those from terrestrial habitats (e.g., Porphyridium). Although diatoms are very diverse in terrestrial habitats, the examined small sample of avail- able diatom strains (18) does by far not adequately represent this group which is probably the most species- rich algal group. Also, for each of the two classes of Stramenopiles (heterokont algae), Phaeothamniophyceae and Raphidophyceae, just two strains are maintained at the SAG and, therefore, are not further discussed here. Similarly, there is only a single strain of Chlorarachnio- phyta (Rhizaria supergroup) in the SAG. The very long chain PUFA docosahexaenoic acid (DHA, 22:6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)) was the third Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 5 of 16 Figure 1 Representative gas chromatograms of fatty acid methyl esters from four species belonging to different algal groups. a) Cyanobacteria, Chroococcus minutus SAG 41.79; b) Chlorophyta, Closteriopsis acicularis SAG 11.86; c) Rhodophyta, Pseudochantransia spec. SAG 14.96; d) Chromalveolates (Haptophyta), Prymnesium parvum SAG 127.79. Fatty acid methyl esters: a) 14:0, b) 14:1n-5, c) 16:0, d) 16:1n-9, e) 16:1n-7, f) 16:2n-6, g) 16:4n-3, h) 18:0, i) 18:1n-9, j) 18:1n-7, k) 18:2n-6, l) 18:3n-6, m) 18:3n- 3, n) 18:4n-3, o) 18:5n-3, p) 20:3n-6, q) 20:4n-6, r) 20:5n-3, s) 22:5n-3, t) 22:6n-3. novae-angliae SAG 5.85, followed by the trebouxiophyte Prototheca zopfii SAG 263-8 with 14.2%. Together these findings are in accordance with DHA amounts described before for specific groups of alga [3,4,14,15]. p g p g Eicosapentaenoic acid (EPA, 20:5(5Z, 8Z, 11Z, 14Z, 17Z)) was one of the most common PUFAs, found in all of the 17 groups covered by our study (Table 3). EPA- containing strains were particularly frequent in the Eustigmatophyceae, Glaucophyta, Xanthophyceae and Rhodophyta. The highest EPA proportions of total FA content were in the Rhodophyta, with about 81% of the strains exhibiting more than 10% EPA. The highest values were 52.4% in Compsopogonopsis leptoclados SAG 106.79 and 44.9% in Acrochaetium virgatulum SAG 1.81. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection Also strains of three species of Porphyridium contained high amounts of EPA (31.2% in P. sordidum SAG O 500, 27.5% in P. aerugineum SAG 110.79, 26.7% in P. purpureum SAG 1380-1a). This is in agreement with a report on P. cruentum suggesting that red algae are a rich source of EPA [16]. Despite EPA was rather frequently found in the Glaucophyta, only about half of all strains had EPA proportions greater than 10% (maxi- mum 31.1% in Glaucocystis nostochinearum SAG 28.80). This is in agreement with another study which showed high amounts of EPA (besides ARA) in the glaucophyte Cyanophora paradoxa [17]. The highest percentage (87%) of strains with an EPA proportion of greater than 10% was in the Dinophyta, but with a maximum of just 24.3% in Pyrocystis lunula SAG 2014. In the Euglenoids, Xanthophyceae and Eustigmatophyceae about 67% of all strains had an EPA proportion of greater than 10% with maximum values of about 31% (31.4% in Heterococcus fuornensis SAG 835-5, 31.6% in Euglena proxima SAG 1224-11a) and 34.6% in Goniochloris sculpta SAG 29.96. EPA was rarely found and mostly in insignificant amounts (< 5%) in most green algae, but three strains had an exceptionally higher content of about 20% of total FAs (24.2%, Chlorella sp. SAG 242.80; 24.0%, Chla- mydomonas allensworthii SAG 28.98; 22.3%, Cylindro- capsa involuta SAG 314-1). EPA was the only FA recovered from Chlorarachnion repens SAG 26.97 (Chlorarachniophyta). That Xanthophyceae and Eustig- matophyceae contain EPA in relatively high proportions while green algae rarely accumulate EPA supports pre- vious studies [3,4,14,15,18]. EPA was rarely found and mostly in insignificant amounts (< 5%) in most green algae, but three strains had an exceptionally higher content of about 20% of total FAs (24.2%, Chlorella sp. SAG 242.80; 24.0%, Chla- mydomonas allensworthii SAG 28.98; 22.3%, Cylindro- capsa involuta SAG 314-1). EPA was the only FA recovered from Chlorarachnion repens SAG 26.97 (Chlorarachniophyta). That Xanthophyceae and Eustig- matophyceae contain EPA in relatively high proportions while green algae rarely accumulate EPA supports pre- vious studies [3,4,14,15,18]. Figure 1 Representative gas chromatograms of fatty acid methyl esters from four species belonging to different algal groups a) Cyanobacteria Chroococcus minutus SAG 41 79; b) Figure 1 Representative gas chromatograms of fatty acid methyl esters from four species belonging to different algal groups. a) Cyanobacteria, Chroococcus minutus SAG 41.79; b) Chlorophyta, Closteriopsis acicularis SAG 11.86; c) Rhodophyta, Pseudochantransia spec. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection Table 3 Frequency of four selected PUFAs in 17 taxonomic groups of microalgae on which the examined 2071 strains of the SAG culture collection were distributed, and the size of each group (in total number of strains) The frequency of PUFAs is shown as the percentage of the total number of strains examined per group. more than 5% and in the Eustigmatophyceae even no strain reached 5%. ARA was rarely found in the green algae, i.e. with an average frequency of about 14% in the phyla Chlorophyta and Streptophyta, except for prasino- phyte green algae where ARA was present in 42.9% of all strains (Table 3). However, there were a few single green algal examples with extraordinarily high ARA contents, i.e. 73.8% (corresponding to 102 μg/mg of dry weight, the highest ARA content detected in all investi- gated SAG strains) in the chlorophyte Palmodictyon var- ium SAG 3.92, followed by 52.9% in the chlorophyte Trochisciopsis tetraspora SAG 19.95 and 51.8% in the trebouxiophyte Myrmecia bisecta SAG 2043. That a high ARA content was found in the latter strain is in agreement with that it has been found a close relative with Parietochloris incisa (syn. Lobosphaeropsis incisa, Myrmecia incisa) [19]. P. incisa has been assigned an “oleaginous microalga” and the richest plant source of ARA known so far due to its capability to accumulate high amounts of ARA (up to 59% of its total FA con- tent) [20]. Interestingly, the SAG strain of P. incisa (Lobosphaera incisa SAG 2007) had with 13.2% a much lower ARA content (Table 2). more than 5% and in the Eustigmatophyceae even no strain reached 5%. ARA was rarely found in the green algae, i.e. with an average frequency of about 14% in the phyla Chlorophyta and Streptophyta, except for prasino- phyte green algae where ARA was present in 42.9% of all strains (Table 3). However, there were a few single green algal examples with extraordinarily high ARA contents, i.e. 73.8% (corresponding to 102 μg/mg of dry weight, the highest ARA content detected in all investi- gated SAG strains) in the chlorophyte Palmodictyon var- ium SAG 3.92, followed by 52.9% in the chlorophyte Trochisciopsis tetraspora SAG 19.95 and 51.8% in the trebouxiophyte Myrmecia bisecta SAG 2043. That a high ARA content was found in the latter strain is in agreement with that it has been found a close relative with Parietochloris incisa (syn. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection SAG 14.96; d) Chromalveolates (Haptophyta), Prymnesium parvum SAG 127.79. Fatty acid methyl esters: a) 14:0, b) 14:1n-5, c) 16:0, d) 16:1n-9, e) 16:1n-7, f) 16:2n-6, g) 16:4n-3, h) 18:0, i) 18:1n-9, j) 18:1n-7, k) 18:2n-6, l) 18:3n-6, m) 18:3n- 3, n) 18:4n-3, o) 18:5n-3, p) 20:3n-6, q) 20:4n-6, r) 20:5n-3, s) 22:5n-3, t) 22:6n-3. Figure 1 Representative gas chromatograms of fatty acid methyl esters from four species belonging to different algal groups. a) Cyanobacteria, Chroococcus minutus SAG 41.79; b) Chlorophyta, Closteriopsis acicularis SAG 11.86; c) Rhodophyta, Pseudochantransia spec. SAG 14.96; d) Chromalveolates (Haptophyta), Prymnesium parvum SAG 127.79. Fatty acid methyl esters: a) 14:0, b) 14:1n-5, c) 16:0, d) 16:1n-9, e) 16:1n-7, f) 16:2n-6, g) 16:4n-3, h) 18:0, i) 18:1n-9, j) 18:1n-7, k) 18:2n-6, l) 18:3n-6, m) 18:3n- 3, n) 18:4n-3, o) 18:5n-3, p) 20:3n-6, q) 20:4n-6, r) 20:5n-3, s) 22:5n-3, t) 22:6n-3. Arachidonic acid (ARA, 20:4(5Z, 8Z, 11Z, 14Z)) was most frequently found in the Phaeophyceae where it was present in all strains except one investigated strain (Table 3); in about 54% of all Phaeophyceae strains the proportion of ARA was higher than 10%, but with a maximum of just 17.7% in Halopteris filicina SAG Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 6 of 16 Table 3 Frequency of four selected PUFAs in 17 taxonomic groups of microalgae on which the examined 2071 strains of the SAG culture collection were distributed, and the size of each group (in total number of strains) no. of strains DHA EPA ARA GLA Cyanobacteria 223 1.3 0.9 0.4 12.1 Plantae Glaucophyta 15 80.0 46.7 6.7 Chlorophyta Chlorophyceae 927 5.1 6.9 5.7 26.2 Trebouxiophyceae 253 4.3 16.6 22.9 6.3 Ulvophyceae 70 4.3 22.9 12.9 7.1 prasinophytes 21 14.3 33.3 42.9 57.1 Charophyta 159 1.3 17.6 13.8 31.4 Rhodophyta 78 70.5 67.9 3.8 Excavates Euglenoids 131 42.7 44.3 51.1 Chromalveolates Stramenopiles Bacillariophyceae 18 22.2 44.4 11.1 11.1 Xanthophyceae 81 4.9 75.3 49.4 16.1 Eustigmatophyceae 17 88.2 41.2 5.9 Phaeophyceae 12 58.3 91.7 16.7 Chryso-/Synurophyceae 12 16.7 33.3 8.3 16.7 Haptophyta 13 84.6 61.5 7.7 Cryptophyta Cryptophyta 27 22.2 66.7 3.7 3.7 Alveolates Dinophyta 14 64.3 57.1 14.3 2071 The frequency of PUFAs is shown as the percentage of the total number of strains examined per group. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection Rela- tively high percentages of GLA were found in species/ strains of Closterium (16.5% in C. baillyanum SAG 50.89, 8% in C. lunula SAG 7.84), but GLA was not found in the other 12 strains of that genus. Similarly, in the many strains available for Cosmarium (25) and Micrasterias (16), GLA was found in only 11 and 2 strains, respec- tively. The highest percentages of GLA were found in the green algal class Chlorophyceae (29.9% in Deasonia mul- tinucleata SAG 25.95, 28.5% in Desmodesmus multifor- mis SAG 26.91) and in Cyanobacteria (24.8% in Spirulina maxima SAG 84.79). In about one third (32%) of all chlorophyte GLA strains this FA had precentages of 5% and higher. Distribution of GLA in the cyanobacteria was rather patchy, i.e. the 27 cyanobacteria strains with GLA were mainly restricted to three genera, Calothrix (8 strains), Microcystis (7 strains) and Spirulina (6 strains). Also within each of these genera the GLA percentages were quite variable, e.g. in Spirulina it varied from 4.6% to 24.8%, and three strains where without GLA. FA com- position has previously been used to discriminate cyano- bacteria in isolates and natural samples at the generic level [21,22]. To discriminate species of cyanobacteria, as an additional marker the hydrocarbon composition was used in an earlier study, but in our study we failed to detect any substance out of this group [23]. Interestingly, GLA was the only FA that was detected in more than three out of the 223 examined strains. Therefore, the SAG cyanobacteria strains may be roughly divided into those with GLA present (few genera) and those where almost no PUFAs were present. This corresponds to the earlier findings that described a bipartition of cyanobac- teria, independent of their taxonomic position, into gen- era producing C-18 PUFA and those which do not [24,25]. The prasinophyte genus Tetraselmis presented an y Higher taxonomic levels analyses It was tested whether distribution patterns of FA composition on the investi- gated strains delineate the three “super groups” of eukaryotic algae, Plantae, Chromalveolates and Exca- vates (Euglenoids), and the cyanobacteria from each other. The Plantae super group comprises exclusively eukaryotes with plastids derived from primary endocyto- biosis, i.e. a cyanobacterium was transformed into an organelle through uptake and retention by the host cell followed by the loss of much of its genome [26]. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 7 of 16 whether certain patterns of FA distribution among the various investigated algal groups are present that may correspond to their phylogenetic relationships. In a first set of three analyses (higher taxonomic levels) it was tested 1) whether FA distribution patterns may reflect differences among algal phyla derived from primary (Plantae supergroup) or secondary endocytobiosis (Chro- malveolates, Euglenoids) compared to cyanobacteria representing the plastid origin, 2) the distinction of phyla within the Plantae supergroup (Chlorophyta, Strepto- phyta, Rhodophyta/Glaucophyta) and 3) major evolution- ary lineages (classes) within the Chlorophyta. A second set of analyses focused at the generic level, i.e.it was tested whether separation of genera as based on previous 18S rDNA sequence analyses suggested for Chlamydo- monas s.l., Chlorella s.l. and Scenedesmus s.l. are reflected in the FA distribution patterns. For the first set of ana- lyses the many species (266) which were represented as multiple strains (e.g., Chlamydomonas moewusii, 28) had to be reduced to only a single strain per species to avoid biases. This included also the multiple strains unidenti- fied at the species level, i.e. labelled with “sp.” instead a species name (e.g., Chlorogonium sp., 26). The SAG’s Chlorophyta strains were particularly rich in such multi- ple strains. Also excluded were those strains where only a single FA was detected. This reduced the total number of strains considered in our calculations to 1193. The strains were then divided into eleven groups roughly cor- responding to phyla or classes (Additional file 2). Strains belonging to the Chlorophyta (61% of all investigated strains) were further subdivided into the three classes, Chlorophyceae, Trebouxiophyceae, and Ulvophyceae, whereas the prasinophyte SAG green algal strains (1.7% of all considered Chlorophyta strains) were excluded from the analyses because they comprised only very few species (10). The strains of Glaucophyta (15) and Rhodo- phyta (81) were collectively treated as one composite unit. The Rhizaria - Chlorarachniophyta, was represented just by a single strain and, thus, was omitted from the statistical analyses. group, Tetraselmis, and there in 12 out of the 17 available strains and with variable proportions, i.e. 0.5 - 7.3% of total FA content. In the Streptophyta, GLA was more widely distributed, i.e. it was detected in 17 out of 41 examined genera. GLA distribution was rather variable within strains and species of a certain streptophyte genus, similar to findings of ARA in other genera. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection Lobosphaeropsis incisa, Myrmecia incisa) [19]. P. incisa has been assigned an “oleaginous microalga” and the richest plant source of ARA known so far due to its capability to accumulate high amounts of ARA (up to 59% of its total FA con- tent) [20]. Interestingly, the SAG strain of P. incisa (Lobosphaera incisa SAG 2007) had with 13.2% a much lower ARA content (Table 2). 10.96. ARA had the highest proportion of total FA in the Rhodophyta; there even about 77% of all strains had an ARA content of more than 10% with a maximum of 68.3% in Pseudochantransia sp. SAG 19.96. Interestingly, the ARA content was rather high but variable among the eight examined multiple isolates of the rhodophyte Porphyridium purpureum. While the average ARA pro- portion was about 31% in six strains, it was just 3.8% in SAG 1380-1d, but 44.5% in SAG 1380-1e. We have no explanation for this variation yet; both strains were iso- lated from marine habitats and are kept under the same culture conditions. High proportions of ARA (as well as EPA) were already found characteristic of another spe- cies of Porphyridium cruentum [16]. ARA was present in about half of all investigated Euglenoid strains and with relatively high proportions of total FA content, i.e. about one third of the strains exhibited more than 5% ARA with extraordinarily high values of 41.3% and 34.3% in Rhabdomonas incurva SAG 1271-8 and Khaw- kinea quartana SAG 1204-9. Interestingly, another strain of the same species K. quartana, SAG 1204-9, had less than half (13.3%) of ARA content and in five other species of Rhabdomonas no ARA was detected. This demonstrates that FA contents may be rather vari- able between species of the same genus and even among multiple isolates of the same species. Although about half of all examined strains for the Xanthophyceae and Eustigmatophyceae contained ARA (Table 3), they had this FA in relatively low proportions. Only one fourth of the ARA-containing Xanthophyceae strains exhibited g-Linolenic acid (GLA, 18:3(6Z, 9Z, 12Z)) was the third most common FA in the studied sample of SAG microal- gal strains, missing only in the Haptophyta, Dinophyta and Euglenoids (Table 3). It was most frequently detected in two lineages of green algae, the prasinophytes and the Streptophyta. In prasinophytes, however, GLA was pre- sent only in one out of five genera available for that Lang et al. 2.1 Distribution of four important PUFAs among strains of the SAG algal culture collection Chro- malveolate algae as well as the Euglenoids (the only algal lineage of Excavates) acquired their plastids through secondary endocytobiosis from rhodophyte and a green alga, respectively [26,27]. To consider almost The prasinophyte genus Tetraselmis presented an interesting example to test for FA variation among clo- sely related isolates. Nine strains assigned to that genus have been isolated from the same (marine) locality and regarded as the same species by the isolator (U.G. Schlösser, pers. comm.). Only in two strains DHA was present, but in very small traces (0.3% and 0.4%). In contrast, ARA and GLA were found in all isolates with percentages varying from 0.8% to 2.7% and 0.5% to 7.3%, respectively. y 2.2 Analysis of FA distribution patterns y 2.2 Analysis of FA distribution patterns The detected fatty acid (FA) composition of the 2076 investigated strains was statistically analyzed to test Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 8 of 16 equal numbers of strains for all four groups, 100 strains of Plantae, Chromalveolates and Cyanobacteria were randomly selected which closely amounts the total num- ber of considered euglenoid strains (73). The ordination which resulted from CVA (Canonical Variates Analysis, multigroup discriminant analysis) pointed out a strong difference between cyanobacteria/primary endocytobiosis (Plantae) and the two groups representing secondary endocytobiosis (Chromalveolates/Euglenoids) (Figure 2). The observed difference was without exception supported by non-parametric significance tests for mul- tidimensional data (NP-MANOVA and ANOSIM). Fol- lowing SIMPER, the lowest observed dissimilarity (63.55%) was between Cyanobacteria and Plantae, while the highest (77.29%) was between Plantae and Chromal- veolates. The first canonical variate (CV1) involved 99.99% of all possible differences among the four groups, hence we examined for possible correlations between this axis and FAs. Four FAs were significantly and exclusively correlated with the first canonical variate supported by non-parametric significance tests for mul- tidimensional data (NP-MANOVA and ANOSIM). Fol- lowing SIMPER, the lowest observed dissimilarity (63.55%) was between Cyanobacteria and Plantae, while the highest (77.29%) was between Plantae and Chromal- veolates. The first canonical variate (CV1) involved 99.99% of all possible differences among the four groups, hence we examined for possible correlations between this axis and FAs. Four FAs were significantly and exclusively correlated with the first canonical variate Figure 2 Discrimination of cyanobacteria and three algal eukaryotic supergroups (Plantae, Chromalveolates, Excavates/Euglenoids) as based on fatty acid distribution patterns of 373 investigated cyanobacterial and algal strains using Canonical Variates Analysis. The two vectors shown indicate FAs significantly correlated with canonical axis 1. Lines encircle 95% of members of a particular group. Circles, Cyanobacteria; crosses, Plantae; arrowheads, Excavates/Euglenoids; diamonds, Chromalveolates. Figure 2 Discrimination of cyanobacteria and three algal eukaryotic supergroups (Plantae, Chromalveolates, Excavates/Euglenoids) as based on fatty acid distribution patterns of 373 investigated cyanobacterial and algal strains using Canonical Variates Analysis. The two vectors shown indicate FAs significantly correlated with canonical axis 1. Lines encircle 95% of members of a particular group. Circles, Cyanobacteria; crosses, Plantae; arrowheads, Excavates/Euglenoids; diamonds, Chromalveolates. Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 9 of 16 Page 9 of 16 (CV1), i.e. y 2.2 Analysis of FA distribution patterns 16:0 (rCV1 = -0.61/p < 0.001), 18:2(9Z, 12Z) (rCV1 = -0.46/p < 0.001), 9-octadecanamid (rCV1 = 0.41/ p < 0.001), and 18:1(9Z) (rCV1 = -0.17/p = 0.001). In a second analysis it was tested whether FA distribution patterns distinguish phyla of the Plantae super group, i.e. the two lineages of green algae, Chlorophyta and Streptophyta [28,29], and the composite Rhodophyta/ Glaucophyta group. Because the latter was with 54 strains the smallest group, it was compared with equally large random samples from each the Chlorophyta and Streptophyta (Table 3). The ordination diagram from a CVA of the total of 162 investigated strains clearly sepa- rated the Rhodophyta/Glaucophyta group from both green algal phyla (Figure 3). CV1 involved 79% of all pos- sible differences and even CV2 was with 21% not negligi- ble. The significance tests, NP-MANOVA and ANOSIM, supported the distinction of all three groups. SIMPER showed the Rhodophyta/Glaucophyta composite group large random samples from each the Chlorophyta and Streptophyta (Table 3). The ordination diagram from a CVA of the total of 162 investigated strains clearly sepa- rated the Rhodophyta/Glaucophyta group from both green algal phyla (Figure 3). CV1 involved 79% of all pos- sible differences and even CV2 was with 21% not negligi- ble. The significance tests, NP-MANOVA and ANOSIM, supported the distinction of all three groups. SIMPER showed the Rhodophyta/Glaucophyta composite group Figure 3 Discrimination of 162 algal strains of the Plantae supergroup into three subgroups representing the Rhodophyta/ Glaucophyta composite group (arrowheads) and both green algal phyla, Chlorophyta (diamonds) and Streptophyta (circles) as based on their fatty acid distribution patterns using Canonical Variates Analysis. The vectors shown indicate FAs significantly correlated with CV1 and CV2 Lines encircle 95% of members of a particular group Figure 3 Discrimination of 162 algal strains of the Plantae supergroup into three subgroups representing the Rhodophyta/ Glaucophyta composite group (arrowheads) and both green algal phyla, Chlorophyta (diamonds) and Streptophyta (circles) as based on their fatty acid distribution patterns using Canonical Variates Analysis. The vectors shown indicate FAs significantly correlated with CV1 and CV2. Lines encircle 95% of members of a particular group. Figure 3 Discrimination of 162 algal strains of the Plantae supergroup into three subgroups representing the Rhodophyta/ Glaucophyta composite group (arrowheads) and both green algal phyla, Chlorophyta (diamonds) and Streptophyta (circles) as based on their fatty acid distribution patterns using Canonical Variates Analysis. y 2.2 Analysis of FA distribution patterns Also in contrast to the rDNA phylogenies, the FA analyses split the genus Lobochlamys, i.e. L. culleus was part of the “Chloromonas“ group while L. segnis belonged to the “Reinhardtii“ group. Strains of Oogamochlamys were also separated on both FA groups, in contrast to their species assignments as based on the 18S rDNA analyses. rather dissimilar from both green algal phyla, i.e. there were dissimilarities of 70.55% and 71.53% with the Chlor- ophyta and Streptophyta, respectively. The lowest dissim- ilarity (55.41%) among the three tested groups was between Chlorophyta and Streptophyta. There were five FAs significantly and exclusively correlated with CV1, i.e. 18:3(9Z, 12Z, 15Z) (rCV1 = 0.77/p < 0.001), 20:4 (rCV1 = -0.49/p < 0.001), 20:5(5Z, 8Z, 11Z, 14Z, 17Z) (rCV1 = -0.59/p < 0.001), 18:1(9Z) (rCV1 = 0.30/p = 0.001) and 16:0 (rCV1 = -0.56/p = 0, 001). Two FAs were correlated exclusively with CV2, i.e. they discriminated Chlorophyta and Streptophyta, 18:1(9Z) (rCV2 = -0.4477/p < 0.001) and 9-octadecanamid (rCV2 = 0.34/p < 0.001). The by far largest fraction of all considered strains (60.3%) were from the Chlorophyta which made it interesting to test whether FA distribution patterns can discriminate between the three classes of Chlorophyta, the Chlorophy- ceae, Trebouxiophyceae and Ulvophyceae. Ulvophyceae was the smallest of the three with just 49 strains and, therefore, random samples of almost the same size (54) from each of the other two classes were used for the sta- tistical analyses. The CVA did not reveal any distinct groups, i.e. the analyzed strains tended to form three groups corresponding to the three green algal classes, but with a considerable overlap among them (Figure 4). However, the three classes were found significantly dis- tinct from each other in both employed significance tests and SIMPER. The latter and correlation analyses allowed to consider 9-octadecanamid (rCV1 = -0.58/p < 0.001; rCV2 = -0.22/p < 0.010) and the FA 18:2(9Z, 12Z) (rCV1 = -0.44/p < 0.001; rCV2 = -0.53/p < 0.001) as the only vari- ables to discriminate well Ulvophyceae from Chlorophy- ceae/Trebouxiophyceae and Trebouxiophyceae from Ulvophyceae/Chlorophyceae, respectively. Species and strains formerly assigned to a single genus Scenedesmus were shown to be actually distributed on sev- eral genera by rRNA gene sequence analyses. For example, the genus Acutodesmus has been segregated from Scene- desmus [31,32]. y 2.2 Analysis of FA distribution patterns A NMDS ordination plot of FA distribu- tion patterns revealed a tendency among the studied strains to be distributed on two clusters, i.e. one cluster of 8 strains of Acutodesmus (mainly including multiple strains of A. obliquus) was clearly separated from another cluster containing mainly strains of Scenedesmus s.str. (Figure 6). The multiple strains of S. vacuolatus were grouped together with four other strains of the genus, except for SAG 211-11n which was close to the Acutodes- mus cluster. The multiple strains of A. obliquus, however, were distributed on both clusters (Figure 6). Seven strains of A. obliquus mainly formed up the Acutodesmus cluster, whereas five other A. obliquus strains grouped together with strains of Scenedesmus s.str. This means that within the same green algal species, A. obliquus, two distinct FA patterns exist. AFLP fingerprints already showed extensive genetic variation among the multiple strains of A. obliquus while ITS2 rDNA sequence comparisons demonstrated conspecificity of the multiple strains, except for SAG 276- 20 (T. Friedl, unpubl. observation). Therefore, the finding of A. obliquus strains being separated in two FA pattern groups favours the view that genetic differences resolved by AFLPs may correspond to different phenotypic proper- ties. Consequently, it may be crucial to carefully record which strain has been used in any application [33]. Though strain SAG 276-20 was found not to belong to the same species, A. obliquus, its FA pattern suggests that it may still be a member of Acutodesmus because it was grouped in the Acutodesmus cluster (Figure 6). Generic level analyses The three previous analyses showed that phylogenetic relationships at the level of phyla and classes among algal groups were reflected in FA distribution patterns using a large sample of strains. Therefore, in a second group of analyses, we tested whether differences in FA distribution patterns may resolve the same distinction of genera as in rRNA gene sequence analyses. To test this, we selected three genera which are widely used in biotechnological applications and well represented by SAG strains, i.e. Chlorella s.l., Scene- desmus s.l. and Chlamydomonas s.l.. Recent18S rRNA gene sequence analyses revealed each of the three as para- or polyphyletic assemblages encompassing several distinct genera. For Chlamydomonas we selected 17 species (53 strains), out of which 9 were represented by multiple strains (e.g., C. reinhardtii, 16), which were distributed on five independent lineages/clades (= genera) in the 18S rDNA phylogeny [30]. y 2.2 Analysis of FA distribution patterns The vectors shown indicate FAs significantly correlated with CV1 and CV2. Lines encircle 95% of members of a particular group. Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 10 of 16 rather dissimilar from both green algal phyla, i.e. there were dissimilarities of 70.55% and 71.53% with the Chlor- ophyta and Streptophyta, respectively. The lowest dissim- ilarity (55.41%) among the three tested groups was between Chlorophyta and Streptophyta. There were five FAs significantly and exclusively correlated with CV1, i.e. 18:3(9Z, 12Z, 15Z) (rCV1 = 0.77/p < 0.001), 20:4 (rCV1 = -0.49/p < 0.001), 20:5(5Z, 8Z, 11Z, 14Z, 17Z) (rCV1 = -0.59/p < 0.001), 18:1(9Z) (rCV1 = 0.30/p = 0.001) and 16:0 (rCV1 = -0.56/p = 0, 001). Two FAs were correlated exclusively with CV2, i.e. they discriminated Chlorophyta and Streptophyta, 18:1(9Z) (rCV2 = -0.4477/p < 0.001) and 9-octadecanamid (rCV2 = 0.34/p < 0.001). The by far largest fraction of all considered strains (60.3%) were from the Chlorophyta which made it interesting to test whether FA distribution patterns can discriminate between the three classes of Chlorophyta, the Chlorophy- ceae, Trebouxiophyceae and Ulvophyceae. Ulvophyceae was the smallest of the three with just 49 strains and, therefore, random samples of almost the same size (54) from each of the other two classes were used for the sta- tistical analyses. The CVA did not reveal any distinct groups, i.e. the analyzed strains tended to form three groups corresponding to the three green algal classes, but with a considerable overlap among them (Figure 4). However, the three classes were found significantly dis- tinct from each other in both employed significance tests and SIMPER. The latter and correlation analyses allowed to consider 9-octadecanamid (rCV1 = -0.58/p < 0.001; rCV2 = -0.22/p < 0.010) and the FA 18:2(9Z, 12Z) (rCV1 = -0.44/p < 0.001; rCV2 = -0.53/p < 0.001) as the only vari- ables to discriminate well Ulvophyceae from Chlorophy- ceae/Trebouxiophyceae and Trebouxiophyceae from Ulvophyceae/Chlorophyceae, respectively. (2213, 1753) were included. The NMDS ordination clearly separated the members of the “Reinhardtii“ clade (upper right in Figure 5), except for three strains, from those of the “Chloromonas“ clade (lower left in Figure 5). However, the “Chloromonas“ group as revealed by the FA patterns also included the three investigated strains of the “Moewu- sii“ and four of the “Oogamochlamys“ clades which was in contrast to the 18S rDNA phylogenies of [30]. y 2.2 Analysis of FA distribution patterns To better represent the “Oogamo- chlamys“ clade also two strains from the UTEX collection Chlorella vulgaris forms another example where exten- sive genetic variation among multiple strains of the same species has been detected by AFLP analyses [33]. The 15 multiple SAG strains of C. vulgaris were compared to 19 other Chlorella and Chlorella-like strains, i.e. their closest relatives as seen in 18S rDNA phylogenies, C. sorokiniana Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 11 of 16 Figure 4 Discrimination of 162 algal strains of the Chlorophyta into three subgroups representing the three green algal classes Chlorophyceae (diamonds), Trebouxiophyceae (arrowheads) and Ulvophyceae (circles) as based on their fatty acid distribution patterns using Canonical Variates Analysis. Both vectors correspond to variables (fatty acids) correlated with both canonical axes. Lines Figure 4 Discrimination of 162 algal strains of the Chlorophyta into three subgroups representing the three green algal classes Chlorophyceae (diamonds), Trebouxiophyceae (arrowheads) and Ulvophyceae (circles) as based on their fatty acid distribution patterns using Canonical Variates Analysis. Both vectors correspond to variables (fatty acids) correlated with both canonical axes. Lines encircle 65% of members of a particular group. Figure 4 Discrimination of 162 algal strains of the Chlorophyta into three subgroups representing the three green algal classes Chlorophyceae (diamonds), Trebouxiophyceae (arrowheads) and Ulvophyceae (circles) as based on their fatty acid distribution patterns using Canonical Variates Analysis. Both vectors correspond to variables (fatty acids) correlated with both canonical axes. Lines encircle 65% of members of a particular group. and C. lobophora, members of the Parachlorella clade sensu [34] as well as more distantly related strains, i.e. from the Watanabea and Prasiola clades sensu [35]. NMDS ordination based on FA distribution pattern showed almost no variation within the multiple strains of C. vulgaris and clustered them together, except for strain SAG 211-1e (Figure 7). Another cluster distant from C. vulgaris was formed by members of the Watanabea- clade, whereas Chlorella-like algae of the Prasiola-clade were not clustered together. Conclusion The algae collection at the SAG represents a valuable resource of natural products as shown in the present study for FAs and other hydrophobic metabolites. Sev- eral general trends in FA distribution reflect phyloge- netic relationships among phyla and classes as seen in genomic and molecular phylogenies and this makes FA distribution patterns an additional feature to define taxa of higher rank in algae. However the FA profile alone may be no useful marker to distinguish among different Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 12 of 16 Figure 5 Distinction of 54 strains previously assigned to Chlamydomonas s.l. (Chlorophyceae), into the “Reinhardtii“ (upper right) and “Chloromonas“ (lower left) groups as based on fatty acid distribution patterns (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.17). Symbols indicate the lineages and genera as resolved in the rDNA analyses of Pröschold et al. (2001); circles, “Reinhardtii“ clade; empty arrowheads, Lobochlamys; filled circles, Oogamochlamys; filled arrowheads, Chloromonas; filled arrowhead down, “Moewusii“ clade. Figure 5 Distinction of 54 strains previously assigned to Chlamydomonas s.l. (Chlorophyceae), into the “Reinhardtii“ (upper right) and “Chloromonas“ (lower left) groups as based on fatty acid distribution patterns (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.17). Symbols indicate the lineages and genera as resolved in the rDNA analyses of Pröschold et al. (2001); circles, “Reinhardtii“ clade; empty arrowheads, Lobochlamys; filled circles, Oogamochlamys; filled arrowheads, Chloromonas; filled arrowhead down, “Moewusii“ clade. Figure 5 Distinction of 54 strains previously assigned to Chlamydomonas s.l. (Chlorophyceae), into the “Reinhardtii“ (upper right) and “Chloromonas“ (lower left) groups as based on fatty acid distribution patterns (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.17). Symbols indicate the lineages and genera as resolved in the rDNA analyses of Pröschold et al. (2001); circles, “Reinhardtii“ clade; empty arrowheads, Lobochlamys; filled circles, Oogamochlamys; filled arrowheads, Chloromonas; filled arrowhead down, “Moewusii“ clade. Methods genera and species. For this, the comparison of further metabolites, like sterols, entire lipids and hydrocarbons should be considered. Thus, PUFA contents in microal- gae are rather difficult to predict at the levels of genera and species, making it difficult to select appropriate strains for biotechnological research/applications which aim at yielding high lipid contents. Therefore, each addi- tional or novel isolate will be worth of examination for its PUFA content. Preparation of microalgal cultures Preparation of microalgal cultures The microalgal cells were harvested from cultures at the stationary phase and stored at -20°C. Stationary phase was reached after different periods of culturing ranging from three months to about one year, depending on the strain-specific SAG’s standard maintenance protocols. Before FA extraction the algal material was lyophilised for two days until the cell pellets were totally dry. Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 13 of 16 Alkaline hydrolysis, transesterification and extraction of FA methyl esters (FAMEs) corresponding FAMEs was accomplished by adding 150 μl sodium methoxide [36]. After 20 min shaking a Figure 6 Separation of Acutodesmus (empty circles) from Scenedesmus s.str. strains (filled arrowheads) as seen in FA pattern distribution. Multiple strains of A. obliquus are indicated by abbreviation “Aobl”, those of S. vacuolatus by “Svac”. E, P, T, strains of the genera Enallax, Pectinodesmus and Tetradesmus (Non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.16). Figure 6 Separation of Acutodesmus (empty circles) from Scenedesmus s str strains (filled arrowheads) as seen in FA pattern Figure 6 Separation of Acutodesmus (empty circles) from Scenedesmus s.str. strains (filled arrowheads) as seen in FA pattern distribution. Multiple strains of A. obliquus are indicated by abbreviation “Aobl”, those of S. vacuolatus by “Svac”. E, P, T, strains of the genera Enallax, Pectinodesmus and Tetradesmus (Non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.16). Alkaline hydrolysis, transesterification and extraction of FA methyl esters (FAMEs) Alkaline hydrolysis, transesterification and extraction of FA methyl esters (FAMEs) Alkaline hydrolysis, transesterification and extraction of FA methyl esters (FAMEs) corresponding FAMEs was accomplished by adding 150 μl sodium methoxide [36]. After 20 min shaking at RT the FAMEs were extracted two times with 500 μl n- hexane and 500 μl 1 M NaCl. The hexane phases were transferred into a 1.5 ml tube and dried under streaming nitrogen. Finally the FAMEs were redissolved in 10 μl acetonitrile and analysed by GC. Prior to FAME extraction the dry weight of lyophilised algal material was determined and then the samples were transferred into a 2 ml tube. The samples were extracted by adding 405 μl of methanol/toluol 2:1 (v/v) followed by homogenisation of the cells with a potter (Heidolph RZR 2020, Schwabach) for 30 s. To avoid autoxidation, the samples were overlaid with argon. As internal standard, 10 μg of tripentadecanoate (diluted in 10 μl toluol) was added. Transesterification of lipid bound FAs to their Preparation of 4, 4-dimethyloxaline (DMOX) derivatives The position of double bonds of unknown FAME iso- mers was determined by analysing the corresponding DMOX derivatives to allow identification by MS [37]. FAMEs were prepared as described, but the hexane phases were transferred into a 1.5 ml glass tube. Sam- ples were dried under streaming nitrogen and 200 μl 2 redissolved with 50 μl chloroform. The DMOX deriva- tives were separated on a 20 cm × 20 cm silica gel 60 TLC plate (Merck) with petrol ether/diethyl ether 2:1 (v/v) as a developing solvent The plate was sprayed Figure 7 Comparison of FA patterns of multiple strains of Chlorella vulgaris (arrowheads) and their closer relatives (filled circles) with more distantly related Chlorella-like green algae of the Watanabea- (empty circles) and Prasiola-clades (diamonds) sensu Darienko et al., 2010 (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.12). Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 14 of 16 Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 14 of 16 Figure 7 Comparison of FA patterns of multiple strains of Chlorella vulgaris (arrowheads) and their closer relatives (filled circles) with more distantly related Chlorella-like green algae of the Watanabea- (empty circles) and Prasiola-clades (diamonds) sensu Darienko et al., 2010 (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.12). Alkaline hydrolysis, transesterification and extraction of FA methyl esters (FAMEs) Figure 7 Comparison of FA patterns of multiple strains of Chlorella vulgaris (arrowheads) and their closer relatives (filled circles) with more distantly related Chlorella-like green algae of the Watanabea- (empty circles) and Prasiola-clades (diamonds) sensu Darienko et al., 2010 (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.12). DMOX derivatives to allow identification by MS [37]. FAMEs were prepared as described, but the hexane phases were transferred into a 1.5 ml glass tube. Sam- ples were dried under streaming nitrogen and 200 μl 2- alkenyl-4, 4-dimethyloxazoline (Sigma, München) were added. After incubation at 180°C over night in a heating block, the samples were cooled to RT and transferred with 2 ml dichloromethane into a 12 ml glass tube and reextracted with 5 ml hexane and 2 ml water. The hex- ane phase was dried under streaming nitrogen and DMOX derivatives to allow identification by MS [37]. FAMEs were prepared as described, but the hexane phases were transferred into a 1.5 ml glass tube. Sam- ples were dried under streaming nitrogen and 200 μl 2- alkenyl-4, 4-dimethyloxazoline (Sigma, München) were added. After incubation at 180°C over night in a heating block, the samples were cooled to RT and transferred with 2 ml dichloromethane into a 12 ml glass tube and reextracted with 5 ml hexane and 2 ml water. The hex- ane phase was dried under streaming nitrogen and redissolved with 50 μl chloroform. The DMOX deriva- tives were separated on a 20 cm × 20 cm silica gel 60 TLC plate (Merck) with petrol ether/diethyl ether 2:1 (v/v) as a developing solvent. The plate was sprayed with 0.2% 8-anilino-1-naphthalene-sulfonic acid to visualize the DMOX derivatives under UV-light. The blue/yellow band of the DMOX derivatives was scraped out and the derivatives extracted by consecutive addition of 0.4 ml water, 2 ml methanol, 2 ml chloroform and 2 ml saturated NaCl solution. Between each step the Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Page 15 of 16 Page 15 of 16 Metric Multidimensional Scaling (NMDS). The signifi- cance of the differences among a priori predefined algal groups were tested using non-parametric multidimen- sional significance tests (Non-Parametric Multivariate Analysis of Variance, Analysis of Similarity) and visua- lised as ordinations from multigroup discriminant analy- sis (Canonical Variates Analysis). The percentages of dissimilarity between group pairs were investigated con- ducting SIMPER analysis. Alkaline hydrolysis, transesterification and extraction of FA methyl esters (FAMEs) To link the significant differ- ences among algal groups with particular variables/fatty acids possibly contributing to the observed difference, correlation analyses were conducted (Spearman’s rank correlation coefficient, r/rho), permutation significance tests). All statistical analyses and graphical visualisations have been conducted in PAST version 2.07 software package. Final graphical attributes required for publica- tion were adapted in vector graphics editor Inkscape ver- sion 4.7 and CorelDraw X3 Graphic suite. sample was vigorously mixed and finally centrifuged for 5 min at 3220 × g to separate phases. The lower phase was transferred into a new glass tube and dried under nitrogen stream, redissolved in 10 μl acetonitrile and analysed with GC/MS. Statistical analyses of FA distribution patterns For each detected fatty acid (FA) its percentage of the total FA content of a strain was used as variable. For the investigation of the general structure of the data sets, common indirect ordination techniques were used, i.e. Principal Components Analysis (PCA), Correspondence and Detrended Correspondence Analysis, and Non- Identification of FAMEs by GC with flame ionisation detection (FID) The prepared FAMEs were analysed by GC/FID using a capillary DB-23 column (30 m × 0.25 mm, 0.25 μm coat- ing thickness, J&W, Scientific, Agilent, Waldbronn) according to Hornung et al. (2002). Helium was used as carrier gas with a flow of 0.1 ml/min. The temperature gradient was 150°C for 1 min, 150-200°C at 4 K/min, 200- 250°C at 5 K/min and 250°C for 6 min. Tripentadecanoate was added to each sample for quantification and the FAMEs were identified according to the retention time of the corresponding peaks in the standard “F.A.M.E. Mix C4-C24” (Sigma, München), which was injected before every 50th run. The injection volume depended on the concentration of FAMEs within in the sample. Identification of FAMEs by GC/MS FID signals which were not identified by their retention times on GC and either represented FAMEs or other unpolar substances were further analysed by their mass spectra using a 6890 Gas Chromatograph/5973 Mass Selective Detector system (Agilent, Waldbronn). The GC/MS conditions were the same as for GC-analysis. The electron energy was 70 eV, the ion source tempera- ture 230°C, and the temperature for the transfer line added up to 260°C. The identification of unknown sub- stances was done by comparison of the obtained mass spectra with the mass spectra library NIST98 and the “Lipid Library” of the Scottish Crop Science research Institute http://www.lipidlibrary.co.uk/index.html. Additional file 2: Reduced FAME database for statistical analyses. The database contains information about clade, phylum, class, genus and species identification (1st to 5th column) as well as SAG strain number (6th column) and the amount of the different substances given as relative proportion (following columns). Acknowledgements and Funding All chromatograms of the microalgal samples were ana- lysed by using the ChemStation software version 9.03 (Agilent, Waldbronn). All peaks spanning a peak area of more than 50 units were integrated. The amount of each FAME was calculated using a defined amount (1 μg) of the internal standard tripentadecanoate and the dry weight (DW) of each sample: area of peak × 1 μg/area of tripenta- decanoate/mg d.w = μg FAME/mg DW The authors are grateful to Dr. Fredi Brühlmann (Geneva) and Dr. Cornelia Göbel (Göttingen) for their continuous support with analytical methods and interpretation of data, Dr. Maike Lorenz (Göttingen) for continuous support with microalgal handling, database work and interpretation of data, Prof. Dr. Rüdiger Hardeland (Göttingen) for providing with dinophyte strains. This work was supported by Firmenich SA, Geneva. List of abbreviations ALA li l i id AR ALA: α-linolenic acid; ARA: Arachidonic acid; CVA: canonical variance analysis; DHA: docosahexaenoic acid; DMOX: 4, 4-dimethyloxaline; EPA: Eicosapentaenoic acid; FA: fatty acid; FAME: fatty acid methyl ester; GC: gas chromatography; GLA: γ-Linolenic acid; MS: mass spectrometry; NMDS: non- metric multidimensional scaling; PA: palmitic acid; PUFAs: polyunsaturated fatty acids; SAG: culture collection of microalgae in Göttingen; SDA: stearidonic acid. ALA: α-linolenic acid; ARA: Arachidonic acid; CVA: canonical variance analysis; DHA: docosahexaenoic acid; DMOX: 4, 4-dimethyloxaline; EPA: Additional material Additional file 1: FAME database established of all SAG microalgal strains screened. The database contains information about clade, phylum, class, genus and species identification (1st to 5th column) as well as SAG strain number (6th column) and the amount of the different substances given as relative proportion (following columns). Additional file 2: Reduced FAME database for statistical analyses. The database contains information about clade, phylum, class, genus and species identification (1st to 5th column) as well as SAG strain number (6th column) and the amount of the different substances given as relative proportion (following columns). Additional file 1: FAME database established of all SAG microalgal strains screened. The database contains information about clade, phylum, class, genus and species identification (1st to 5th column) as well as SAG strain number (6th column) and the amount of the different substances given as relative proportion (following columns). Additional file 2: Reduced FAME database for statistical analyses. The database contains information about clade, phylum, class, genus and species identification (1st to 5th column) as well as SAG strain number (6th column) and the amount of the different substances given as relative proportion (following columns). Additional file 1: FAME database established of all SAG microalgal strains screened. The database contains information about clade, phylum, class, genus and species identification (1st to 5th column) as well as SAG strain number (6th column) and the amount of the different substances given as relative proportion (following columns). Author details 1 1Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Biochemistry, Göttingen, Germany. 2Cyano-Biofuels GmbH, Magnussstrasse 11, 12489 Berlin, Germany. 3Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences, Department of Experimental Phycology and Culture Collection of Algae in Göttingen (EPSAG), Göttingen, Germany. References 24. Kenyon CN: Fatty acid composition of unicellular strains of blue-green algae. J Bacteriol 1972, 109:827-834. 1. 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J Phycol 2005, 41:311-321. 37. Fay L, Richli U: Location of double bonds in polyunsaturated fatty acids by gas chromatography-mass spectrometry after 4, 4-dimethyloxazoline derivatization. J Chromatogr A 1991, 541:89-98. 14. Dunstan GA, Brown MR, Volkman JK: Cryptophyceae and Rhodophyceae; chemotaxonomy, phylogeny, and application. Phytochemistry 2005, 66:2557-2570. doi:10.1186/1471-2229-11-124 Cite this article as: Lang et al.: Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC Plant Biology 2011 11:124. doi:10.1186/1471-2229-11-124 Cite this article as: Lang et al.: Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC Plant Biology 2011 11:124. 15. Authors’ contributions IL carried out the fatty acid analysis of all algal strains and drafted the manuscript. LH performed the statistical analysis. IF and TF conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Page 16 of 16 Lang et al. BMC Plant Biology 2011, 11:124 http://www.biomedcentral.com/1471-2229/11/124 Received: 6 May 2011 Accepted: 6 September 2011 Published: 6 September 2011 Received: 6 May 2011 Accepted: 6 September 2011 Published: 6 September 2011 23. Dembitsky VM, Srebnik M: Variability of hydrocarbon and fatty acid components in cultures of the filamentous cyanobacterium Scytonema sp. isolated from microbial community “black cover” of limestone walls in Jerusalem. Biochemistry (Mosc) 2002, 67:1276-1282. References Shiran D, Khozin I, Heimer YM, Cohen Z: Biosynthesis of eicosapentaenoic acid in the microalga Porphyridium cruentum.1. The use of externally supplied fatty acids. Lipids 1996, 31:1277-1282. 16. Cohen Z: The production potential of eicosapentaenoic and arachidonic acids by the red alga Porphyridium cruentum. J Am Oil Chem Soc 1990, 67:916-920. 17. Zook D, Schenk HEA: Lipids in cyanophora paradoxa. III. Lipids in cell compartments. Endocyt C Res 1986, 3:203-211. 18. Cavalier-Smith T: The origin of eukaryotic and archaebacterial cells. Ann N Y Acad Sci 1987, 503:17-54. 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 19. Neustupa J, Elias M, Skaloud P, Nemcova Y, Sejnohova L: Xylochloris irregularis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel subaerial coccoid green alga. Phycologia 2011, 50:57-66. 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 20. Bigogno C, Khozin-Goldberg I, Boussiba S, Vonshak A, Cohen Z: Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid. Phytochemistry 2002, 60:497-503. 21. Caudales R, Wells JM: Differentiation of free-living Anabaena and Nostoc cyanobacteria on the basis of fatty-acid composition. Int J Syst Bacteriol 1992, 42:246-251. 22. Krüger G, De Wet H, Kock J, Pieterse A: Fatty acid composition as a taxonomic characteristic for Microcystis and other coccoid cyanobacteria (blue-green alga) isolates. Hydrobiologia 1995, 308:145-151.
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A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability
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Digital Object Identifier (DOI): 10.1016/j.chom.2014.01.006 Published In: Cell Host & Microbe Publisher Rights Statement: Open Access funded by Medical Research Council License: http://creativecommons.org/licenses/by/3.0/ General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. 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. Edinburgh Research Explorer A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability Edinburgh Research Explorer Citation for published version: Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host & Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 Citation for published version: Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host & Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 Citation for published version: Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host & Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 Citation for published version: Citation for published version: Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host & Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 SUMMARY et al., 2009; 2012; Wild et al., 2011; Zheng et al., 2009). Binding partners of LC3/ATG8 family members typically contain an LC3-interacting region (LIR). LIRs form intermolecular b sheets with LC3/ATG8 family members by virtue of a consensus W/FxxI/L motif, often preceded by acidic residues (Johansen and Lamark, 2011). et al., 2009; 2012; Wild et al., 2011; Zheng et al., 2009). Binding partners of LC3/ATG8 family members typically contain an LC3-interacting region (LIR). LIRs form intermolecular b sheets with LC3/ATG8 family members by virtue of a consensus W/FxxI/L motif, often preceded by acidic residues (Johansen and Lamark, 2011). Autophagy recycles cellular components and de- fends cells against intracellular pathogens. While viruses must evade autophagocytic destruction, some viruses can also subvert autophagy for their own benefit. The ability of influenza A virus (IAV) to evade autophagy depends on the Matrix 2 (M2) ion-channel protein. We show that the cytoplasmic tail of IAV M2 interacts directly with the essential autophagy protein LC3 and promotes LC3 relocali- zation to the unexpected destination of the plasma membrane. LC3 binding is mediated by a highly conserved LC3-interacting region (LIR) in M2. The M2 LIR is required for LC3 redistribution to the plasma membrane in virus-infected cells. Mutations in M2 that abolish LC3 binding interfere with fila- mentous budding and reduce virion stability. IAV therefore subverts autophagy by mimicking a host short linear protein-protein interaction motif. This strategy may facilitate transmission of infection be- tween organisms by enhancing the stability of viral progeny. While autophagy restricts the growth of pathogens poorly adapted to life in the cytosol (e.g., Salmonella enterica), profes- sional cytosol-dwelling bacteria, such as Shigella flexneri, evade restriction by autophagy (Boyle and Randow, 2013; Mostowy and Cossart, 2012). Other pathogenic microbes have evolved mechanisms to actively benefit from host autophagy. Multiple viruses deploy such subversion strategies, but the molecular mechanisms involved are unclear (Feng et al., 2013; Heaton and Randall, 2010; Klein and Jackson, 2011; Reggiori et al., 2010). The sizes of RNA virus genomes are evolutionarily constrained (Belshaw et al., 2008). Their need to hijack cellular machinery while evading host immune responses requires complex coding capacities and multifunctional viral proteins. Multiple distinct peptide products are often produced from the same gene (e.g., Firth and Brierley, 2012; Thomas et al., 1988; Wise et al., 2012). A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability Rupert Beale,1,2,5 Helen Wise,3,4,5 Amanda Stuart,4 Benjamin J. Ravenhill,1 Paul Digard,3,4,* and Felix Randow1,2,* 1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK 2University of Cambridge, Department of Medicine, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK 3Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, UK 4Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 0QQ, UK 5These authors contributed equally to this work C @ ( ) @ ( ) *Correspondence: paul.digard@roslin.ed.ac.uk (P.D.), randow@mrc-lmb.cam.ac.uk (F.R.) http://dx.doi.org/10.1016/j.chom.2014.01.006 SUMMARY In addition, viral proteins subvert host physiology by encoding short linear motifs (SLiMs) that mimic host protein interaction interfaces, but which require little coding capacity (Davey et al., 2011). No viral SLiMs have yet been described to subvert autophagy. Take down policy Download date: 24. Oct. 2024 Cell Host & Microbe Short Article Cell Host & Microbe Sh A Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 2 INTRODUCTION One important human pathogen affecting host cell autophagy is influenza A virus (IAV). This enveloped, segmented negative sense RNA virus infects a wide range of vertebrate species and causes seasonal epidemics and sporadic pandemics in hu- mans as well as outbreaks in domestic animals. Epidemic IAV causes significant annual global mortality and in pandemic years can result in millions of deaths (Taubenberger and Kash, 2010). One important human pathogen affecting host cell autophagy is influenza A virus (IAV). This enveloped, segmented negative sense RNA virus infects a wide range of vertebrate species and causes seasonal epidemics and sporadic pandemics in hu- mans as well as outbreaks in domestic animals. Epidemic IAV causes significant annual global mortality and in pandemic years can result in millions of deaths (Taubenberger and Kash, 2010). The ability of IAV to subvert autophagy is dependent on the Matrix 2 (M2) ion-channel protein, which blocks fusion of auto- phagosomes with lysosomes (Gannage´ et al., 2009). M2 is a tetrameric integral membrane protein, made from a spliced tran- script (Holsinger et al., 1994; Lamb et al., 1985). M2 comprises a 25 amino acid N-terminal ectodomain, a transmembrane a helix and a 50 amino acid cytoplasmic domain. This cytoplasmic tail contains a membrane-proximal amphipathic a helix and a Macroautophagy (hereafter autophagy) can generate raw materials at times of cellular stress by degrading cytoplasmic contents (Mizushima and Komatsu, 2011). During starvation- induced autophagy, cytoplasm is nonselectively engulfed into double-membrane vesicles (autophagosomes), whose content is digested upon fusion with lysosomes. This process has been adapted for host defense, where invading pathogens are selectively targeted by autophagy (Deretic, 2012; Levine et al., 2011; Randow et al., 2013). Selective antimicrobial autophagy relies on cargo receptors that simultaneously detect path- ogen-associated ‘‘eat me’’ signals and bind to members of the ATG8/LC3 family of ubiquitin-related proteins residing on auto- phagosomal membranes (Boyle and Randow, 2013; Thurston The ability of IAV to subvert autophagy is dependent on the Matrix 2 (M2) ion-channel protein, which blocks fusion of auto- phagosomes with lysosomes (Gannage´ et al., 2009). M2 is a tetrameric integral membrane protein, made from a spliced tran- script (Holsinger et al., 1994; Lamb et al., 1985). M2 comprises a 25 amino acid N-terminal ectodomain, a transmembrane a helix and a 50 amino acid cytoplasmic domain. Plasma Membrane To observe how IAV subverts the cellular autophagocytic ma- chinery, we investigated the distribution of GFP-tagged LC3 upon virus infection. Human cell lines, stably transduced with GFP-LC3, were infected with IAV strain A/PR/8/34 (H1N1) (Fig- ure 1A and Figure S1 available online). In uninfected cells, GFP-LC3 was distributed diffusely throughout the cytoplasm. Following infection, LC3 accumulated in the perinuclear region, as previously described (Gannage´ et al., 2009). In addition, GFP-LC3 strikingly relocalized to the cell periphery. To investi- gate the subcellular localization of LC3 in infected cells, we stained nonpermeabilized cells with wheat-germ agglutinin (WGA) to label extracellular glycans. In uninfected cells, there was little colocalization of GFP-LC3 and WGA, while in infected cells GFP-LC3 and WGA colocalized at the plasma membrane, but not on perinuclear autophagosomes (Figure 1B). To verify the presence of LC3 at the plasma membrane and examine the subcellular distribution of autophagosomes in IAV-infected cells, we performed immunoelectron microscopy (Kabeya et al., 2000). As expected, in both mock and IAV-infected cells, LC3 was present in double-membrane vesicles corresponding to au- tophagosomes (Figure 1C). Infected but not uninfected cells also demonstrated staining of the plasma membrane, both at sites of active viral budding and in areas where less budding was taking place (Figure 1C). LC3 G120A, a mutant that cannot be lipidated, did not localize to autophagosomes or the plasma membrane. Taken together, these data demonstrate that IAV directs LC3 to the plasma membrane, an unusual and unexpected destination. Strong conservation of motifs frequently indicates biological importance. We therefore examined conservation of the putative M2 LIR motif (Squires et al., 2012). Of 2,685 unique M2 se- quences, 2,661 have an FVSI or FVNI motif (Figure 3C). This near-absolute conservation of the hydrophobic residues renders the predicted b strand a strong candidate LIR motif. To test if IAV M2 binds LC3 via its putative LIR motif, we per- formed LUMIER binding assays (Barrios-Rodiles et al., 2005; von Muhlinen et al., 2013) with purified GST-LC3 and the cyto- solic domain of M2 fused to luciferase. WT M2 and the naturally occurring variant S93N bound LC3, whereas M2DLIR, M2 F91S, M2 V92S, and M2 I94S failed to bind (Figure 3D). M2 and the LIR-containing p62 compete for binding to LC3, indi- cating that M2 binds the same site as the classical p62 LIR (Figure S2A). Plasma Membrane To determine the monomeric binding affinity of the interaction, a fluorescently labeled peptide corresponding to the last 13 amino acids of M2 and purified LC3 protein were used in fluorescence anisotropy equilibrium measure- ments. A dissociation constant (KD) of 9.5 ± 1.2 mM was deter- mined (Figure 3E). In vivo this interaction would be enhanced by the tetrameric nature of M2, and reduced dimensionality imposed by the presence of both proteins in membranes. To test whether M2 bound LC3 in vivo in a LIR-dependent manner, we performed GFP-trap pull-down experiments in GFP-LC3- expressing cells infected with WT, DM2, or M2 F91S IAV. WT M2, but not the M2 LIR mutant F91S, coprecipitated with LC3 (Figure 3F). We conclude that the M2 cytosolic tail con- tains a conserved LIR motif capable of directly interacting with LC3 in infected cells. Influenza A Virus M2 Protein Drives Plasma Membrane Localization of LC3 Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability membrane-distal region of unknown structure (Schnell and Chou, 2008; Sharma et al., 2010; Stouffer et al., 2008). M2 has multiple important roles in the virus life cycle. During viral entry its ion channel activity is required to trigger disassembly in response to lowered endosomal pH, and its cytoplasmic tail con- tributes to virus assembly, budding, and morphogenesis (Ross- man and Lamb, 2011). here DM2) due to mutation of the M2 splice donor site (Hutch- inson et al., 2008; Wise et al., 2012) did not relocalize LC3 to the plasma membrane or induce its perinuclear accumulation (Figure 2B). GFP-LC3 G120A failed to relocalize in cells in- fected with WT virus (Figure 2B), confirming the electron microscopy data (Figure 1C) and indicating that the autophagy machinery is required for IAV to redistribute LC3 in infected cells. here DM2) due to mutation of the M2 splice donor site (Hutch- inson et al., 2008; Wise et al., 2012) did not relocalize LC3 to the plasma membrane or induce its perinuclear accumulation (Figure 2B). GFP-LC3 G120A failed to relocalize in cells in- fected with WT virus (Figure 2B), confirming the electron microscopy data (Figure 1C) and indicating that the autophagy machinery is required for IAV to redistribute LC3 in infected cells. membrane-distal region of unknown structure (Schnell and Chou, 2008; Sharma et al., 2010; Stouffer et al., 2008). M2 has multiple important roles in the virus life cycle. During viral entry its ion channel activity is required to trigger disassembly in response to lowered endosomal pH, and its cytoplasmic tail con- tributes to virus assembly, budding, and morphogenesis (Ross- man and Lamb, 2011). Here, we report a LIR motif in the cytoplasmic tail of M2. The M2 LIR motif causes the relocalization of LC3 to the plasma membrane in IAV-infected cells at the time of virus budding, and is essential to generate stable viral progeny. We propose that in addition to blocking autophagosome maturation (Gan- nage´ et al., 2009), IAV hijacks the autophagy machinery via the M2 LIR motif to provide suitable resources for viral budding and to enhance virion stability. Influenza M2 Contains a Conserved LIR Motif in Its Cytoplasmic Tail Because IAV M2 is required for redistribution of LC3 in infected cells, we speculated that it binds LC3 directly. LIR motifs are b strands that form an intermolecular b sheet on binding to LC3 (Johansen and Lamark, 2011). To identify potential LIRs in M2, we used JPRED to predict secondary structures in its cytosolic tail. One a-helix and one b strand were predicted (Figure 3A). The predicted b strand contains a FVSI motif that matches the consensus LIR motif (Johansen and Lamark, 2011). Among bio- chemically proven LIR motifs, the M2 FVSI motif most closely corresponds to those found in optineurin and ATG13, which both contain FVxI as their hydrophobic core (Alemu et al., 2012; Wild et al., 2011) (Figure 3B). Preceding the putative M2 LIR motif are three acidic residues, another typical feature of LC3/ATG8 binding proteins. INTRODUCTION This cytoplasmic tail contains a membrane-proximal amphipathic a helix and a 239 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 239 Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability Influenza A Virus M2 Protein Drives Plasma Membrane Localization of LC3 The pattern of LC3 immunogold staining (Figure 1C) parallels the known distribution of M2, which, though abundant at the plasma membrane during budding, is incorporated into virions at low levels (Leser and Lamb, 2005). Furthermore, GFP-LC3 and M2 closely colocalized at the plasma membrane (Figures 2A and S1). This finding prompted us to test whether M2 is required for the relocalization of LC3. In contrast to wild-type (WT) IAV, infection with a virus deficient in M2 (V7-T9; named Plasma Membrane Targeting of LC3 Depends on the M2 LIR Motif Plasma Membrane Targeting of LC3 Depends on the M2 LIR Motif To test whether relocalization of LC3 in infected cells depends on the M2 LIR motif, cells expressing GFP-LC3 were infected 240 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Cell Host & Microbe Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability A B C 10µm 10µm IAV, LC3 IAV, LC3 G120A mock, LC3 GFP-LC3 DAPI NP merge GFP-LC3 DAPI WGA merge IAV mock IAV e 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells 49 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. CT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. CT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with gold-labeled anti-GFP anti arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. so Figure S1. A GFP-LC3 DAPI NP merge IAV NP B 10µm 10µm GFP-LC3 DAPI WGA merge IAV mock IAV B IAV C IAV, LC3 IAV, LC3 G120A mock, LC3 e 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells 49 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. T116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. C IAV, LC3 IAV, LC3 G120A mock, LC3 Figure 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells C3 to the Plasma Membrane in Influenza-Infected Cells Figure 1. Relocalization of LC3 to the Plasma Membrane in Influenza Infected Cells (A) A549 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. (B) HCT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. (C) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with gold-labeled anti-GFP antibody. Black arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. Figure 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells (A) A549 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. (B) HCT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. (C) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with gold-labeled anti-GFP antibody. Black arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability A B A A A A Figure 2. Relocalization of LC3 to the Plasma Membrane Requires Influenza M2 (A) HCT116 cells expressing GFP-LC3 were in- fected with IAV, fixed at 16 hr p.i., and stained with anti-M2. Scale bar, 10 mm. (B) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with WT or DM2 mutant IAV, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. See also Figure S1. with IAV encoding WT or mutant M2 polypeptides (Figures 3G and S2B) and scored for the accumulation of LC3 at the plasma membrane and in perinuclear autophagosomes (Figures 3H and 3I). Both phenomena depended on M2. However, plasma membrane relocalization of LC3 occurred efficiently only in cells infected with viruses encoding functional M2 LIR motifs (WT or M2 S93N), but not in those infected with viruses encoding LIR motifs unable to bind LC3 (M2 F91S and I94S) (Figure 3H). This phenomenon was not due to a delay in replication of mutant viruses (Figure S2C). In contrast, the proportion of cells accumulating perinuclear autophagosomes was not signifi- cantly different between WT and LIR mutants (Figure 3I). LC3 unable to become lipidated (LC3 G120A) did not redistribute to either the perinuclear region or plasma membrane in IAV- infected cells (Figures 3H and 3I). Thus, while IAV M2 causes both the accumulation of autophagosomes and the relocaliza- tion of LC3 to the plasma membrane, these functions are sepa- B core autophagy prot and Komatsu, 2011 decreased the numb However, upon infec for the accumulatio translocation of LC3 gest that influenza st in a manner distinct Taken together, o lates the autophagy the accumulation of shown (Gannage´ et of LC3 to the plas in an ATG16L1-depe contrast, the reloca but not its perinuclea interaction of LC3 w rable, since the M2 LIR motif is required for the relocalization of LC3 to the plasma membrane, but not its perinu- clear accumulation. B B B Further evidence for M2-mediated but LIR-independent manipulation of the autophagy machinery came from the analysis of LC3 lipidation in infected cells (Figure 3J; also evident in Figure 3F). LC3 undergoes ATG5/ATG12/ATG16- dependent lipidation with phosphatidyl ethanolamine, which provides a mem- brane anchor to LC3 and is a critical step in autophagosome biogenesis (Ichi- mura et al., 2000; Kabeya et al., 2000). Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability Lipidated LC3 (LC3-II) migrates faster in SDS-PAGE. Cells infected with WT IAV or the control M2 mutant S93N con- tained more LC3-II than mock-infected cells, consistent with the virus triggering autophagy (Figure 3J). Enhanced LC3-II levels were dependent on M2 but only partially dependent on the functionality of its LIR motif, as infection with M2 F91S produced intermediate amounts of LC3-II. To understand how M2 subverts auto- phagy, cells expressing GFP-LC3 were depleted of FIP200, a component of the ULK kinase complex, or ATG16L1, a core autophagy protein required for LC3 conjugation (Mizushima and Komatsu, 2011) (Figure S3). Depletion of either protein decreased the number of starvation-induced autophagosomes. However, upon infection with IAV, only ATG16L1 was required for the accumulation of perinuclear autophagosomes and the translocation of LC3 to the plasma membrane. These data sug- gest that influenza stimulates autophagy downstream of FIP200, in a manner distinct from starvation. with IAV encoding WT or mutant M2 polypeptides (Figures 3G and S2B) and scored for the accumulation of LC3 at the plasma membrane and in perinuclear autophagosomes (Figures 3H and 3I). Both phenomena depended on M2. However, plasma membrane relocalization of LC3 occurred efficiently only in cells infected with viruses encoding functional M2 LIR motifs (WT or M2 S93N), but not in those infected with viruses encoding LIR motifs unable to bind LC3 (M2 F91S and I94S) (Figure 3H). This phenomenon was not due to a delay in replication of mutant viruses (Figure S2C). In contrast, the proportion of cells accumulating perinuclear autophagosomes was not signifi- cantly different between WT and LIR mutants (Figure 3I). LC3 unable to become lipidated (LC3 G120A) did not redistribute to either the perinuclear region or plasma membrane in IAV- infected cells (Figures 3H and 3I). Thus, while IAV M2 causes both the accumulation of autophagosomes and the relocaliza- tion of LC3 to the plasma membrane, these functions are sepa- Taken together, our data demonstrate that IAV M2 manipu- lates the autophagy machinery in two separate ways: by causing the accumulation of perinuclear autophagosomes, as previously shown (Gannage´ et al., 2009), and by inducing the translocation of LC3 to the plasma membrane. Both phenomena occur in an ATG16L1-dependent and FIP200-independent manner. In contrast, the relocalization of LC3 to the plasma membrane, but not its perinuclear accumulation, requires the LIR-dependent interaction of LC3 with M2. Figure 2. Relocalization of LC3 to the Plasma Membrane Requires Influenza M2 (A) HCT116 cells expressing GFP-LC3 were in- fected with IAV, fixed at 16 hr p.i., and stained with anti-M2. Scale bar, 10 mm. (B) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with WT or DM2 mutant IAV, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. See also Figure S1. Plasma Membrane Targeting of LC3 Depends on the M2 LIR Motif See also Figure S1 g ) A549 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-N ) HCT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. ) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with go ack arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 241 Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability 242 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability A B C D G E F H I J 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. parison of M2 LIR motif with established LIRs. o derived from 2,685 unique IAV M2 sequences. A B C D E E F E D G H I J Figure 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization (A) M2 cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. (B) Comparison of M2 LIR motif with established LIRs. (C) Logo derived from 2,685 unique IAV M2 sequences. (D) LUMIER binding assay. Binding of luciferase-tagged cytoplasmic regions of the indicated IAV M2 variants expressed in 293ET cells to beads coate purified GST or GST-LC3. (E) Fluorescence anisotropy of hydroxycoumarin-labeled M2 C terminus (DADDGHFVSIELE) against purified LC3. (F) Lysates of HCT116 cells expressing GFP-LC3 and infected with the indicated IAVs were immunoprecipitated with GFP-TRAP resin. Input and bound fra were blotted with the indicated antibodies. Dotted line, irrelevant lanes removed. (G) HCT116 cells expressing GFP-LC3 infected with the indicated virus, fixed at 16 hr p.i., and stained with anti-HA. (H and I) Plasma membrane localization (H) or perinuclear accumulation (I) of GFP-LC3 in HCT116 cells expressing the indicated LC3 alleles and infected w indicated viruses. Coverslips were assessed blindly in triplicate at 16 hr p.i. Mean and SD. **p < 0.01 by ANOVA. (J) Lysates of HCT116 cells expressing GFP-LC3 were blotted for indicated antigens at 16 hr p.i. with indicated IAV. See also Figures S2 and S3. G H I J e 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization H H G I Figure 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization (A) M2 cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. (B) Comparison of M2 LIR motif with established LIRs. (C) Logo derived from 2,685 unique IAV M2 sequences. (D) LUMIER binding assay. Figure 4. M2 LIR Motif Required for Fila- mentous Budding and Virus Stability Consis a role for autophagy in filamentous budding, both dep ATG16L1 and LIR motif mutation reduced the formatio ment bundles (Figure S4). We conclude that the M2 L is required for normal filamentous budding of IAV. 244 Cell Host & Microbe 15 239 247 February 12 2014 ª2014 Elsevier Inc (B) MDCK cells were infected with the indicated PR8 MUd viruses, fixed at 10 hr p.i., and stained without permeabilization with antiserum against PR8 virus. (Top) Maximum intensity z stack pro- jection of confocal images. (Bottom) Side-on view of z stacks. Scale bar, 10 mm. (C) A549 cells infected with WT or mutant PR8 or PR8 MUd viruses at an moi of 1. Cell-free super- natants (16 hr p.i.) were harvested and frozen either immediately or after having been left at room temperature for 1 or 2 days. Experiments were performed in parallel. Titers were determined in triplicate after thawing by plaque assay; mean and standard deviation are shown. **p < 0.01 by ANOVA. S93N I94S See also Figure S4. in both a spherical and a filamentous fashion, depending on the genetic back- ground of the virus and the host-cell type (Bourmakina and Garcı´a-Sastre, 2003; Elleman and Barclay, 2004; Roberts and Compans, 1998). Most human-path- ogenic isolates of IAV form up to 10 mm long filamentous particles that require extensive membrane resources. Since M2 influences filament formation (Iwat- suki-Horimoto et al., 2006; Roberts and Compans, 1998; Rossman et al., 2010), we hypothesized that accumulation of LC3 at the plasma membrane might represent the virus mobilizing lipid re- sources for budding. Accordingly, we introduced the M2 LIR mutations into a filamentous strain of IAV, PR8-MUd (Noton et al., 2007). PR8-MUd produces a profusion of micrometer-length fila- ments arrayed in parallel bundles. While the M2 S93N LIR variant resembled WT virus, cells infected with the M2 F91S or I94S virus encoding nonfunctional LIRs produced fewer filaments with a lower tendency to form bundles (Figure 4A). Confocal microscopy, which biases to- ward detection of long filament bundles (Simpson-Holley et al., 2002), revealed a large difference in the propensity of cells infected with WT or F91S PR8 MUd to project filament bundles away from the cell surface (Figure 4B). Consistent with in filamentous budding, both depletion of otif mutation reduced the formation of fila- e S4). We conclude that the M2 LIR motif filamentous budding of IAV. Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability Binding of luciferase-tagged cytoplasmic regions of the indicated IAV M2 variants expressed in 293ET cells to beads coated with purified GST or GST-LC3. (E) Fluorescence anisotropy of hydroxycoumarin-labeled M2 C terminus (DADDGHFVSIELE) against purified LC3. (F) Lysates of HCT116 cells expressing GFP-LC3 and infected with the indicated IAVs were immunoprecipitated with GFP-TRAP resin. Input and bound fractions were blotted with the indicated antibodies. Dotted line, irrelevant lanes removed. (G) HCT116 cells expressing GFP-LC3 infected with the indicated virus, fixed at 16 hr p.i., and stained with anti-HA. (H and I) Plasma membrane localization (H) or perinuclear accumulation (I) of GFP-LC3 in HCT116 cells expressing the indicated LC3 alleles and infected with the indicated viruses. Coverslips were assessed blindly in triplicate at 16 hr p.i. Mean and SD. **p < 0.01 by ANOVA. (J) Lysates of HCT116 cells expressing GFP-LC3 were blotted for indicated antigens at 16 hr p.i. with indicated IAV. See also Fig res S2 and S3 Figure 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization (A) M2 cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. (B) Comparison of M2 LIR motif with established LIRs. (C) Logo derived from 2,685 unique IAV M2 sequences. (D) LUMIER binding assay. Binding of luciferase-tagged cytoplasmic regions of the indicated IAV M2 variants expressed in 293ET cells to beads coated with purified GST or GST-LC3. (E) Fluorescence anisotropy of hydroxycoumarin-labeled M2 C terminus (DADDGHFVSIELE) against purified LC3. (F) Lysates of HCT116 cells expressing GFP-LC3 and infected with the indicated IAVs were immunoprecipitated with GFP-TRAP resin. Input and bound fractions were blotted with the indicated antibodies. Dotted line, irrelevant lanes removed. (G) HCT116 cells expressing GFP-LC3 infected with the indicated virus, fixed at 16 hr p.i., and stained with anti-HA. (H and I) Plasma membrane localization (H) or perinuclear accumulation (I) of GFP-LC3 in HCT116 cells expressing the indicated LC3 alleles and infected with the indicated viruses. Coverslips were assessed blindly in triplicate at 16 hr p.i. Mean and SD. **p < 0.01 by ANOVA. (J) Lysates of HCT116 cells expressing GFP-LC3 were blotted for indicated antigens at 16 hr p.i. with indicated IAV. See also Figures S2 and S3 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 243 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 243 Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability Formation of Filamentous Virions Depends on the M2 LIR The strong conservation of the M2 LIR motif and its requirement for the accumulation of LC3 at the plasma membrane suggest that LC3 relocalization benefits the viral life cycle. IAV can bud A B 0 1 2 5 6 7 days 0 1 2 4 5 6 days IAV S93N I94S ** F91S ** log10 PFU/ml log10 PFU/ml IAV S93N I94S ** F91S ** F91S IAV IAV I94S S93N F91S C PR8 PR8 MUd a role for autophag ATG16L1 and LIR m ment bundles (Figu is required for norm Figure 4. M2 LIR Motif Required for Fila- mentous Budding and Virus Stability Figure 4. M2 LIR Motif Required for Fila- mentous Budding and Virus Stability (A) MDCK cells were infected with the indicated PR8 MUd viruses at an moi of 5, fixed at 16 hr p.i., and visualized by scanning electron microscopy. Scale bar 10 mm Figure 4. M2 LIR Motif Required for Fila- mentous Budding and Virus Stability A A IAV S93N I94S F91S F91S IAV (A) MDCK cells were infected with the indicated PR8 MUd viruses at an moi of 5, fixed at 16 hr p.i., and visualized by scanning electron microscopy. Scale bar, 10 mm. Formation of Filamentous Virions Depends on the M2 LIR The strong conservation of the M2 LIR motif and its requirement for the accumulation of LC3 at the plasma membrane suggest that LC3 relocalization benefits the viral life cycle. IAV can bud B 0 1 2 5 6 7 days 0 1 2 4 5 6 days IAV S93N I94S ** F91S ** log10 PFU/ml log10 PFU/ml IAV S93N I94S ** F91S ** F91S IAV I94S S93N C PR8 PR8 MUd Scale bar, 10 mm. (B) MDCK cells were infected with the PR8 MUd viruses, fixed at 10 hr p.i., a without permeabilization with antiseru PR8 virus. (Top) Maximum intensity z jection of confocal images. (Bottom) Sid of z stacks. Scale bar, 10 mm. (C) A549 cells infected with WT or mut PR8 MUd viruses at an moi of 1. Cell-f natants (16 hr p.i.) were harvested and fr immediately or after having been lef temperature for 1 or 2 days. Experim performed in parallel. Titers were det triplicate after thawing by plaque as and standard deviation are shown. **p ANOVA. See also Figure S4. in both a spherical and a fila fashion, depending on the gene ground of the virus and the type (Bourmakina and Garcı´a 2003; Elleman and Barclay, 2004 and Compans, 1998). Most hum ogenic isolates of IAV form up t long filamentous particles that extensive membrane resource M2 influences filament formatio suki-Horimoto et al., 2006; Rob Compans, 1998; Rossman et a we hypothesized that accumu LC3 at the plasma membran represent the virus mobilizing sources for budding. Accordin introduced the M2 LIR mutati a filamentous strain of IAV, P (Noton et al., 2007). PR8-MUd p a profusion of micrometer-len ments arrayed in parallel bundle the M2 S93N LIR variant resem virus, cells infected with the M2 I94S virus encoding nonfunctio produced fewer filaments with tendency to form bundles (Fig Confocal microscopy, which b ward detection of long filament (Simpson-Holley et al., 2002), re large difference in the propensit infected with WT or F91S PR8 project filament bundles away cell surface (Figure 4B). DISCUSSION We describe a functional, highly conserved LC3-binding/LIR motif in the C terminus of IAV M2. This LIR is required for the generation of stable virions, which correlates with the M2 LIR- dependent recruitment of the autophagosomal marker LC3 to the plasma membrane of infected cells. In contrast, the M2 LIR is not required for the induction of autophagy per se, since viruses bearing a mutant M2 LIR enhanced the proportion of lipidated LC3 in infected cells similar to WT IAV. In contrast, viruses lacking M2 scarcely stimulated LC3-II accumulation. We therefore propose a model whereby M2 subverts autophagy by two different means: (1) via a LIR-independent mechanism that, in accordance with a previous report (Gannage´ et al., 2009), is not dependent on the M2 cytosolic domain containing the LIR motif, and (2) via the LIR motif identified here, which redirects LC3 to the plasma membrane. The two activities may not be independent, since M2 may stimulate autophagy via beclin-1 (Gannage´ et al., 2010) and utilize its LIR motif to transport lipids to the plasma membrane. This mobilization of resources via the autophagy machinery may be important to prevent the depletion of the plasma membrane due to budding virus particles. Transmission of infection between individuals and species depends on survival of virions outside the host. The relative contribution of respiratory droplets, fomite, and aerosol trans- mission for IAV is still under debate, but the fomite route in particular is a time-demanding process (Brankston et al., 2007; Hall, 2013). Significantly, IAV with mutations in its LIR is less stable. We have demonstrated that filament formation is affected by LIR mutations. Ultrastructural studies of viruses lacking the M2 C terminus (and thus the LIR motif) showed that even spherical virions had decreased structural integrity (Sugita et al., 2011). We conclude that the LIR motif is involved in budding and is required for morphologically normal and sta- ble viral progeny. Whereas decreased stability is detrimental to the spread of the virus, it may have a technological use. There are significant con- cerns surrounding the manipulation of highly pathogenic strains of IAV, especially in potential gain-of-function experiments (Lan- glois et al., 2013; Patterson et al., 2013). LIR mutations could potentially provide enhanced biosafety by ensuring decreased environmental stability. Motif To test whether the M2 LIR influences virus replication, we examined growth of the PR8 and PR8 MUd viruses. As ex- pected (Simpson-Holley et al., 2002), the nonfilamentous PR8 virus replicated to higher titers than the filamentous PR8 MUd strain (Figure 4C). Within the two backgrounds, viruses encod- ing functional and nonfunctional M2 LIR motifs replicated indis- tinguishably, suggesting the M2 LIR does not directly affect the production of infectious particles (Figures 4C and S2C). How- ever, when viral supernatants were left at room temperature for 24 or 48 hr, viruses encoding WT M2 or M2 S93N remained stable, while viruses encoding nonfunctional LIRs (M2 I94S or F91S) lost infectivity. Filamentous virus was especially sensitive to LIR mutations; PR8 MUd encoding nonbinding M2 LIR mutants suffered a 10-fold drop in titer after just 24 hr at room temperature. M2 is largely excluded from mature virus particles (Rossman and Lamb, 2011), and immunogold staining for LC3 was observed only rarely in virions. This is consistent with the LIR motif predominantly restoring the depleted plasma membrane of host cells as virus particles bud, rather than contributing directly to the viral membrane. Cells produce up to 104 IAV par- ticles (Mo¨ hler et al., 2005). Even if only 1,000 spherical virus par- ticles were produced, their surface corresponds to a substantial fraction (20%) of the apical plasma membrane. The far greater amount of membrane required to produce a filamentous virion suggests that, as seen here, LIR function is of particular impor- tance in supporting filament formation. Given that many other RNA viruses (especially respiratory pathogens) produce filamen- tous particles (Ba¨ chi and Howe, 1973; von Magnus, 1953; Yao and Compans, 2000), it may be that other enveloped viruses have also evolved mechanisms to mobilize lipid resources from host cells via autophagy. We therefore conclude that the LIR motif in M2 is of functional importance to IAV, as indicated by its high degree of conserva- tion and its essential role in producing stable virions, a prerequi- site for the transmissibility of infection. Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Figure 4. M2 LIR Motif Required for Fila- mentous Budding and Virus Stability B IAV B B F91S F91S 0 1 2 5 6 7 days 0 1 2 4 5 6 days IAV S93N I94S ** F91S ** log10 PFU/ml log10 PFU/ml IAV S93N I94S ** F91S ** C PR8 PR8 MUd C 5 log10 PFU/ml Formation of Filamentous Virions Depends on the M2 LIR The strong conservation of the M2 LIR motif and its requirement for the accumulation of LC3 at the plasma membrane suggest that LC3 relocalization benefits the viral life cycle. IAV can bud 244 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 244 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability Stability of Influenza A Virions Depends on the M2 LIR Motif Stability of Influenza A Virions Depends on the M2 LIR Motif Stability of Influenza A Virions Depends on the M2 LIR Motif the plasma membrane. The only other known physiological instance of LC3 localizing to the plasma membrane occurs in os- teoclasts during lysosomal secretion (DeSelm et al., 2011). Our electron micrographs revealed autophagosomes close to the plasma membrane, consistent with the idea that the LC3 at the plasma membrane results from fusion of vesicles. If true, this mechanism suggests that the primary biological purpose of the M2 LIR motif is to deliver appropriate membrane resources for the generation of virus particles. On this account, the M2 pro- tein would ensure both the availability of new transport vesicles by manipulating autophagy downstream of FIP200, and the cor- rect delivery of that lipid resource to the plasma membrane via the LIR motif. siRNA A549 cells expressing GFP-LC3 were transfected on days 1 and 3 with siRNA and were challenged with either infection or starvation on day 5. Gannage´ , M., Ra¨ mer, P.C., and Mu¨ nz, C. (2010). Targeting Beclin 1 for viral subversion of macroautophagy. 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(G0700815, P.D.), the BBSRC (BB/J004324/1, P.D.), and the Academy of Medical Sciences (R.B.). We thank Drs. Akutsu, Ye, Virdee, Howard, and Skepper for assistance and members of our laboratories for discussions. Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus. J. Virol. 82, 11869–11879. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., et al. (2000). A ubiqui- tin-like system mediates protein lipidation. Nature 408, 488–492. Received: August 29, 2013 Revised: December 20, 2013 Accepted: January 16, 2014 Published: February 12, 2014 Received: August 29, 2013 Revised: December 20, 2013 Accepted: January 16, 2014 Published: February 12, 2014 Iwatsuki-Horimoto, K., Horimoto, T., Noda, T., Kiso, M., Maeda, J., Watanabe, S., Muramoto, Y., Fujii, K., and Kawaoka, Y. (2006). The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly. J. 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Two distinct mechanisms might target LC3 to the plasma mem- brane: LC3 may become directly conjugated to the plasma membrane, a scenario that can be demonstrated experimentally but for which no physiological equivalent is known (Fujita et al., 2008), or it may occur by the union of LC3-positive vesicles with This study reveals that IAV utilizes a SliM to hijack autophagy. How this process enhances virion stability remains open, but it likely involves delivery of appropriate resources to the plasma membrane during budding, as evidenced by the highly unusual appearance of LC3 at the plasma membrane. 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Genetic diversity and population structure analysis based on the high density SNP markers in Ethiopian durum wheat (Triticum turgidum ssp. durum)
BMC genomic data
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Alemu et al. BMC Genetics (2020) 21:18 https://doi.org/10.1186/s12863-020-0825-x Alemu et al. BMC Genetics (2020) 21:18 https://doi.org/10.1186/s12863-020-0825-x Open Access © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Genetic diversity and population structure analysis based on the high density SNP markers in Ethiopian durum wheat (Triticum turgidum ssp. durum) Admas Alemu1,2* , Tileye Feyissa1, Tesfaye Letta3 and Bekele Abeyo4 Abstract Background: Ethiopia has been considered as a center of diversity and the second possible center of domestication of durum wheat. Genetic diversity and population structure analysis in the existing Ethiopian durum wheat germplasm have enormous importance in enhancing breeding effort and for sustainable conservation. Hence, 192 Ethiopian durum wheat accessions comprising 167 landraces collected from major wheat-growing areas of the country and 25 improved varieties released from Debre Zeit and Sinana Agricultural Research Centers, Ethiopia in different years (1994–2010) were assembled for the current study. Results: The panel was genotyped with a High-density 90 K wheat SNP array by Illumina and generated 15,338 polymorphic SNPs that were used to analyze the genetic diversity and to estimate the population structure. Varied values of genetic diversity indices were scored across chromosomes and genomes. Genome-wide mean values of Nei’s gene diversity (0.246) and polymorphism information content (0.203) were recorded signifying the presence of high genetic diversity within this collection. Minor allele frequency of the genome varied with a range of 0.005 to 0.5 scoring a mean value of 0.175. Improved varieties clustered separately to landraces in population structure analysis resulted from STRUCTURE, PCA and neighbor joining tree. Landraces clustering was irrespective of their geographical origin signifying the presence of higher admixture that could arise due to the existence of historical exchanges of seeds through informal seed system involving regional and countrywide farming communities in Ethiopia. Conclusions: Sustainable utilization and conservation of this rich Ethiopian durum wheat genetic resource is an irreplaceable means to cope up from the recurrent climate changes and biotic stresses happening widely and thereby able to keep meeting the demand of durum productivity for the ever-growing human population. Keywords: Ethiopian durum wheat, Genetic diversity, Population structure, SNPs, Landraces, Improved varieties Background for about 95% of world wheat production and durum covers the other 5% [1]. Modern wheat cultivars generally refer to two species: hexaploid bread wheat, Triticum aestivum (2n = 6x = 42, AABBDD), and tetraploid/hard or durum wheat, T. durum (2n = 4x = 28, AABB) used for pasta, macaroni, couscous and low-rising bread, and the former accounts In Ethiopia, wheat is the second most widely produced cereal crop after corn and the third most important staple food behind corn and sorghum [2]. Hard red wheat accounts for about 75–80% of the national pro- duction, while durum makes up roughly 10–15% [2]. Wheat has versatile uses in making various foods and drinks, such as bread, ‘Kolo’ (traditional Ethiopian snack made from wheat mixed with barley, chickpea and other legumes and roasted in a clay griddle), ‘Tella’ (traditional * Correspondence: adth14@gmail.com 1Department of Microbial, Cellular and Molecular Biology, Addis Ababa University, P.O.Box 1176, Addis Ababa, Ethiopia 2Department of Biology, Debre Tabor University, Debre Tabor, Ethiopia Full list of author information is available at the end of the article * Correspondence: adth14@gmail.com 1Department of Microbial, Cellular and Molecular Biology, Addis Ababa University, P.O.Box 1176, Addis Ababa, Ethiopia 2Department of Biology, Debre Tabor University, Debre Tabor, Ethiopia Full list of author information is available at the end of the article Page 2 of 12 Page 2 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 maintained more than 60,000 accessions of different crops in its gene bank and of these, 7000 are durum wheat accessions accounting 12% from the total [7, 8]. Besides, up to recent time, agricultural research centers and institutions have been involved in collecting and conserving Ethiopian durum wheat landrace accessions in the country. Due to its uniqueness, Ethiopian durum wheat has been served as a center of focus for genetic studies and the source of novel alleles [9–14]. Vavilov [4] and Zohary [15] reported the presence of high genetic diversity in Ethiopian durum wheat and recent studies specified uniqueness of Ethiopian durum landraces form the Fertile Crescent collections (primary center of domes- tication) and considered as the possible second center of domestication for the crop [3]. Durum wheat is long established in the country and it was likely introduced into the northern highlands of Ethiopia around 3000 BC [16]. Previous studies indicated the existence of high genetic variation of cultivated durum wheat in Ethiopia that arises Ethiopian beer), pasta, macaroni, biscuit, cake, and others. Additionally, wheat straw is commonly used as a roof thatching material and as animal feed in most wheat-growing rural areas of Ethiopia. Hence, increasing wheat production has been a national goal to decrease the gap between production and human consumption especially in view of the fastest-growing population as compared to production. Durum wheat is the result of two successful domesti- cation events by ancient farmers: first, from wild emmer (Triticum turgidum ssp. dicoccoides) to domesticated emmer (T. turgidum ssp. dicoccum) with the loss of fra- gility of spikes (disarticulation into spikelets), and sec- ond, from cultivated emmer to durum with the appearance of naked kernels or free threshing kernel [3]. Ethiopia is one of the few countries that has been served as the center of primary gene pool for various crops [4– 6]. Ethiopian biodiversity institute (EBI), formerly known as Institute of biodiversity conservation (IBC), has Fig. 1 Distribution of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes (B) in 192 Ethiopian durum wheat accessions Fig. 1 Distribution of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes (B) in 192 Ethiopian of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes Fig. 1 Distribution of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes (B) in 192 Ethiopian durum wheat accessions Page 3 of 12 Page 3 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Single nucleotide polymorphisms (SNPs) are the most abundant class of DNA markers. Lower rates of recurrent mutation make them evolutionarily stable. They are excellent markers for studying complex gen- etic traits and for understanding the genomic evolu- tion. They have been widely used in genome-wide association studies, genetic resource characterization, marker-assisted breeding and genomic selection [26]. Hybridization arrays/microarrays have been used as a preeminent solution to develop SNPs in complex polyploid genomes such as wheat [27]. Once a com- prehensive SNP data set is available for a species, a well-designed microarray can be produced; and gener- ally, the technology is then cost-efficient and the process is relatively convenient. The technology avoids the risk of miscalling diversity on homoeolo- gous genomes and its power recently increased 100- fold in wheat moving from 9 K [28] to 820 K [29] genome-wide SNPs. The 90 K wheat SNP array [27] due to the wide range of agro-ecological conditions coupled with diverse farmers’ culture [11, 12, 17–22]. Ethiopian farmers have grown durum wheat since imme- morial time, mostly under adverse environmental condi- tions and they developed a broad gene pool of durum wheat landraces adapted to various environmental condi- tions [23]. In Ethiopia, durum wheat is commonly planted on heavy black clay soils (vertisols) of the highlands be- tween 1800 and 2800 masl [23]. Mechanisms of detecting and analyzing genetic diver- sity have gradually progressed from Mendelian survey of discrete morphological traits to molecular examinations of DNA variation [24]. Genetic diversity analysis is a critical component of plant genetics, breeding, conserva- tion and evolution [25]. Understanding the existing gen- etic divergence and distribution of crop species has paramount importance for conservation and selection of parents with diverse genetic backgrounds, thereby ren- dering crop improvement more efficient [22]. Fig. 2 Frequency distribution of Nei’s gene diversity (A), polymorphic information content (PIC) (B) and minor allelic frequency (MAF) (C) of 15,338 polymorphic SNPs generated from Ethiopian durum wheat accessions Fig. 2 Frequency distribution of Nei’s gene diversity (A), polymorphic information content (PIC) (B) and minor allelic frequency (MAF) (C) of 15,338 polymorphic SNPs generated from Ethiopian durum wheat accessions Fig. 2 Frequency distribution of Nei’s gene diversity (A), polymorphic information content (PIC) (B) and minor allelic frequency (MAF) (C) of 15,338 polymorphic SNPs generated from Ethiopian durum wheat accessions Page 4 of 12 Page 4 of 12 Page 4 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Results has been successfully used for genetic diversity ana- lysis, genome-wide association mapping and construc- tion of high-density consensus maps in both bread and durum wheat [12, 30–32]. SNP markers distribution Chromo- some 1A scored the highest PIC (0.229) and gene di- versity (0.282) (Table 1). In contrast, the lowest PIC and genetic diversity score was observed on chromo- some 7A (PIC = 0.181; gene diversity = 0.217). Chro- mosomes 2A, 2B, 3A, 3B, 7A and 7B showed slightly lower polymorphic information content than the aver- age PIC values of the whole genome. On the other hand, homoeologous chromosome groups 1, 4, and 5 scored higher Nei’s genetic diversity than the average genome-wide value. The highest gene diversity, PIC and MAF were on homoeologous chromosome group five. Comparable mean values of genetic diversity, PIC and MAF were scored on A and B genomes. SNPs of which 84.43% was polymorphic. Higher number polymorphic SNP markers were recorded on B genome (9013 SNPs) than the A genome (6325 SNPs) in Ethiop- ian durum wheat accessions (Fig. 1-b). genome was 0.175 ranged from 0.005 to 0.5. Chromo- some 1A scored the highest PIC (0.229) and gene di- versity (0.282) (Table 1). In contrast, the lowest PIC and genetic diversity score was observed on chromo- some 7A (PIC = 0.181; gene diversity = 0.217). Chro- mosomes 2A, 2B, 3A, 3B, 7A and 7B showed slightly lower polymorphic information content than the aver- age PIC values of the whole genome. On the other hand, homoeologous chromosome groups 1, 4, and 5 scored higher Nei’s genetic diversity than the average genome-wide value. The highest gene diversity, PIC and MAF were on homoeologous chromosome group five. Comparable mean values of genetic diversity, PIC and MAF were scored on A and B genomes. SNP markers distribution From 81,587 SNP probes available on the chip, 30,510 SNP calls (23,354 polymorphic SNPs) were reproducible in the current Ethiopian durum wheat panel. From these markers, 18,788 SNPs had a known position but only 15, 338 (81.63%) were polymorphic and used for the current study (Additional file 2: Table S2). The smallest number of SNP markers were recorded on chromosome 1A (263 SNPs) while the highest on chromosome 2B (2253 SNPs) (Fig. 1-a). Chromosome 2B also contributed the highest number of polymorphic SNP markers (1755 SNPs) while the smallest on chromosome 1A (236 SNPs). Consider- ing the distribution of SNPs across homoeologous chro- mosomes, group two scored the highest number of SNP markers (3639 SNPs of which 78.38% is polymorphic) while the smallest number on group one with 1709 Molecular characterization of Ethiopian durum wheat accessions has been investigated in DNA markers with a very limited number, such as microsatellites [11, 20, 21]. However, except in a single attempt that has made to characterize Ethiopian durum wheat landraces collected by EBI siding with Mediterranean durum wheat [12], the germplasm has not been extensively investigated with a high density SNP markers. Hence, the present study aimed to assess the genetic diversity and population structure of 167 landraces and 25 improved varieties col- lected and maintained at Debre Zeit and Sinana Agricul- tural Research Centers, Ethiopia with a 90 K wheat SNP array. Table 1 Mean values of diversity indices and minor allelic frequency distribution of SNP markers across chromosomes in 192 Ethiopian durum wheat accessions Chromosome Nei’s Gene diversity Polymorphic information content (PIC) Minor allelic frequency (MAF) A-Genome 1A 0.282 0.229 0.208 2A 0.221 0.185 0.150 3A 0.246 0.202 0.175 4A 0.248 0.204 0.176 5A 0.272 0.221 0.200 6A 0.265 0.215 0.194 7A 0.217 0.181 0.150 B-Genome 1B 0.254 0.208 0.185 2B 0.229 0.190 0.158 3B 0.233 0.194 0.159 4B 0.279 0.226 0.209 5B 0.265 0.217 0.191 6B 0.246 0.203 0.172 7B 0.235 0.193 0.164 Homoeologous 1 0.259 0.211 0.188 2 0.226 0.188 0.155 3 0.238 0.197 0.166 4 0.264 0.215 0.193 5 0.268 0.218 0.194 6 0.255 0.209 0.182 7 0.226 0.187 0.157 A-Genome 0.2448 0.2015 0.1744 B-Genome 0.2471 0.2035 0.1750 Whole Genome 0.2462 0.2027 0.1748 Page 5 of 12 Page 5 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 genome was 0.175 ranged from 0.005 to 0.5. Genetic diversity analysis The SNP markers exhibited a wide range of poly- morphic information content (PIC) and Nei’s gene di- versity across chromosomes and genomes of Ethiopian durum wheat. Frequency distribution of SNPs for gene diversity, polymorphic information content, and frequency of the minor allele values of the genome is presented in Fig. 2-a, Fig. 2-b, and Fig. 2-c, respectively. While a detail of the frequency dis- tribution of SNP markers across chromosomes is pre- sented for values of gene diversity (Additional file 4: Figure S2), PIC (Additional file 5: Figure S3) and minor allelic frequency (Additional file 6: Figure S4). The overall mean value of polymorphic information content was 0.203 ranged from 0.01 to 0.375. Nei’s gene diversity score was varied from 0.01 to 0.5 with a mean value of 0.246 and the mean MAF of the Genetic stratification and principal component analysis Hence, accessions were grouped into three clusters based on the STRUCTURE-inferred clus- tering result with 75, 27 and 90 accessions came to- gether for sub-populations 1, 2 and 3, respectively (Additional file 1: Table S1). Landraces gathered on cluster-one and cluster-three while all improved var- ieties, except one variety (Selam) that was under cluster one, assembled on sub-population two. The neighbor- joining based clustering analysis (Fig. 4) also identified three clear clusters and except one accession all are grouped based on the STRUCTURE based stratification. 24.29, 6.61 and 3.74% of the total variance, respectively. The smaller numbers of variance explained by the sec- ond and consecutive PCs indicated that only few PCs couldn’t encapsulate the existing genetic variance in Ethiopian durum wheat. The first PC (PC1) distantly clustered varieties from landraces and the second PC grouped the two landrace subgroups (Fig. 5-a). The first two PCs (PC1 and PC2) clearly clustered the three sub- populations. However, clustering gets distorted when additional principal components were considered (Fig. 5-b). Genetic stratification and principal component analysis Genetic stratification and principal component analysis The optimal sub-population of accessions was inferred through two approaches: The first method was the STRUCTURE-based clustering approach that was in- ferred based on the second order rate of change of the likelihood (ΔK) (Table 3). The result indicated a clear peak at K = 3 signifying the optimal sub-populations in Fig. 3 Inference of the optimal numbers of sub-populations (clusters) in Ethiopian durum wheat panel with the Bayesian clustering model in STRUCTURE (A) and by the discriminant analysis of principal components (DAPC) using adegenet package (B) Fig. 3 Inference of the optimal numbers of sub-populations (clusters) in Ethiopian durum wheat panel with the Bayesian clustering model in STRUCTURE (A) and by the discriminant analysis of principal components (DAPC) using adegenet package (B) Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Page 6 of 12 the panel (Fig. 3-a). The second approach was based on the discriminant analysis of principal components (DAPC) and the result couldn’t show a clear lowest Bayesian information criterion (BIC) on a specific K value above which BIC values decreased spontaneously with simultaneous increment making an elbow at the optimal K value (Fig. 3-b). However, in this case, it pro- vided a clue in which somehow less than five clusters could be optimal. Hence, accessions were grouped into three clusters based on the STRUCTURE-inferred clus- tering result with 75, 27 and 90 accessions came to- gether for sub-populations 1, 2 and 3, respectively (Additional file 1: Table S1). Landraces gathered on cluster-one and cluster-three while all improved var- ieties, except one variety (Selam) that was under cluster one, assembled on sub-population two. The neighbor- joining based clustering analysis (Fig. 4) also identified three clear clusters and except one accession all are grouped based on the STRUCTURE based stratification. the panel (Fig. 3-a). The second approach was based on the discriminant analysis of principal components (DAPC) and the result couldn’t show a clear lowest Bayesian information criterion (BIC) on a specific K value above which BIC values decreased spontaneously with simultaneous increment making an elbow at the optimal K value (Fig. 3-b). However, in this case, it pro- vided a clue in which somehow less than five clusters could be optimal. Genetic divergence between landraces and improved varieties The Bayesian model based stratification and PCA clustering methods grouped landraces and varieties in distinct places except in a single variety, Selam, which was clustered alongside landraces. The numbers of polymorphic SNPs were slightly higher in varieties than landraces. Varieties scored higher gene diversity (0.297), PIC (0.240) and the mean frequency of minor Principal component analysis (PCA) was analyzed with all Polymorphic SNPs generated from the panel. The first, second and third principal components explained Fig. 4 Neighbor-joining tree generated based on simple matching dissimilarity coefficients using SNP markers from 192 Ethiopian durum wheat accessions. Colors of accessions are clusters inferred from STRUCTURE-based analysis Fig. 4 Neighbor-joining tree generated based on simple matching dissimilarity coefficients using SNP markers from 192 Ethiopian durum wheat accessions. Colors of accessions are clusters inferred from STRUCTURE-based analysis Page 7 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics Fig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A) Varieties cluster (green, K-2) showing a clear separation from the other two landrace clusters (K1 & K2) using PC1 and PC2. (B) Clustering got disrupted when considering other PCs Fig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A) Varieties cluster (green, K-2) showing a clear separation from the other two landrace clusters (K1 & K2) using PC1 and PC2. (B) Clustering got disrupted when considering other PCs ig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A) Fig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A) Varieties cluster (green, K-2) showing a clear separation from the other two landrace clusters (K1 & K2) using PC1 and PC2. (B) Clustering got disrupted when considering other PCs allele (0.218) than landraces (gene diversity = 0.213; PIC = 0.173 & MAF = 0.154) (Table 2). Further analysis of molecular variance between the 25 varieties and 167 landrace accessions indicated higher genetic variation between the two groups (61.02%) than individuals within the group (38.98%) (Table 5). Genetic divergence between landraces and improved varieties However, the number of landraces included in the present study was higher by six folds than the number of varieties and that could affect the reliability of the PIC, genetic diversity and the minor allelic frequency scores reported in the current study. Genetic clustering via geographic origin The current Ethiopian durum wheat germplasm com- prises landrace accessions collected from major wheat- producing areas of the country (Additional file 3: Figure S1) including Bale, Gondar, Gojjam, Shewa, Tigray, and Wollo, and 12 Ethiopian durum wheat landraces cur- rently cultivated in the USA. Genetic variation among clusters Landraces collected from Bale (South- eastern Ethiopia) grouped in both clusters (44 land- races in cluster 1 and 24 landraces in cluster 3). The two landraces collected from Tigray region (North Ethiopia) were clustered in sub-population one. How- ever, a landrace collected from Gondar, adjacent to Tigray, was sub-grouped in cluster three. All twelve Ethiopian landraces that are now cultivated in the USA were gathered in sub-population three. Table 4 Analysis of molecular variance (AMOVA) for Ethiopian du STRUCTURE clustering result Source of variation DF Sum of squares Variance c Among populations 2 166,172.20 700.94 Va Among individuals Within populations 189 240,563.25 636.41 Vb Total 191 406,735.45 1337.35 Discussions into the different sub-groups irrespective to their geo- graphic origin. For instance, eight landraces collected from northeastern Ethiopia (Wollo) were grouped in sub-population one while 25 landraces from the same origin clustered in sub-population three (Additional file 1: Table S1). Landraces collected from central Ethiopia (Akaki and Shewa) clustered in both sub- groups; 4 landraces in sub-population one while 19 landraces in sup-population three. However, from the total eight landraces collected in Bichena (a town in East Gojjam Zone, west-central Ethiopia), seven were grouped in sub-population three and the other one landrace altogether with four landraces collected from other parts of Gojjam were grouped in sub- population one. Landraces collected from Bale (South- eastern Ethiopia) grouped in both clusters (44 land- races in cluster 1 and 24 landraces in cluster 3). The two landraces collected from Tigray region (North Ethiopia) were clustered in sub-population one. How- ever, a landrace collected from Gondar, adjacent to Tigray, was sub-grouped in cluster three. All twelve Ethiopian landraces that are now cultivated in the USA were gathered in sub-population three. into the different sub-groups irrespective to their geo- graphic origin. For instance, eight landraces collected from northeastern Ethiopia (Wollo) were grouped in sub-population one while 25 landraces from the same origin clustered in sub-population three (Additional file 1: Table S1). Landraces collected from central Ethiopia (Akaki and Shewa) clustered in both sub- groups; 4 landraces in sub-population one while 19 landraces in sup-population three. However, from the total eight landraces collected in Bichena (a town in East Gojjam Zone, west-central Ethiopia), seven were grouped in sub-population three and the other one landrace altogether with four landraces collected from other parts of Gojjam were grouped in sub- population one. Landraces collected from Bale (South- eastern Ethiopia) grouped in both clusters (44 land- races in cluster 1 and 24 landraces in cluster 3). The two landraces collected from Tigray region (North Ethiopia) were clustered in sub-population one. How- ever, a landrace collected from Gondar, adjacent to Tigray, was sub-grouped in cluster three. All twelve Ethiopian landraces that are now cultivated in the USA were gathered in sub-population three. Genetic variation among clusters The analysis of molecular variance (AMOVA) re- vealed the presence of higher genetic variance be- tween STRUCTURE-inferred sub-populations (52.41%) than among individuals within clusters (47.59%) (Table 4). The clustering analysis indicated that the SNPs data couldn’t group landraces clearly based on their geo- graphical background and accessions were admixed Table 2 Mean of diversity indices and minor allelic frequency distribution of SNPs in landraces and varieties of Ethiopian durum wheat Accession Type Sample size No. of polymorphic SNPs Nei’s Gene diversity Polymorphic information content (PIC) Minor allelic frequency (MAF) Landrace 167 13,466 0.213 0.173 0.154 Cultivar 25 13,725 0.297 0.240 0.218 Alemu et al. BMC Genetics (2020) 21:18 Page 8 of 12 Alemu et al. BMC Genetics Table 3 Inference of the optimal numbers of clusters existed in Ethiopian durum wheat panel using Delta K Statistics K L(K) stdev L’(K) L”(K) |L”(K)| Delta K 1 −157,376 3.369 – – – – 2 −138,930 1370.013 18,446.93 −3224.75 3224.75 2.35 3 −123,708 543.0884 15,222.19 −5458.76 5458.76 10.05 4 −113,944 1149.967 9763.425 −1419.46 1419.46 1.23 5 −105,600 708.175 8343.965 −3511.28 3511.28 4.96 6 −100,768 677.594 4832.685 −1326.03 1326.025 1.96 7 −97,261 1353.421 3506.66 580.6019 580.6019 0.43 8 −93,173 1398.676 4087.262 −1491.05 1491.054 1.07 9 −90,577 1708.376 2596.208 635.7119 635.7119 0.37 10 −87,345 2170.333 3231.92 Where; K = Subpopulations; Ln (PD) = The log likelihood for each K; L (K) = An average of 20 values of Ln P(D); stdev = Standard deviation for 20 values of Ln P(D); L’(K) = L(K) n – L(K) n-1; L”(K) = L′(K) n+ 1 −L′(K); ΔK = |L”(K)|/Stdev; The gray color of the third line designates the optimal sub-populations of the panel based on the highest delta K value into the different sub-groups irrespective to their geo- graphic origin. For instance, eight landraces collected from northeastern Ethiopia (Wollo) were grouped in sub-population one while 25 landraces from the same origin clustered in sub-population three (Additional file 1: Table S1). Landraces collected from central Ethiopia (Akaki and Shewa) clustered in both sub- groups; 4 landraces in sub-population one while 19 landraces in sup-population three. However, from the total eight landraces collected in Bichena (a town in East Gojjam Zone, west-central Ethiopia), seven were grouped in sub-population three and the other one landrace altogether with four landraces collected from other parts of Gojjam were grouped in sub- population one. Genetic diversity of Ethiopian durum wheat Genetic diversity of Ethiopian durum wheat y p Genetic diversity is imperative to provide a robust food security system capable of adapting to recurrent biotic and abiotic stresses. Genetic diversity analysis is a crucial step in noticing alleles that could be used as the source of novel traits with high yielding, resilient for biotic and/ or abiotic stresses and yet delivers satisfied productivity or in meeting the end-user demands in plant breeding. Ethiopian durum wheat landraces have especially proven to show a relevant variation for various traits derived from their potential in adapting to changing environ- mental conditions [33]. Due to this, Ethiopian durum wheat germplasm has served as a center of focus for genetic studies and served as the source of novel QTLs, genes and gene complexes for many traits [9–14, 34]. Slightly higher number of SNPs (30,510) were repro- duced in the present study from the 90 K wheat SNP array than previously reported by Mengistu et al. [12] on Ethiopian durum wheat (30,155 SNPs) and in Mediterra- nean durum wheat collections (21,069 SNPs). Genomes of A and B did not show a significant difference in diver- sity indices, indicating that they have followed similar Table 4 Analysis of molecular variance (AMOVA) for Ethiopian durum wheat accessions with and without grouping according to STRUCTURE clustering result Source of variation DF Sum of squares Variance components Percentage of variation Fixation indices P value Among populations 2 166,172.20 700.94 Va 52.41 FST = 0.52 Va and FST = 0.000 Among individuals Within populations 189 240,563.25 636.41 Vb 47.59 FIS = 1.00 Vb and FIS = 0.000 Total 191 406,735.45 1337.35 Page 9 of 12 Alemu et al. BMC Genetics (2020) 21:18 Table 5 Analysis of molecular variance) between landraces and varieties Source of variation DF Sum of squares Variance components Percentage of variation Fixation indices P value Among populations 1 108,435.72 1228.63 Va 61.02 FST = 0.61 Va and FST = 0.000 Among individuals Within populations 190 298,299.73 784.99 Vb 38.98 FIS = 1.00 Vb and FIS = 0.000 Total 191 406,735.45 2013.62 evolutionary histories in Ethiopian durum wheat land- races and improved varieties [12]. by simultaneous increment creating an elbow shape [41]. However, it provided a clue in which somehow less than five clusters could be optimal. Varieties showed a single distinct cluster and landraces distributed into two distinct clusters. Genetic diversity of Ethiopian durum wheat Both principal component analysis (PCA) with the first two components and the neighbor joining cluster- ing based on simple matching dissimilarity coefficient proved the former clustering result was optimal showing three clear clusters. In the current study, clustering was not based on their geographic origin where landrace ac- cessions were originally collected in Ethiopia. Mengistu et al. [12] reported a similar result on a study conducted in 311 Ethiopian durum wheat accessions (287 landraces and 24 varieties) collected from major wheat producing areas of the country. This admixture could be due to the existence of historical and current exchange of seeds through informal seed system involving regional and countrywide farming communities [34]. Ren et al. [36] re- ported neither geographical nor ecological evidence was detected in grouping 150 durum wheat accessions with world-wide origin and noted that the possible reason could be the existence of gene flow via germplasm ex- changes among different regions occurred frequently or that human transfer of genes in history made a very big admixture. Kabbaj et al. [3] found higher admixtures be- tween 370 durum wheat accessions included landraces, varieties and elite lines collected from more than 30 coun- tries including Ethiopia. However, they observed a very limited admixture between Ethiopian landraces with other collections originated world-wide and Ethiopian durum wheat landraces made a separate cluster and proved the presence of a unique morphology [10, 34] and represent a separate sub-species under the name Triticum durum subs. Abyssinicum or T. aethiopicum [22]. This phenom- ena placed Ethiopia as a secondary center of origin and di- versity for durum wheat since the germplasm is distinct from the primary region of origin of durum wheat, the Fertile Crescent countries [3]. The 90 K wheat SNP array was a platform made to cap- ture the most reliable gene-associated SNP markers avail- able in the wheat genome worldwide and could not enable to mine new loci. The less number of SNPs reproduced from the array in the current panel indicates the possibil- ity of existence of novel alleles and further studies would be benefited from the employment of both hybridization and sequencing techniques to provide a thorough descrip- tion of Ethiopian durum wheat genome. Genetic diversity of Ethiopian durum wheat Comparing with previous reports, higher genetic diver- sity indices were scored in Ethiopian durum wheat panel (Table 1) that strengthens the unresolved and ongoing ar- gument of Ethiopia as the center of origin or domestication of durum wheat [3]. The result unveiled the presence of higher genetic diversity in Ethiopian durum wheat that could arise because of various causes including adaptation to wider agro-ecology [23], natural crossings due to culti- vating mixed genotypes in a field and diverse farmers’ cul- ture of agricultural practices [22, 35]. For instance, Ren et al. [36] reported mean polymorphic information content (0.18) and Nei’s gene diversity (0.22) from world-wide col- lected 150 durum wheat accessions genotyped with 1536 SNP markers. Kabbaj et al. [3] obtained a mean PIC value of 0.119 from 337 durum wheat accessions included land- races, varieties and elite lines collected from more than 30 countries genotyped with 35 K Affymetrix Axiom wheat breeders array. Eltaher et al. [37] reported slightly higher mean gene diversity (0.3) and PIC (0.23) in 250 winter wheat accessions genotyped with Genotyping-By- Sequencing (GBS) platform. However, unlike the present study, they only included SNP markers having less than 20% missing information and with minor allelic frequency (MAF) greater than 5%. As expected, higher PIC and gen- etic diversity scores were reported in studies using multi- allelic markers such as SSR, unlike SNPs, that could go be- yond 0.5 values [38, 39]. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12863-020-0825-x. Additional file 1 : Table S1. Accession names and types, cultivated areas, seed sources and population structure of 192 Ethiopian durum wheat accessions used for the current study. Additional file 1 : Table S1. Accession names and types, cultivated areas, seed sources and population structure of 192 Ethiopian durum wheat accessions used for the current study. Additional file 2 : Table S2. List of polymorphic SNPs, their alleles and chromosomes used for genetic diversity analysis in Ethiopian durum wheat accessions. Genetic structure In this study, 192 Ethiopian durum wheat accessions comprising 167 landraces and 25 improved varieties were assembled and genotyped with a high density 90 K wheat SNP array to analyze the existing genetic diversity and population structure within accessions. Clustering analysis showed a higher genetic admixture between landraces despite their geographic origin resulted from Genetic stratification analysis based on the Bayesian clus- tering model of the second order rate of change of the likelihood [40] revealed the presence of three subpopula- tions. However, discriminant analysis of principal compo- nents based on the Bayesian information criterion (BIC) couldn’t show the smallest BIC on a specific K value above which the BIC values spontaneously decreased followed Page 10 of 12 Page 10 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 the probability of finding polymorphisms between any two random samples while Nei’s gene diversity defined as the probability of two randomly chosen alleles from the population is different. Principal component analysis (PCA) [43] for the genetic relationships among individ- uals was calculated using a package “SNPrelate” [44] in R studio [45]. Neighbor-Joining tree based on simple matching dissimilarity coefficient was constructed using DARwin var. 6.0.14 [46] and the resulting trees were dis- played using FigTree var. 1.4.3 [47]. A software package Arlequin v.3.5.2.2 [48] was used to assess the molecular variance (AMOVA) between clusters based on STRUCT URE-inferred subpopulations and between landraces and varieties. the existence of higher rate of historical seed exchange throughout the country. Diversity indices revealed the presence of higher genetic diversity in Ethiopian durum wheat accessions. Landraces adapted to wider agroecol- ogy and with the genetic capacity to tolerate various stresses could be used as a source of unique alleles in the enhancement of durum breeding through marker assisted selection or marker assisted backcrossing. Hence, sustainable conservation and utilization of Ethi- opian durum wheat genetic resource is key for future breeding strategies in Ethiopia and worldwide. Genetic structure analysis Two approaches were implemented to infer the optimal clusters/subpopulations existed in 192 Ethiopian durum wheat accessions. First, a Bayesian model-based cluster- ing approach was used to estimate the optimal subpopu- lations and the membership probability of each genotype to the subpopulations using STRUCTURE v.2.3 [49]. To infer the optimal clusters, an ad hoc quantity (ΔK) ap- proach was applied that was calculated based on the sec- ond order rate of change of the likelihood [40]. For this analysis, 10 sub-populations with 20 independent itera- tions for each sub-population was done under the ad- mixture model of population structure with correlated allele frequencies and 50,000 lengths burn-in period and 100,000 Markov Chain Monte Carlo (MCMC) replica- tions after burn-in was applied for each iteration. Landraces were originally collected from major wheat- producing areas of Ethiopia (Additional file 3: Figure S1) including Bale, Gondar, Gojjam, Shewa, Tigray, and Wollo, as well as twelve lines, which are originally from Ethiopia but currently cultivated in the USA. A detail of accessions is summarized in Additional file 1: Table S1. DNA extraction and SNP genotyping The second approach was based on the discriminant analysis of principal components (DAPC) implemented using a package “adegenet” [41] in R studio. In this method, the optimal clustering solution corresponded to the lowest Bayesian Information Criterion (BIC) and the number of clusters determined as the value of K above which BIC values decreased with simultaneous incre- ment making an elbow at the optimal cluster [41]. A pooled tissue sample of twenty five one-week-old seedlings was taken for genomic DNA extraction for each accession. The DNA extraction was done with DNeasy 96 Plant Kit (Qiagen GmbH, Hilden, Germany). SNP markers were generated using the Illumina iSe- lect® 90 K wheat SNP assay comprising 81,587 gene- associated SNPs [27]. Marker genotypes were called with the GenomeStudio v2011.1 software package (Illumina, San Diego, CA, USA) and calls showing residual hetero- zygosity were entered as missing values before exporting genotype data from the GenomeStudio. A high-density consensus map of tetraploid wheat generated by Macca- ferri et al. [32] was used to identify chromosome posi- tions of SNPs. The SNPs data used for diversity analysis is available in Additional file 2: Table S2. Plant material One hundred sixty seven Ethiopian durum landrace ac- cessions collected from major wheat growing areas of the country and twenty five improved varieties released in different years and have been cultivated in Ethiopia were assembled for the present study. Improved varieties were released by Debre Zeit Agricultural Research Cen- ter (DZARC) and Sinana Agricultural Research Center (SARC), Ethiopia in different years (1994–2010). All landrace accessions and varieties are maintained by these two agricultural research centers as a single seed descent (SSD) progenies. Funding Fingerprinting of accessions was made possible with the financial support from Bill and Melinda Gates Foundation, the Department for International Development of the United Kingdom, and the AGER Agroalimentare e Ricerca-Project “From seed to pasta-Multidisciplinary approaches for a more sustainable and high quality durum wheat production”. The role of the fund- ing bodies is limited to direct funding of the fingerprinting of genotypes that result in this manuscript. 13. Liu W, Maccaferri M, Rynearson S, Letta T, Zegeye H, Tuberosa R, et al. Novel Sources of Stripe Rust Resistance Identified by Genome-Wide Association Mapping in Ethiopian Durum Wheat (Triticum turgidum ssp. durum). Front Plant Sci. 2017;8. https://doi.org/10.3389/fpls.2017.00774. 14. Maccaferri M, Harris NS, Twardziok SO, Pasam RK, Gundlach H, Spannagl M, et al. Durum wheat genome highlights past domestication signatures and future improvement targets. Nat Genet. 2019;51:885–95. https://doi.org/10. 1038/s41588-019-0381-3. Abbreviations AMOVA A l i AMOVA: Analysis of molecular variance; BIC: Bayesian information content; DAPC: Discriminant analysis of principal components; DZARC: Debre Zeit agricultural research center; EBI: Ethiopian biodiversity institute; MAF: Minor allele frequency; MCMC: Markov chain Monte Carlo; PCA: Principal AMOVA: Analysis of molecular variance; BIC: Bayesian information content; DAPC: Discriminant analysis of principal components; DZARC: Debre Zeit agricultural research center; EBI: Ethiopian biodiversity institute; MAF: Minor allele frequency; MCMC: Markov chain Monte Carlo; PCA: Principal component analysis; PIC: Polymorphic information content; SARC: Sinana agricultural research center; SNP: Single nucleotide polymorphism 8. Mengistu DK, Kidane YG, Fadda C, Pè ME. Genetic diversity in Ethiopian durum wheat ( Triticum turgidum var durum ) inferred from phenotypic variations. Plant Genet Resour Charact Util. 2018;16:39–49. https://doi.org/10. 1017/S1479262116000393. component analysis; PIC: Polymorphic information content; SARC: Sinana agricultural research center; SNP: Single nucleotide polymorphism 9. Porceddu E, Perrino P, Olita G. Preliminary information on an Ethiopian wheat germplasm collecting mission. In: Scarascia Mugnozza GT, editor. Genetics and breeding of durum wheat. Bari, Italy: University of Bari; 1973. p. 181–200. Genetic diversity analysis Numbers and percent of polymorphic loci, polymorph- ism information content (PIC), Nei’s gene diversity and minor allelic frequency (MAF) were calculated using Power Marker v 3.25 [42]. PIC was estimated based on Additional file 3 : Figure S1. Map of Ethiopia showing the major wheat producing areas where 167 landraces were originally collected (Source: Atlas of the Ethiopian rural Economy (2006); http://www.ifpri.org/ node/3763). Page 11 of 12 Page 11 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 3. Kabbaj H, Sall AT, Al-Abdallat A, Geleta M, Amri A, Filali-Maltouf A, et al. Genetic diversity within a global panel of durum wheat (Triticum durum) landraces and modern Germplasm reveals the history of alleles exchange. Front Plant Sci. 2017;8. https://doi.org/10.3389/fpls.2017.01277. Additional file 4 : Figure S2. Frequency distributions for Nie’s gene diversity score of polymorphic SNPs across chromosomes in Ethiopian durum wheat accessions. Front Plant Sci. 2017;8. https://doi.org/10.3389/fpls.2017.01277. Additional file 5 : Figure S3. Frequency distributions for polymorphic information content (PIC) values of polymorphic SNPs across chromosomes. 4. Vavilov NI. The origin, variation, immunity, and breeding of cultivated plants. Cambridge: Cambridge University Press; 1951. 4. Vavilov NI. The origin, variation, immunity, and breeding of cultivated plants. Cambridge: Cambridge University Press; 1951. g g y 5. Westphal E. Agricultural systems in Ethiopia. Wageningen: Centre for Agricultural Publishing and Documentation; 1975. http://edepot.wur.nl/361350 6. Harlan JR. Ethiopia: a center of diversity. Econ Bot. 1969;23:309–14. https:// doi.org/10.1007/BF02860676. 5. Westphal E. Agricultural systems in Ethiopia. Wageningen: Centre for Agricultural Publishing and Documentation; 1975. http://edepot.wur.nl/361350 5. Westphal E. Agricultural systems in Ethiopia. Wageningen: Centre for Agricultural Publishing and Documentation; 1975. http://edepot.wur.nl/361350 5. Westphal E. Agricultural systems in Ethiopia. Wageningen: Centre for Agricultural Publishing and Documentation; 1975. http://edepot.wur.nl/361350 6. Harlan JR. Ethiopia: a center of diversity. Econ Bot. 1969;23:309–14. https:// doi.org/10.1007/BF02860676. Additional file 6 : Figure S4. Frequency distributions for minor allele frequency (MAF) values of polymorphic SNPs across chromosomes. Additional file 6 : Figure S4. Frequency distributions for minor allele frequency (MAF) values of polymorphic SNPs across chromosomes. 6. Harlan JR. Ethiopia: a center of diversity. Econ Bot. 1969;23:309–14. https:// doi.org/10.1007/BF02860676. 7. IBC (Institute of Biodiversity Conservation). Ethiopia: Second Country Report on the State of PGRFA to FAO. Addis Ababa, Ethiopia; 2008. http://www. pgrfa.org. 7. IBC (Institute of Biodiversity Conservation). Ethiopia: Second Country Report on the State of PGRFA to FAO. Addis Ababa, Ethiopia; 2008. http://www. pgrfa.org. Received: 11 November 2019 Accepted: 5 February 2020 Received: 11 November 2019 Accepted: 5 February 2020 20. Alamerew S, Chebotar S, Huang X, Röder M, Börner A. Genetic diversity in Ethiopian hexaploid and tetraploid wheat germplasm assessed by microsatellite markers. Genet Resour Crop Evol. 2004;51:559–67. https://doi. org/10.1023/B:GRES.0000024164.80444.f0. Competing interests 17. Bechere E, Belay G, Mitiku D, Merker A. Phenotypic diversity of tetraploid wheat landraces from northern and north-central regions of Ethiopia. Hereditas. 2004;124:165–72. https://doi.org/10.1111/j.1601-5223.1996.00165.x. The authors declare that they have no competing interests. Authors’ contributions AA, TF and TL conceived and designed the study. TL and AA assembled the panel and participated in genotyping. AA prepared the manuscript and carried out the data analysis. All co-authors participated in interpreting the data, revising and editing the manuscript and approved the final version of the manuscript. 12. Mengistu DK, Kidane YG, Catellani M, Frascaroli E, Fadda C, Pè ME, et al. High-density molecular characterization and association mapping in Ethiopian durum wheat landraces reveals high diversity and potential for wheat breeding. Plant Biotechnol J. 2016;14:1800–12. https://doi.org/10. 1111/pbi.12538. Acknowledgments The authors are grateful to Debre zeit and Sinana Agricultural Research Centers for their generous provision of landraces and improved varieties used in this study. The first author further obliged to Addis Ababa University and University of Bologna for the training provided. 10. Pecetti L, Annicchiarico P, and Damania AB. Biodiversity in a germplasm collection of durum wheat. Euphytica. 1992;60:229–238. doi: https://doi.org/ https://doi.org/10.1007/BF00039403. 11. Haile JK, Hammer K, Badebo A, Nachit MM, Röder MS. Genetic diversity assessment of Ethiopian tetraploid wheat landraces and improved durum wheat varieties using microsatellites and markers linked with stem rust resistance. Genet Resour Crop Evol. 2013;60:513–27. https://doi.org/10.1007/ s10722-012-9855-1. Author details 1D f 18. Tesemma T, Bechere E. Developing elite durum wheat landrace selections (composites) for Ethiopian peasant farm use: raising productivity while keeping diversity alive. 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A Radar Object-Based Examination of Rain System Climatology and Including Climate Variability Hooman Ayat  (  h.ayat@student.unsw.edu.au ) University of New South Wales https://orcid.org/0000-0001-9499-5894 Jason P. Evans  University of New South Wales Steven C. Sherwood  University of New South Wales Joshua Soderholm  Australian Bureau of Meteorology Research Article Keywords: radar object-based examination, rain system, climatology, climate variability, warming, storms, intensity, rainfall statistics Posted Date: August 11th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-783979/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License A Radar Object-Based Examination of Rain System Climatology and Including Climate Variability Hooman Ayat  (  h.ayat@student.unsw.edu.au ) University of New South Wales https://orcid.org/0000-0001-9499-5894 Jason P. Evans  University of New South Wales Steven C. Sherwood  University of New South Wales Joshua Soderholm  Australian Bureau of Meteorology Research Article Keywords: radar object-based examination, rain system, climatology, climate variability, warming, storms, intensity, rainfall statistics Posted Date: August 11th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-783979/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License A radar object-based examination of rain system climatology and including climate variability Hooman Ayat1, Jason P. Evans1, Steven C. Sherwood1, Joshua Soderholm2 1 Climate Change Research Centre and ARC Centre of Excellence for Climate Extremes, University of New South Wales, Sydney, New South Wales, Australia 2 Science and Innovation Group, Australian Bureau of Meteorology, Melbourne, Victoria, Australia 1 Climate Change Research Centre and ARC Centre of Excellence for Climate Extremes, University of New South Wales, Sydney, New South Wales, Australia 2 Science and Innovation Group, Australian Bureau of Meteorology, Melbourne, Victoria, Australia 1 Climate Change Research Centre and ARC Centre of Excellence for Climate Extremes, University of New South Wales, Sydney, New South Wales, Australia 2 Science and Innovation Group, Australian Bureau of Meteorology, Melbourne, Victoria, Australia Corresponding author: Hooman Ayat (h.ayat@student.unsw.edu.au) Research Article Keywords: radar object-based examination, rain system, climatology, climate variability, warming, storms, intensity, rainfall statistics Posted Date: August 11th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-783979/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1. Abstract We know the climate is warming and this is changing some aspects of storms, but we have little knowledge of storm characteristics beyond intensity, which limits our understanding of storms overall. In this study, we apply a cell-tracking algorithm to 20 years of radar data at a mid-latitude coastal-site (Sydney, Australia), to establish a regional storm climatology. The results show that extreme storms in terms of translation-speed, size and rainfall intensity usually occur in the warm season, and are slower and more intense over land between ~10am and ~8pm (AEST), peaking in the afternoon. Storms are more frequent in the cold season and often initiate over the ocean and move northward, leading to precipitation mostly over the ocean. Using clustering algorithms, we have found five storm types with distinct properties, occurring throughout the year but peaking in different seasons. While overall rainfall statistics don't show any link to climate modes, links do appear for some storm types using a multivariate approach. This climatology for a variety of storm characteristics will allow future study of any changes in change. due due to 1 1. Introduction 1 Heavy rainfall is a significant threat to life and property in many parts of the world, 2 especially when it is accompanied by flash floods (Johnson et al. 2016; Allen and Allen 2016). 3 Many studies have shown the potential for climate change to impact rainfall intensity, but how it 4 will affect other storm characteristics (size, translation speed, orientation, etc.) remains largely 5 unexplored. The first step towards exploring the potential future changes is to establish an 6 observed baseline for a wide variety of storm characteristics. 7 Climatological studies can help us to better understand storm characteristics and their 8 (local and remote) drivers in different seasons. Many studies have used gridded datasets (e.g., 9 global climate models, reanalysis data) to perform climatological studies globally and over 10 specific regions; however, the coarse-resolution of these datasets are often unable to properly 11 capture small scale storms like thunderstorms. Therefore, in order to capture these small scale 12 storms using these datasets, researchers have tried to establish thunderstorm climatologies based 13 on the concept of favourable conditions for thunderstorms, which usually includes a combination 14 of convective available potential energy (CAPE) and vertical wind shear in a region (Ahmed et 15 al. 2019; Brooks et al. 2003; Taszarek et al. 2020; Allen et al. 2011; Groenemeijer et al. 2017). 16 By employing this environmental approach, Allen and Karoly (2014) presented a severe 17 thunderstorm climatology over Australia during 2003–2010, using ERA-Interim reanalysis and 18 reported observation data. They showed that these types of storms are more frequent in 19 December during the afternoon, consistent with the seasonal and diurnal cycle of surface 20 temperature and the maximum availability of heating. Although this approach provides valuable 21 information, environmentally favorable conditions do not necessarily lead to a thunderstorm, 22 causing a misestimation of the thunderstorm frequency. In addition, this approach only provides 23 us with storm frequency and it is unable to provide information on other storm characteristics 24 (Allen and Karoly 2014). 25 Using coarse-resolution datasets, previous authors have tried to investigate the effect of 26 natural climate variability (e.g., El Niño–Southern Oscillation (ENSO), the Indian Ocean Dipole 27 (IOD)) on the rainfall over Australia. Ashok et al. (2003) showed that IOD has significant 28 negative partial correlations with rainfall over the western and southern regions of Australia 29 using an atmospheric general circulation model. 1. Introduction 1 2013) and middle-east (Shwehdi 2005). A 57 limitation of this approach is that it is unable to capture storms without lightning. Additionally, it 58 is only able to provide information regarding storm frequency, not other information like rainfall 59 intensity (Walsh et al. 2016). 60 Rain gauge data and reports of hail, tornado and wind gusts are other means of studying 61 thunderstorm climatology used in many studies (Bhardwaj and Singh 2018; Enno et al. 2013; 62 Saha and Quadir 2016; Pinto et al. 2013; Groenemeijer et al. 2017; Kelly et al. 1985; Doswell et 63 al. 2005). The databases of reported tornadoes, hailstorms, and gust winds in some countries like 64 Australia have a long-term record (Bureau of Meteorology 2021), which can provide valuable 65 information for climatological studies. Using these datasets over Australia, some researchers 66 have found that severe thunderstorms are most prevalent between October and April (Niall and 67 Walsh 2005; Schuster et al. 2005; Davis and Walsh 2008) with a peak between 3 and 7 pm 68 (Griffiths et al. 1993; Schuster et al. 2005), consistent with the findings of Allen and Karoly 69 (2014) using environmental approach and ERA-Interim reanalysis data. Higher frequency of 70 heavy rain events over Australia in summer was also reported by Dare and Davidson (2015), 71 using a high resolution gridded daily rain gauge data. Some efforts have also been made to 72 employ these types of data to investigate relationships between ENSO and storm events, globally 73 (Cook and Schaefer 2008; Cook et al. 2017; Lee et al. 2016, 2013; Lepore et al. 2017) and over 74 Australia (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008; Nicholls et 75 al. 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et al. 2013; 76 King et al. 2014; Ashcroft et al. 2019). Based on these studies, winter-spring rainfall is usually 77 reduced during El Niño and enhanced during La Niña over the eastern and southeastern parts of 78 Australia. Although gauge and reported data provide us with valuable information, the 79 the need for a multivariate rather than bivariate approach to this problem. 1. Introduction 1 Maher and Sherwood 40 (2014) applied a multivariate approach to Australian precipitation to disentangle the multiple 41 sources of large-scale variability using the ERA-Interim and Australian Water Availability 42 Project datasets, and they showed that ENSO, blocking, and the intensity and position of the 43 ridge are driving wintertime precipitation in Australia, with a minor role played by the jet 44 intensity and the IOD. All of these studies investigated the effects of natural climate variability 45 on rainfall intensity and frequency, and the relationships between other characteristics of storms 46 (i.e., size, shape, translation speed, etc.) and climate modes are not understood. 47 One way of studying the thunderstorm climatology is by measuring the occurrence of 48 lightning using satellite instruments such as the Optical Transient Detector (OTD) and/or the 49 Lightning Imaging Sensor (LIS). Dowdy and Kuleshov (2014) produced a climatological map of 50 lightning ground flash density over Australia, which details the seasonal variability of the 51 lightning ground flash densities over land and ocean. They found that during the cooler months, 52 a maximum in lightning activity occurs over the ocean to the east of the continent. Earlier studies 53 by Kuleshov et al. (2001; 2012) had found that the second-highest thunderstorm frequency 54 occurs in the southeast part of Australia along the coastline, most often from spring to early 55 autumn. Similar research has been conducted globally for regions such as the United States and 56 Europe (Taszarek et al. 2020), Brazil (Pinto et al. 2013) and middle-east (Shwehdi 2005). A 57 limitation of this approach is that it is unable to capture storms without lightning. Additionally, it 58 is only able to provide information regarding storm frequency, not other information like rainfall 59 intensity (Walsh et al. 2016). 60 Rain gauge data and reports of hail, tornado and wind gusts are other means of studying 61 thunderstorm climatology used in many studies (Bhardwaj and Singh 2018; Enno et al. 2013; 62 Saha and Quadir 2016; Pinto et al. 2013; Groenemeijer et al. 2017; Kelly et al. 1985; Doswell et 63 al. 2005). The databases of reported tornadoes, hailstorms, and gust winds in some countries like 64 Australia have a long-term record (Bureau of Meteorology 2021), which can provide valuable 65 information for climatological studies. 1. Introduction 1 Allen and Karoly (2014) employed the ECMWF 30 Interim Re-Analysis (ERA-Interim) data and have found that ENSO has a substantial impact on 31 the spatial distribution of severe thunderstorm environments over the continent. In another 32 reanalysis-based study, Hauser et al. (2020) investigated the winter-spring rainfall variability in 33 southeastern Australia (SEA) during El Niño events by quantifying the contribution of clustered 34 mid-latitude weather systems to monthly precipitation anomalies. The authors found that the 35 cluster with below-average rainfall is more frequent compared to the other clusters during El 36 Niño, which confirmed the general suppression of SEA rainfall during these events. Since 37 precipitation in some regions is correlated with more than one large scale driver, and indices are 38 often correlated with each other, the interconnected nature of precipitation dependence suggests 39 2 the need for a multivariate rather than bivariate approach to this problem. Maher and Sherwood 40 (2014) applied a multivariate approach to Australian precipitation to disentangle the multiple 41 sources of large-scale variability using the ERA-Interim and Australian Water Availability 42 Project datasets, and they showed that ENSO, blocking, and the intensity and position of the 43 ridge are driving wintertime precipitation in Australia, with a minor role played by the jet 44 intensity and the IOD. All of these studies investigated the effects of natural climate variability 45 on rainfall intensity and frequency, and the relationships between other characteristics of storms 46 (i.e., size, shape, translation speed, etc.) and climate modes are not understood. 47 One way of studying the thunderstorm climatology is by measuring the occurrence of 48 lightning using satellite instruments such as the Optical Transient Detector (OTD) and/or the 49 Lightning Imaging Sensor (LIS). Dowdy and Kuleshov (2014) produced a climatological map of 50 lightning ground flash density over Australia, which details the seasonal variability of the 51 lightning ground flash densities over land and ocean. They found that during the cooler months, 52 a maximum in lightning activity occurs over the ocean to the east of the continent. Earlier studies 53 by Kuleshov et al. (2001; 2012) had found that the second-highest thunderstorm frequency 54 occurs in the southeast part of Australia along the coastline, most often from spring to early 55 autumn. Similar research has been conducted globally for regions such as the United States and 56 Europe (Taszarek et al. 2020), Brazil (Pinto et al. 1. Introduction 1 High spatio-temporal resolution radar rainfall estimates offer the potential to study the 92 thunderstorms occurring over their coverage areas, particularly over the regions in which gauge 93 observations are usually sparse or unevenly distributed (Ghaemi et al. 2017; Ayat et al. 2018; 94 Moazami et al. 2014). Radar records in many parts of the globe are temporally limited to just 95 over a decade (Allen 2018). However, this time frame is long enough to conduct climatological 96 studies. Previous researchers have employed these datasets to conduct thunderstorm climatology 97 studies over specific regions such as the United States (Ghebreyesus and Sharif 2020; Kuo and 98 Orville 1973; Croft and Shulman 1989; Falconer 1984), Europe (Kaltenboeck and Steinheimer 99 2015; Kreklow et al. 2020; Weckwerth et al. 2011; Overeem et al. 2009; Bližňák et al. 2018; 100 Fairman Jr et al. 2015) and Asia (Chen et al. 2012). 101 Hail particles are readily detected by conventional weather radars due to strong scatter 102 produced by their comparatively larger surface area, or via their quasi-spherical shape which 103 produces distinct differential reflectivity values for dual-polarization radars compared to oblate 104 rain droplets. Using these properties, a few radar-based hail products have been produced and 105 employed to study the climatology of hailstorms over different regions. For instance, Warren et. 106 al (2020) studied the hail climatology in multiple cities in Australia (including the Sydney 107 region) using a radar-based hail product (Maximum Expected Size of Hail; MESH), and found 108 that, on average, damaging hail storms over Sydney occur 32 days per year with a peak during 109 the warm season (November-March). Similar radar-based studies have been conducted over 110 other parts of the globe like Europe (Junghänel et al. 2016; Nisi et al. 2016; Fluck et al. 2021; 111 Lukach et al. 2017; Saltikoff et al. 2010) and the United States (Cintineo et al. 2012; Murillo et 112 al. 2021). 113 Most of the previous radar-based studies employed a pixel-based statistical approach which limits the storm properties to rainfall/hail frequency and intensity as a function of position. In order to overcome this limitation, a few studies have employed an object-based approach, where discrete storm objects are identified and characterized. For instance, Haberlie and Ashley (2019), Poujol et al. (2020) and Prein et al. (2017) applied object-based techniques to radar products to study the climatology of convective storms in the United States. 1. Introduction 1 91 High spatio-temporal resolution radar rainfall estimates offer the potential to study the 92 thunderstorms occurring over their coverage areas, particularly over the regions in which gauge 93 observations are usually sparse or unevenly distributed (Ghaemi et al. 2017; Ayat et al. 2018; 94 Moazami et al. 2014). Radar records in many parts of the globe are temporally limited to just 95 over a decade (Allen 2018). However, this time frame is long enough to conduct climatological 96 studies. Previous researchers have employed these datasets to conduct thunderstorm climatology 97 studies over specific regions such as the United States (Ghebreyesus and Sharif 2020; Kuo and 98 Orville 1973; Croft and Shulman 1989; Falconer 1984), Europe (Kaltenboeck and Steinheimer 99 2015; Kreklow et al. 2020; Weckwerth et al. 2011; Overeem et al. 2009; Bližňák et al. 2018; 100 Fairman Jr et al. 2015) and Asia (Chen et al. 2012). 101 Hail particles are readily detected by conventional weather radars due to strong scatter 102 produced by their comparatively larger surface area, or via their quasi-spherical shape which 103 produces distinct differential reflectivity values for dual-polarization radars compared to oblate 104 rain droplets. Using these properties, a few radar-based hail products have been produced and 105 distribution of recorded data is highly influenced by the local population (Allen 2018). In addition, gauge instruments are point measurements and sparse rain gauge networks often fail to observe the maximum rainfall (Ayat et al. 2018). The limitations of previous approaches have led to the application of remotely-sensed datasets, which are a growing area of thunderstorm climatology. Some studies (Cecil and Blankenship 2012; Ni et al. 2017) have applied a satellite-based approach using passive and active microwave sensors to estimate the global climatology of hail storms. However, images from these sensors are available only for a few overpasses per day (Ayat et al. 2021a), which may result in biasing the results depending on the diurnal cycle of convection in the respective regions (Punge and Kunz 2016). In addition, passive microwave products are capturing the upper part of the storms in which there might be more hail particles that melt/evaporate at lower altitudes, leading to false estimation of hail on the ground surface (Ayat et al. 2021b). 1. Introduction 1 Using these datasets over Australia, some researchers 66 have found that severe thunderstorms are most prevalent between October and April (Niall and 67 Walsh 2005; Schuster et al. 2005; Davis and Walsh 2008) with a peak between 3 and 7 pm 68 (Griffiths et al. 1993; Schuster et al. 2005), consistent with the findings of Allen and Karoly 69 (2014) using environmental approach and ERA-Interim reanalysis data. Higher frequency of 70 heavy rain events over Australia in summer was also reported by Dare and Davidson (2015), 71 using a high resolution gridded daily rain gauge data. Some efforts have also been made to 72 employ these types of data to investigate relationships between ENSO and storm events, globally 73 (Cook and Schaefer 2008; Cook et al. 2017; Lee et al. 2016, 2013; Lepore et al. 2017) and over 74 Australia (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008; Nicholls et 75 al. 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et al. 2013; 76 King et al. 2014; Ashcroft et al. 2019). Based on these studies, winter-spring rainfall is usually 77 reduced during El Niño and enhanced during La Niña over the eastern and southeastern parts of 78 Australia. Although gauge and reported data provide us with valuable information, the 79 3 distribution of recorded data is highly influenced by the local population (Allen 2018). In 80 addition, gauge instruments are point measurements and sparse rain gauge networks often fail to 81 observe the maximum rainfall (Ayat et al. 2018). 82 The limitations of previous approaches have led to the application of remotely-sensed 83 datasets, which are a growing area of thunderstorm climatology. Some studies (Cecil and 84 Blankenship 2012; Ni et al. 2017) have applied a satellite-based approach using passive and 85 active microwave sensors to estimate the global climatology of hail storms. However, images 86 from these sensors are available only for a few overpasses per day (Ayat et al. 2021a), which 87 may result in biasing the results depending on the diurnal cycle of convection in the respective 88 regions (Punge and Kunz 2016). In addition, passive microwave products are capturing the upper 89 part of the storms in which there might be more hail particles that melt/evaporate at lower 90 altitudes, leading to false estimation of hail on the ground surface (Ayat et al. 2021b). 1. Introduction 1 122 Although object-based techniques can provide us with more information on storm 123 characteristics, the investigated storm properties in most of these studies were limited to storm 124 number, area, and rainfall intensity, whereas other storm characteristics like storm translation 125 speed, shape and aspect ratio, orientation, direction and volume could also be of interest. In 126 addition, the object-based techniques employed in these studies are limited by the object 127 split/merge issue, which is a common problem in object tracking methods and can lead to 128 calculating misleading storm properties (Muñoz et al. 2018). 129 In this research, we employ the Method of Object-based Diagnostic Evaluation (MODE) 130 Time Domain (MTD), which is modified by the authors so as to handle splitting and merging of 131 objects. We apply this to the Wollongong (near Sydney) radar, which has around 20 years of 132 records, to establish an object-based climatology of precipitation in different seasons over the 133 radar footprint areas (i.e., Greater Sydney, Illawarra and other land/ocean regions within 150 km 134 of the radar). An effort has been made to group the main contributing storms with similar object- 135 based characteristics over this region using clustering algorithms followed by investigating their 136 relationships with different climate modes. 137 This study is presented in seven sections. Section 2 describes the Wollongong radar data 138 and its characteristics. Section 3 introduces the object-based and clustering methods along with 139 the statistics employed in this study. Section 4 describes the study area and section 5 presents the 140 results of the object-based climatology over the study area. Section 6 discusses the results shown 141 in the previous section, and finally, the summary of findings is presented in section 7. 142 Soderholm et al. (2017) employed a cell-tracking algorithm over radar MESH product to study 120 hail climatology in southeast Queensland, Similar efforts have been made in Germany 121 (Thomassen et al. 2020), Italy (Sangiorgio and Barindelli 2020) and Spain (Rigo et al. 2010). 122 Although object-based techniques can provide us with more information on storm characteristics, the investigated storm properties in most of these studies were limited to storm number, area, and rainfall intensity, whereas other storm characteristics like storm translation speed, shape and aspect ratio, orientation, direction and volume could also be of interest. 1. Introduction 1 In addition, the object-based techniques employed in these studies are limited by the object split/merge issue, which is a common problem in object tracking methods and can lead to calculating misleading storm properties (Muñoz et al. 2018). In this research, we employ the Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD), which is modified by the authors so as to handle splitting and merging of objects. We apply this to the Wollongong (near Sydney) radar, which has around 20 years of records, to establish an object-based climatology of precipitation in different seasons over the radar footprint areas (i.e., Greater Sydney, Illawarra and other land/ocean regions within 150 km of the radar). An effort has been made to group the main contributing storms with similar object- based characteristics over this region using clustering algorithms followed by investigating their relationships with different climate modes. This study is presented in seven sections. Section 2 describes the Wollongong radar data 38 and its characteristics. Section 3 introduces the object-based and clustering methods along with 39 the statistics employed in this study. Section 4 describes the study area and section 5 presents the 40 results of the object-based climatology over the study area. Section 6 discusses the results shown 41 in the previous section, and finally, the summary of findings is presented in section 7. 42 1. Introduction 1 In Australia, 4 Soderholm et al. (2017) employed a cell-tracking algorithm over radar MESH product to study 120 hail climatology in southeast Queensland, Similar efforts have been made in Germany 121 (Thomassen et al. 2020), Italy (Sangiorgio and Barindelli 2020) and Spain (Rigo et al. 2010). 122 Although object-based techniques can provide us with more information on storm 123 characteristics, the investigated storm properties in most of these studies were limited to storm 124 number, area, and rainfall intensity, whereas other storm characteristics like storm translation 125 speed, shape and aspect ratio, orientation, direction and volume could also be of interest. In 126 addition, the object-based techniques employed in these studies are limited by the object 127 split/merge issue, which is a common problem in object tracking methods and can lead to 128 calculating misleading storm properties (Muñoz et al. 2018). 129 In this research, we employ the Method of Object-based Diagnostic Evaluation (MODE) 130 Time Domain (MTD), which is modified by the authors so as to handle splitting and merging of 131 objects. We apply this to the Wollongong (near Sydney) radar, which has around 20 years of 132 records, to establish an object-based climatology of precipitation in different seasons over the 133 radar footprint areas (i.e., Greater Sydney, Illawarra and other land/ocean regions within 150 km 134 of the radar). An effort has been made to group the main contributing storms with similar object- 135 based characteristics over this region using clustering algorithms followed by investigating their 136 relationships with different climate modes. 137 This study is presented in seven sections. Section 2 describes the Wollongong radar data 138 and its characteristics. Section 3 introduces the object-based and clustering methods along with 139 the statistics employed in this study. Section 4 describes the study area and section 5 presents the 140 results of the object-based climatology over the study area. Section 6 discusses the results shown 141 in the previous section, and finally, the summary of findings is presented in section 7. 142 Soderholm et al. (2017) employed a cell-tracking algorithm over radar MESH product to study 120 hail climatology in southeast Queensland, Similar efforts have been made in Germany 121 (Thomassen et al. 2020), Italy (Sangiorgio and Barindelli 2020) and Spain (Rigo et al. 2010). 3.1 Radar Data 144 Volumetric rain rates are transformed into 171 a Cartesian grid at a 0.5 km altitude using the Barnes weighting function and a 2.5km constant 172 radius of influence. The final grid has a horizontal resolution of 1 km and a domain size of 300 173 km by 300 km. 174 Rainfall Measuring Mission (TRMM) and the Global Precipitation Measurement Mission 158 (GPM). Satellite overpasses with precipitation are compared with ground radar measurement 159 using the volume matching technique described by Louf et al. (2019), providing a mean 160 calibration value for every pass. Periods of stable calibration are identified, and the mean 161 absolute calibration value for these periods is applied as an offset to the ground radar data. 162 Removal of non-meteorological echoes from reflectivity datasets is challenging. In addition to 163 the ground clutter filtering performed by the signal processor, the technique described by Gabella 164 et al. (2002) is applied using filters for echo continuity and minimum echo area. Unfortunately, 165 this technique is not suitable for removing anomalous propagation which is commonly observed 166 over the adjacent South Pacific Ocean. 167 Reflectivity data is transformed to rain rates using a fitted Z-R relationship derived from 168 9 years of hourly rain gauge data using the Camden Airport AWS (35.04° S, 150.69° E). The A 169 and B coefficients for this relationship were 81 and 1.8 respectively. The maximum rainrate is 170 limited to 100 mm/hr to limit contamination from hail. Volumetric rain rates are transformed into 171 a Cartesian grid at a 0.5 km altitude using the Barnes weighting function and a 2.5km constant 172 radius of influence. The final grid has a horizontal resolution of 1 km and a domain size of 300 173 km by 300 km. 174 Rainfall Measuring Mission (TRMM) and the Global Precipitation Measurement Mission 158 (GPM). Satellite overpasses with precipitation are compared with ground radar measurement 159 using the volume matching technique described by Louf et al. (2019), providing a mean 160 calibration value for every pass. Periods of stable calibration are identified, and the mean 161 absolute calibration value for these periods is applied as an offset to the ground radar data. 162 Removal of non-meteorological echoes from reflectivity datasets is challenging. In addition to 163 the ground clutter filtering performed by the signal processor, the technique described by Gabella 164 et al. 3.1 Radar Data 144 (2002) is applied using filters for echo continuity and minimum echo area. Unfortunately, 165 this technique is not suitable for removing anomalous propagation which is commonly observed 166 over the adjacent South Pacific Ocean. 167 Reflectivity data is transformed to rain rates using a fitted Z-R relationship derived from 168 9 years of hourly rain gauge data using the Camden Airport AWS (35.04° S, 150.69° E). The A 169 and B coefficients for this relationship were 81 and 1.8 respectively. The maximum rainrate is 170 limited to 100 mm/hr to limit contamination from hail. Volumetric rain rates are transformed into 171 a Cartesian grid at a 0.5 km altitude using the Barnes weighting function and a 2.5km constant 172 radius of influence. The final grid has a horizontal resolution of 1 km and a domain size of 300 173 km by 300 km. 174 3.2 Challenges with anomalous propagation 175 Despite all the efforts made in removing non-meteorological echoes, the Wollongong 176 radar site experiences significant anomalous propagation over the ocean within the eastern 177 portion of its coverage. These echoes primarily occur during heat-wave conditions, where strong 178 low-level vertical gradients of humidity and temperature create regions of super refraction for 179 lower elevation scans. The resultant echoes have similar reflectivity gradients, size and shape as 180 precipitation echoes, while also being non-stationary, limiting the effectiveness of any 181 algorithms to remove non-meteorological echoes from reflectivity data (fig S5a; Online 182 Resource 1). In order to reduce the effect of these clutter sources in precipitation estimates, we 183 have opted for a two-step clutter removal process over the whole dataset: 1) Since these clutters 184 often include pixels with low intensity, applying the 3 mm/hr threshold over the convolved data 185 (to detect the storm objects; see section 5.1), effectively removed this clutter throughout the year 186 (fig S5b; Online Resource 1) except some extremes in summer (fig S5d; Online Resource 1). 2) 187 The days with extreme clutter were removed manually from the dataset using maximum daily 188 reflectivity maps (see fig S4; Online Resource 1) such that the days with high values of 189 maximum reflectivity over the ocean and low values over land were considered as a day with 190 extreme clutter over the ocean. 191 3.1 Radar Data 144 This study uses data from a Bureau of Meteorology operational S-band weather radar 145 located near Wollongong, NSW (34.26° S, 150.87° E, 471 m altitude; (Soderholm et al. 2020)). 146 The site experiences partial blocking up to 3 dB in the lowest scan (0.5° elevation) from the 147 northwest to the southeast due to the significant terrain associated with the Great Dividing 148 Range. The archive for this radar started in November 1996 and continues to operate as of 2021. 149 However, the study period is limited to June 2018 in this study. Several hardware and 150 configuration changes have taken place over the last 24 years. Initially, the radar operated on a 151 10-minute volume cycle with 16-level reflectivity data. In December 1999 the number of 152 reflectivity levels was increased to 64. Between October 2010 and January 2021, a major 153 hardware upgrade delivered 160-level reflectivity data and a 6-minute volume cycle. One 154 significant gap is present in the archive from 1/1/1998 to 15/12/1998. 155 To ensure the accuracy of reflectivity values across the entire dataset, an absolute 156 calibration technique is applied using precipitation radar measurements from the Tropical 157 5 Rainfall Measuring Mission (TRMM) and the Global Precipitation Measurement Mission 158 (GPM). Satellite overpasses with precipitation are compared with ground radar measurement 159 using the volume matching technique described by Louf et al. (2019), providing a mean 160 calibration value for every pass. Periods of stable calibration are identified, and the mean 161 absolute calibration value for these periods is applied as an offset to the ground radar data. 162 Removal of non-meteorological echoes from reflectivity datasets is challenging. In addition to 163 the ground clutter filtering performed by the signal processor, the technique described by Gabella 164 et al. (2002) is applied using filters for echo continuity and minimum echo area. Unfortunately, 165 this technique is not suitable for removing anomalous propagation which is commonly observed 166 over the adjacent South Pacific Ocean. 167 Reflectivity data is transformed to rain rates using a fitted Z-R relationship derived from 168 9 years of hourly rain gauge data using the Camden Airport AWS (35.04° S, 150.69° E). The A 169 and B coefficients for this relationship were 81 and 1.8 respectively. The maximum rainrate is 170 limited to 100 mm/hr to limit contamination from hail. 3. Study area 192 The climate of these regions is categorized as humid subtropical, or Cfa based on 195 the Köppen–Geiger classification (Kottek et al. 2006), and is significantly affected by the coastal 196 position, with small interseasonal variations ranging from cool winters to warm and hot summers 197 (Bureau of Meteorology 2016; Dare and Davidson 2015). The mean annual precipitation 198 recorded at Observatory Hill (in Greater Sydney) and Wollongong University (in Illawarra) 199 locations are 1213.4 mm and 1348.6 mm, respectively (Bureau of Meteorology 2013). 200 3. Study area 192 The study area in this research is the radar coverage regions located up to 150 km from 193 the Wollongong radar, which includes coastal regions of Greater Sydney and Illawarra in New 194 6 South Wales. The climate of these regions is categorized as humid subtropical, or Cfa based on 195 the Köppen–Geiger classification (Kottek et al. 2006), and is significantly affected by the coastal 196 position, with small interseasonal variations ranging from cool winters to warm and hot summers 197 (Bureau of Meteorology 2016; Dare and Davidson 2015). The mean annual precipitation 198 recorded at Observatory Hill (in Greater Sydney) and Wollongong University (in Illawarra) 199 locations are 1213.4 mm and 1348.6 mm, respectively (Bureau of Meteorology 2013). 200 Several factors may impact the precipitation over these regions and its seasonality. 201 Generally, precipitation peaks in the first half of the year and decreases in the second half 202 (Bureau of Meteorology 2013). In the summer, the easterly (or inland) trough is a major 203 contributor to rainfall over these regions with a peak in the evening. Its impact can be enhanced 204 by interacting with any upper-level troughs or cold fronts crossing over these regions. Frontal 205 systems also bring rainfall to these regions throughout the year, but mostly in winter when the 206 subtropical ridge moves northward over inland Australia (Bureau of Meteorology 2010). Another 207 source of precipitation over these regions is cut-off lows, which can occur at any time of year but 208 are most common during autumn and winter. These can be intense and last up to a week when 209 formed as part of a blocking pair or east coast lows, in which case they are accompanied by long- 210 lasting heavy rainfall and gusty winds (Bureau of Meteorology 2007). Northwest cloud bands 211 (stretching from northwest to southeast Australia) can also bring precipitation over these regions. 212 They may interact with cold fronts and cut-off lows over southeastern Australia to produce very 213 heavy rainfall over these regions (Reid et al. 2019). Several modes of variability are known to 214 affect precipitation in Australia, However, precipitation over the coastal zone that includes our 215 study area has shown little relationship (Timbal and Hendon 2011; Fita et al. 2017) Method 216 South Wales. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) In 239 addition, with the help of the new approach in tracking the storms, it’s possible to extract storm 240 characteristics with more details from different parts of the storms and better calculate 241 characteristics like translation speed, direction and track length during the lifetime of the storms 242 with high rate of split/merge events. 243 In this research, we are studying the extracted characteristics related to “storms” and 244 “storm objects” using the modified MTD method. The selected threshold to filter the objects is 3 245 mm/hr in the convolved data smoothed by a “3×3-pixel” moving window across the map. The 246 storm-object characteristics of interest in this study include: 1) area: the number of pixels in the 247 storm object; 2) translation speed: the ratio of the distance between the volumetric centroid of 248 two connected objects in time to the temporal resolution of the dataset; 3) maximum intensity: 249 the maximum precipitation rate within a storm object; 4) object average intensity: the average 250 precipitation rate of all cells within a storm object; 5) object volume discharge: the volumetric 251 rain rate that passes through the storm object area during a specified period; 6) aspect ratio: the 252 ratio of the minor and major axis of the fitted ellipse over the storm object; 7) object direction: 253 the compass direction of the line connecting the centroids of two consecutive objects in a 254 sequence, and finally, 8) orientation that is the compass direction of the major axis of the fitted 255 ellipse. 256 Figure 1a represents an example of running the modified MTD on Wollongong radar data 234 during an event that occurred on 2018-10-4. The lines with different colors are showing the 3D- 235 object tracks. In this event, land-originating storms’ parts had a southeastward direction and later 236 were merged with the storms’ parts that had formed over the ocean. Considering split/merge 237 events in this event has shown how successfully this approach could separate storms’ parts over 238 land and ocean before merging which is not possible using the original version of MTD. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD): 217 218 MTD is an extension for MODE to track the storm objects detected in a precipitation map 219 by the MODE algorithm (Clark et al. 2014). Here we employed a modified version of MTD, 220 proposed by Ayat et al. (2021b), that considers split/merge events during the lifetime of a storm. 221 In this method, the “storm objects” at each time step are the connected pixels higher than a 222 specified threshold in the convolved precipitation map (smoothed by an “n⨯n-pixel” moving 223 window across the map). Every “storm object” at each time-step has a unique label number 224 unless it has overlap with another storm object at the previous time-step (each blob in figure 1b). 225 In this case, it takes the label of the storm object at the previous time-step. If a storm-object has 226 overlap with two (or more) storm objects in the previous time step (merging) or two (or more) 227 objects have overlaps with a storm-object at the previous time step (splitting), the storm- 228 object/storm-objects at the current timestep takes/take a new label. Based on these definitions, A 229 “sequence of objects (or 3D objects)” is the connected storm objects in time that has a unique 230 label number and doesn’t have split/merge events during its lifetime (connected blobs with the 231 same colours and numbers in figure 1b). Finally, a “storm” is a group of the 3D objects that are 232 connected via split/merging events (The whole diagram in figure 1b). 233 7 Figure 1a represents an example of running the modified MTD on Wollongong radar data 234 during an event that occurred on 2018-10-4. The lines with different colors are showing the 3D- 235 object tracks. In this event, land-originating storms’ parts had a southeastward direction and later 236 were merged with the storms’ parts that had formed over the ocean. Considering split/merge 237 events in this event has shown how successfully this approach could separate storms’ parts over 238 land and ocean before merging which is not possible using the original version of MTD. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) In 239 addition, with the help of the new approach in tracking the storms, it’s possible to extract storm 240 characteristics with more details from different parts of the storms and better calculate 241 characteristics like translation speed, direction and track length during the lifetime of the storms 242 with high rate of split/merge events. 243 In this research, we are studying the extracted characteristics related to “storms” and 244 “storm objects” using the modified MTD method. The selected threshold to filter the objects is 3 245 mm/hr in the convolved data smoothed by a “3×3-pixel” moving window across the map. The 246 storm-object characteristics of interest in this study include: 1) area: the number of pixels in the 247 storm object; 2) translation speed: the ratio of the distance between the volumetric centroid of 248 two connected objects in time to the temporal resolution of the dataset; 3) maximum intensity: 249 the maximum precipitation rate within a storm object; 4) object average intensity: the average 250 precipitation rate of all cells within a storm object; 5) object volume discharge: the volumetric 251 rain rate that passes through the storm object area during a specified period; 6) aspect ratio: the 252 ratio of the minor and major axis of the fitted ellipse over the storm object; 7) object direction: 253 the compass direction of the line connecting the centroids of two consecutive objects in a 254 sequence, and finally, 8) orientation that is the compass direction of the major axis of the fitted 255 ellipse. 256 The studied storm characteristics in this research include: 1) storm area: the average 257 storm snapshot areas in the storm lifetime; 2) storm volume discharge: the average storm 258 snapshot volume discharge in the storm lifetime; 3) storm average intensity: the average of 259 precipitation rate in the storm lifetime; 4) storm max average intensity: the maximum of storm 260 averaged intensity (calculated at each snapshot) in the storm lifetime; 5) storm translation speed: 261 the area-weighted average translation speed of the storm snapshots in the storm lifetime; 6) storm 262 direction: the area-weighted average direction of the storm snapshots in the storm lifetime; 7) 263 storm contributing objects: The number of root storm objects in the storm graph diagram (e.g. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) in 264 figure 1 storm object numbers 1, 2 and 3 are the root objects in the storm diagram) and 8) storm 265 split/merge event number: the number of split/merge events in the storm lifetime. Note that no 266 thresholds have been applied over the defined storm/storm-object properties in this study. 267 8 8 269 Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (2018-10-04; 270 UTC). Panel (b) illustrates the diagram of a tracked storm with split/merge events. Note that each 271 blob represents a storm object at each time step and each colour shows a sequence (or 3D) 272 object. 273 Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (201 Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (2018-10-04; 270 UTC). Panel (b) illustrates the diagram of a tracked storm with split/merge events. Note that each 271 blob represents a storm object at each time step and each colour shows a sequence (or 3D) 272 object. 273 Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (2018-10-04; 270 UTC). Panel (b) illustrates the diagram of a tracked storm with split/merge events. Note that each 271 blob represents a storm object at each time step and each colour shows a sequence (or 3D) 272 object. 273 4.2. Clustering Analysis 274 The aim of this section is to find storm clusters (types) with similar quantitative 275 characteristics over the study area using both the Agglomerative clustering algorithm and the t- 276 SNE technique. 277 q 4.2.1 t-Distributed Stochastic Neighbor Embedding (t-SNE) 278 279 t-SNE is a statistical technique to visualize high-dimensional data by projecting it on a 280 two or three-dimensional map. Here is a brief overview of the main stages in this method: 1) It 281 starts with constructing a probability distribution of similarities over pairs of events in high- 282 dimensional data such that a similar pair of events have a higher value compared to the one that 283 is less similar; then, 2) another probability distribution of similarities is defined over the points in 284 the low-dimensional map, and finally, 3) the algorithm minimizes the divergence between two 285 distributions using Kullback–Leibler divergence parameter (KL divergence) between the two 286 distributions with respect to the locations of the points in the map. Note that the KL divergence 287 parameter is a measure of how one probability distribution diverges from another using a 288 gradient descent method. For more details, please refer to the original research paper by Maaten 289 and Hinton (2008). 290 4.2.2 Agglomerative clustering 291 292 The agglomerative technique is one of the common types of hierarchical clustering in 293 grouping data based on their similarity. This technique works in a “bottom-up” manner by 294 treating each object as a separate group in the beginning. Next, at each step, the two clusters with 295 4.2.1 t-Distributed Stochastic Neighbor Embedding (t-SNE) 278 279 t-SNE is a statistical technique to visualize high-dimensional data by projecting it on a 280 two or three-dimensional map. Here is a brief overview of the main stages in this method: 1) It 281 starts with constructing a probability distribution of similarities over pairs of events in high- 282 dimensional data such that a similar pair of events have a higher value compared to the one that 283 is less similar; then, 2) another probability distribution of similarities is defined over the points in 284 the low-dimensional map, and finally, 3) the algorithm minimizes the divergence between two 285 distributions using Kullback–Leibler divergence parameter (KL divergence) between the two 286 distributions with respect to the locations of the points in the map. 4.2. Clustering Analysis 274 307 One of the problems with hierarchical clustering is that it doesn’t give information 308 regarding the number of clusters, or where to stop the merging process in the algorithm. In order 309 to overcome this limitation, we have employed the Calinski Harabasz index (CHI) to define the 310 number of clusters which is the optimum value of CHI by increasing the number of clusters. 311 CHI is the ratio of between-cluster variance (VARB) to within-cluster variance (VARW): 312 313 𝐶𝐻𝐼= 𝑉𝐴𝑅𝐵 𝑉𝐴𝑅𝑊× 𝑁−𝐾 𝐾−1 (1) 314 𝑉𝐴𝑅𝐵= ∑ 𝑛𝑘 |𝑧𝑘−𝑧|2 𝑁 𝑘=1 (2) 315 𝑉𝐴𝑅𝑊= ∑ ∑ 𝑛𝑘 |𝑥𝑖−𝑧𝑘|2 𝑛𝑘 𝑘=1 𝐾 𝑘=1 (3) 316 317 Here, N is the population of the data, K denotes the number of clusters, and Zk and z refer 318 to the centroid of cluster k and the entire data, respectively. In the second and third equations, nk 319 is the population of cluster k and xi denotes each member of that cluster (Li et al. 2018). 320 In order to find independent properties as the input for the clustering algorithm, the correlations between all pairs of the storm-object properties have been calculated and the pairs with correlation higher than 0.5 are considered as dependent variables and haven’t been used together as the input in the clustering algorithm. Note that all input data are normalized (ranging from 0-1) by dividing each input storm property by its maximum and standard deviation. One of the problems with hierarchical clustering is that it doesn’t give information regarding the number of clusters, or where to stop the merging process in the algorithm. In order to overcome this limitation, we have employed the Calinski Harabasz index (CHI) to define the number of clusters which is the optimum value of CHI by increasing the number of clusters. 4.2. Clustering Analysis 274 CHI is the ratio of between-cluster variance (VARB) to within-cluster variance (VARW): 𝐶𝐻𝐼= 𝑉𝐴𝑅𝐵 𝑉𝐴𝑅𝑊× 𝑁−𝐾 𝐾−1 (1) 𝑉𝐴𝑅𝐵= ∑ 𝑛𝑘 |𝑧𝑘−𝑧|2 𝑁 𝑘=1 (2) 𝑉𝐴𝑅𝑊= ∑ ∑ 𝑛𝑘 |𝑥𝑖−𝑧𝑘|2 𝑛𝑘 𝑘=1 𝐾 𝑘=1 (3) 𝐶𝐻𝐼= 𝑉𝐴𝑅𝐵 𝑉𝐴𝑅𝑊× 𝑁−𝐾 𝐾−1 (1) 𝑉𝐴𝑅𝐵= ∑ 𝑛𝑘 |𝑧𝑘−𝑧|2 𝑁 𝑘=1 (2) 𝑉𝐴𝑅𝑊= ∑ ∑ 𝑛𝑘 |𝑥𝑖−𝑧𝑘|2 𝑛𝑘 𝑘=1 𝐾 𝑘=1 (3) 𝐶𝐻𝐼= 𝑉𝐴𝑅𝐵 𝑉𝐴𝑅𝑊× 𝑁−𝐾 𝐾−1 𝑉𝐴𝑅𝐵= ∑ 𝑛𝑘 |𝑧𝑘−𝑧|2 𝑁 𝑘=1 𝑉𝐴𝑅𝑊= ∑ ∑ 𝑛𝑘 |𝑥𝑖−𝑧𝑘|2 𝑛𝑘 𝑘=1 𝐾 𝑘=1 (2) (3) (3) Here, N is the population of the data, K denotes the number of clusters, and Zk and z refer 318 to the centroid of cluster k and the entire data, respectively. In the second and third equations, nk 319 is the population of cluster k and xi denotes each member of that cluster (Li et al. 2018). 320 4.2. Clustering Analysis 274 Note that the KL divergence 287 parameter is a measure of how one probability distribution diverges from another using a 288 gradient descent method. For more details, please refer to the original research paper by Maaten 289 and Hinton (2008). 290 The agglomerative technique is one of the common types of hierarchical clustering in 293 grouping data based on their similarity. This technique works in a “bottom-up” manner by 294 treating each object as a separate group in the beginning. Next, at each step, the two clusters with 295 9 the most similarity are merged into a bigger cluster and this process continues until all objects 296 are merged into one single big cluster (Subasi 2020). Here we have used the t-SNE algorithm to 297 project our n-dimensional data on a two-dimension map (see fig 9a) and increase the divergence 298 of potential clusters. Then, the agglomerative technique has been employed over the projected 299 data to find the clusters. Similar process has been repeated by applying KMeans clustering 300 algorithm (see fig S3; Online Resource 1). However, based on the density map, the cluster 301 borders have been better recognized by the agglomerative technique. 302 the most similarity are merged into a bigger cluster and this process continues until all objects 296 are merged into one single big cluster (Subasi 2020). Here we have used the t-SNE algorithm to 297 project our n-dimensional data on a two-dimension map (see fig 9a) and increase the divergence 298 of potential clusters. Then, the agglomerative technique has been employed over the projected 299 data to find the clusters. Similar process has been repeated by applying KMeans clustering 300 algorithm (see fig S3; Online Resource 1). However, based on the density map, the cluster 301 borders have been better recognized by the agglomerative technique. 302 In order to find independent properties as the input for the clustering algorithm, the 303 correlations between all pairs of the storm-object properties have been calculated and the pairs 304 with correlation higher than 0.5 are considered as dependent variables and haven’t been used 305 together as the input in the clustering algorithm. Note that all input data are normalized (ranging 306 from 0-1) by dividing each input storm property by its maximum and standard deviation. 4.3. Statistics 321 F1, n an 341 F2,m refer to empirical distribution functions for both variables, and sup is the supremum 342 function. Note that the supremum function of a subset S of a set K is the least element in K th 343 is greater than or equal to all elements of S. 344 In order to model the relationship between two or more variables, in this research, w 345 have employed the Ordinary least squares (OLS) method: 346 347 𝑦= 𝛽𝑋 348 (8) 349 𝛽= 𝑎𝑟𝑔𝑚𝑖𝑛𝑏𝜖𝑅𝑝 𝑆(𝑏) 350 (9) 351 𝑆(𝑏) = (𝑦−𝑋𝑏)𝑇(𝑦−𝑋𝑏) (10) 352 353 Where n is the number of observations, p is the number of independent variables, y is th 354 n×1 matrix of the dependent parameter, X is the n×p matrix of independent variables. 355 Suppose b is a "candidate" value for the parameter β which is the n×1 matrix o 356 coefficients for independent variables. 357 An effort has been made in this study to investigate the relationships between climat 358 indices (i.e., El Niño–Southern Oscillation (ENSO), Indian Ocean Dipole (IOD), and Souther 359 Annular Mode (SAM)) and storm properties in each cluster (derived from the previous section 360 Since precipitation can be correlated with more than one index, and the indices are ofte 361 correlated with each other (Maher and Sherwood 2014; Taschetto et al. 2011; Mekanik et a 362 2013; Mekanik and Imteaz 2012), we have utilized a multivariate approach rather than 363 bivariate approach to consider the dependency of the climate modes on each other. Equation 1 364 is the model representing the relationship between the three selected indices and each objec 365 based storm properties (in each cluster) for every month using a multiple linear analysis t 366 consider the dependency of the selected indices and include the annual variations. 367 368 𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀 (11) 369 370 (5) (5) (7) (7) Where Dn,m denotes the KS statistic and n,m are the sizes of the two datasets. F1, n and F2,m refer to empirical distribution functions for both variables, and sup is the supremum function. Note that the supremum function of a subset S of a set K is the least element in K that is greater than or equal to all elements of S. 4.3. Statistics 321 Here we employed the non-parametric Kendall’s Tau rank correlation coefficient (tau) to 322 investigate the strength of relationships between variables. This value is derived from the 323 following equation: 324 325 𝑡𝑎𝑢= 𝐶−𝐷 𝐶+𝐷 (4) 326 327 In this equation, C is the number of matched pairs and D is the number of mismatched 328 pairs of the two variables. 329 To determine the significance of the difference between the two data distributions, the 330 nonparametric statistical Kolmogorov-Smirnov test (KS-test) has been employed. This technique 331 is a non-parametric test which doesn’t need the input data distributions to be normal. The null 332 Here we employed the non-parametric Kendall’s Tau rank correlation coefficient (tau) to 322 investigate the strength of relationships between variables. This value is derived from the 323 following equation: 324 𝑡𝑎𝑢= 𝐶−𝐷 𝐶+𝐷 (4) 𝑡𝑎𝑢= 𝐶−𝐷 𝐶+𝐷 (4) In this equation, C is the number of matched pairs and D is the number of mismatched 328 pairs of the two variables. 329 In this equation, C is the number of matched pairs and D is the number of mismatched 328 pairs of the two variables. 329 To determine the significance of the difference between the two data distributions, the 330 nonparametric statistical Kolmogorov-Smirnov test (KS-test) has been employed. This technique 331 is a non-parametric test which doesn’t need the input data distributions to be normal. The null 332 10 hypothesis in this test is that both samples come from a population with the same distribution. 333 This null hypothesis is rejected at the level of ⍺ if: 334 335 hypothesis in this test is that both samples come from a population with the same distribution. 333 This null hypothesis is rejected at the level of ⍺ if: 334 335 hypothesis in this test is that both samples come from a population with the same distribution. 333 This null hypothesis is rejected at the level of ⍺ if: 334 335 𝐷𝑛,𝑚> 𝐶(𝛼)√ 𝑛+𝑚 𝑛×𝑚 𝐷𝑛,𝑚= 𝑠𝑢𝑝𝑥|𝐹1,𝑛(𝑥) −𝐹2.𝑚(𝑥)| (6) 𝐶𝛼= √−𝑙𝑛( 𝛼 2) × 1 2 𝐷𝑛,𝑚> 𝐶(𝛼)√ 𝑛+𝑚 𝑛×𝑚 (5) 336 𝐷𝑛,𝑚= 𝑠𝑢𝑝𝑥|𝐹1,𝑛(𝑥) −𝐹2.𝑚(𝑥)| 337 (6) 338 𝐶𝛼= √−𝑙𝑛( 𝛼 2) × 1 2 (7) 339 340 𝐷𝑛,𝑚> 𝐶(𝛼)√ 𝑛+𝑚 𝑛×𝑚 (5) 336 𝐷𝑛,𝑚= 𝑠𝑢𝑝𝑥|𝐹1,𝑛(𝑥) −𝐹2.𝑚(𝑥)| 337 (6) 338 𝐶𝛼= √−𝑙𝑛( 𝛼 2) × 1 2 (7) 339 340 Where Dn,m denotes the KS statistic and n,m are the sizes of the two datasets. 4.3. Statistics 321 In order to model the relationship between two or more variables, in this research, we have employed the Ordinary least squares (OLS) method: 𝑦= 𝛽𝑋 (8) 𝛽= 𝑎𝑟𝑔𝑚𝑖𝑛𝑏𝜖𝑅𝑝 𝑆(𝑏) (9) 𝑆(𝑏) = (𝑦−𝑋𝑏)𝑇(𝑦−𝑋𝑏) (10) 𝑦= 𝛽𝑋 (8) 𝛽= 𝑎𝑟𝑔𝑚𝑖𝑛𝑏𝜖𝑅𝑝 𝑆(𝑏) (9) 𝑆(𝑏) = (𝑦−𝑋𝑏)𝑇(𝑦−𝑋𝑏) (10) Where n is the number of observations, p is the number of independent variables, y is the 354 n×1 matrix of the dependent parameter, X is the n×p matrix of independent variables. 355 Where n is the number of observations, p is the number of independent variables, y is the 354 n×1 matrix of the dependent parameter, X is the n×p matrix of independent variables. 355 Suppose b is a "candidate" value for the parameter β which is the n×1 matrix of 356 coefficients for independent variables. 357 An effort has been made in this study to investigate the relationships between climate 358 indices (i.e., El Niño–Southern Oscillation (ENSO), Indian Ocean Dipole (IOD), and Southern 359 Annular Mode (SAM)) and storm properties in each cluster (derived from the previous section). 360 Since precipitation can be correlated with more than one index, and the indices are often 361 correlated with each other (Maher and Sherwood 2014; Taschetto et al. 2011; Mekanik et al. 362 2013; Mekanik and Imteaz 2012), we have utilized a multivariate approach rather than a 363 bivariate approach to consider the dependency of the climate modes on each other. Equation 11 364 is the model representing the relationship between the three selected indices and each object- 365 based storm properties (in each cluster) for every month using a multiple linear analysis to 366 consider the dependency of the selected indices and include the annual variations. 367 𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀 (11) (11) 𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀 𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀 11 Where C0 is the Month constant and C1, C2, C3 are Niño 3.4, IOD and SAM coefficients, 371 respectively. Note that, in this equation, each object-based property at every timestep is matched 372 with its monthly climate indices. 4.3. Statistics 321 373 The monthly time series of Niño 3.4, DMI and AAO from NOAA, which refer to ENSO, 374 IOD and Southern Annular Mode (SAM) are accessible from the NOAA Physical Sciences 375 Laboratory (information available online at https://psl.noaa.gov/data/climateindices/list/) 376 4. Results 377 378 The object-based storm properties are compared in different seasons in section 5.1 379 followed by the detailed analyses of the storms originating on land and ocean. Then, the diurnal 380 cycles of storm/storm-object properties are analyzed in section 5.2. Finally, in section 5.3 the 381 main contributing storms with similar object-based properties are clustered using clustering 382 analysis, and the effect of climate variability (i.e., ENSO, IOD and SAM) on these clusters is 383 investigated. Note that by applying the object-based technique over the study period, 1,218,787 384 storm-objects and 35,445 storms have been identified. From the detected storm-objects, 252,636, 385 277406, 371389 and 317356 objects are detected in spring (SON), summer (DJF), autumn 386 (MAM) and winter (JJA), respectively. The corresponding numbers for storms are 8233, 8091, 387 10080, 9041. 388 Where C0 is the Month constant and C1, C2, C3 are Niño 3.4, IOD and SAM coefficients, 371 respectively. Note that, in this equation, each object-based property at every timestep is matched 372 with its monthly climate indices. 373 The monthly time series of Niño 3.4, DMI and AAO from NOAA, which refer to ENSO, 374 IOD and Southern Annular Mode (SAM) are accessible from the NOAA Physical Sciences 375 Laboratory (information available online at https://psl.noaa.gov/data/climateindices/list/) 376 12 5.1. Seasonal analyses of storm/storm-object properties 5.1. Seasonal analyses of storm/storm-object properties 5.1. Seasonal analyses of storm/storm-object properties 389 5.1. Seasonal analyses of storm/storm-object properties Fig. 2 Storm/storm-object property distributions for different seasons. Note that the bins in the x- axis are equally spaced in the logarithmic scale except panels e-l. Panels e, h and l are also presented in polar coordinates. Green, red, brown and blue lines refer to spring, summer, autumn and winter, respectively. The y-axis in cartesian plots and radius in polar plots show the normalized frequency ranging from 0-100. Note that storm direction in panel (l) refers to the direction of the storm motion, and all the angles shown in panels e, h and l are measured from the positive direction of the x-axis. Figure 2 represents the PDFs of the storm and storm-object properties in different seasons extracted from the radar data. Storms in summer and spring tend to move towards the east- southeast (fig 2i) and are larger in size (fig 2a 2p) and volume (fig 2p 2q) compared to the Fig. 2 Storm/storm-object property distributions for different seasons. Note that the bins in the x- axis are equally spaced in the logarithmic scale except panels e-l. Panels e, h and l are also presented in polar coordinates. Green, red, brown and blue lines refer to spring, summer, autumn and winter, respectively. The y-axis in cartesian plots and radius in polar plots show the normalized frequency ranging from 0-100. Note that storm direction in panel (l) refers to the direction of the storm motion, and all the angles shown in panels e, h and l are measured from the positive direction of the x-axis. Figure 2 represents the PDFs of the storm and storm-object properties in different seasons 98 extracted from the radar data. Storms in summer and spring tend to move towards the east- 99 southeast (fig 2i), and are larger in size (fig 2a, 2p) and volume (fig 2p, 2q) compared to the 00 autumn and winter storms which usually move northward. Comparing the mode of PDFs of 01 13 rainfall intensity (fig 2c, 2d, 2m, 2o) shows that typical storms in autumn are more intense 02 compared to the other seasons. 5.1. Seasonal analyses of storm/storm-object properties However, extreme rain intensity is higher the warmer the season, 03 since storms with maximum intensity above 40 mm/hr (top 10% of storms in maximum 04 intensity) during spring, summer, autumn and winter have occurred 1194, 1662, 1182 and 441 05 times, respectively, during the study timeframe. 06 rainfall intensity (fig 2c, 2d, 2m, 2o) shows that typical storms in autumn are more intense 402 compared to the other seasons. However, extreme rain intensity is higher the warmer the season, 403 since storms with maximum intensity above 40 mm/hr (top 10% of storms in maximum 404 intensity) during spring, summer, autumn and winter have occurred 1194, 1662, 1182 and 441 405 times, respectively, during the study timeframe. 406 In autumn, storms tend to move slower (fig 2f, 2n) and look more symmetric (fig 2i) than 407 in other seasons. In summer, storm objects are mostly oriented near 315∘ from the positive x-axis 408 while in autumn and spring the object orientation angles mostly change to 330∘ and in winter 409 storm objects are mostly oriented west-east (fig 2h). Along with having larger and more severe 410 storms in summer than in winter, summertime storms often include more contributing objects 411 and split/merge events during their lifetimes (fig 2j, 2k). Although all of the mentioned 412 differences are statistically significant based on Kolmogorov–Smirnov test, some storm 413 properties clearly vary with seasons such as rainfall intensity and storm direction. However, 414 there are some properties that look about the same in all four seasons like storm-object areas (fig 415 2a). Note that the differences between the storm size in different seasons are clearer in the storm 416 area and volume PDFs (fig 2p, 2q). 417 418 rainfall intensity (fig 2c, 2d, 2m, 2o) shows that typical storms in autumn are more intense 402 compared to the other seasons. However, extreme rain intensity is higher the warmer the season, 403 since storms with maximum intensity above 40 mm/hr (top 10% of storms in maximum 404 intensity) during spring, summer, autumn and winter have occurred 1194, 1662, 1182 and 441 405 times, respectively, during the study timeframe. 406 In autumn, storms tend to move slower (fig 2f, 2n) and look more symmetric (fig 2i) than 407 in other seasons. 5.1. Seasonal analyses of storm/storm-object properties In summer, storm objects are mostly oriented near 315∘ from the positive x-axis 408 while in autumn and spring the object orientation angles mostly change to 330∘ and in winter 409 storm objects are mostly oriented west-east (fig 2h). Along with having larger and more severe 410 storms in summer than in winter, summertime storms often include more contributing objects 411 and split/merge events during their lifetimes (fig 2j, 2k). Although all of the mentioned 412 differences are statistically significant based on Kolmogorov–Smirnov test, some storm 413 properties clearly vary with seasons such as rainfall intensity and storm direction. However, 414 there are some properties that look about the same in all four seasons like storm-object areas (fig 415 2a). Note that the differences between the storm size in different seasons are clearer in the storm 416 area and volume PDFs (fig 2p, 2q). 417 418 In autumn, storms tend to move slower (fig 2f, 2n) and look more symmetric (fig 2i) than 407 in other seasons. In summer, storm objects are mostly oriented near 315∘ from the positive x-axis 408 while in autumn and spring the object orientation angles mostly change to 330∘ and in winter 409 storm objects are mostly oriented west-east (fig 2h). Along with having larger and more severe 410 storms in summer than in winter, summertime storms often include more contributing objects 411 and split/merge events during their lifetimes (fig 2j, 2k). Although all of the mentioned 412 differences are statistically significant based on Kolmogorov–Smirnov test, some storm 413 properties clearly vary with seasons such as rainfall intensity and storm direction. However, 414 there are some properties that look about the same in all four seasons like storm-object areas (fig 415 2a). Note that the differences between the storm size in different seasons are clearer in the storm 416 area and volume PDFs (fig 2p, 2q). 417 418 14 Fig. 3 PDFs of storm properties for storms originating on land (solid lines) and ocean (dashed lines). All differences between land and ocean distributions are statistically significant based on the Kolmogorov–Smirnov test. NL indicates the number of storms originating on land and NO refers to the storms originating on the ocean. 419 420 421 422 423 424 425 426 427 428 429 430 431 Fig. 3 PDFs of storm properties for storms originating on land (solid lines) and ocean (dashed lines). All differences between land and ocean distributions are statistically significant based on the Kolmogorov–Smirnov test. NL indicates the number of storms originating on land and NO refers to the storms originating on the ocean. Fig. 3 PDFs of storm properties for storms originating on land (solid lines) and ocean (dashed lines). All differences between land and ocean distributions are statistically significant based on the Kolmogorov–Smirnov test. NL indicates the number of storms originating on land and NO refers to the storms originating on the ocean. We have also compared the typical and extreme storms’ properties originating on land vs. 426 ocean in different seasons by comparing their PDFs shown in figure 3. The results show that 427 storms in spring and summer mostly initiate over land. However, in autumn and winter storms 428 often originate over the South Pacific Ocean. Land-originating storms are typically larger (fig 3a- 429 d) and move faster (fig 3n-q) than ocean-originating storms in all seasons. In summer and spring, 430 typical land-originating storms have higher maximum (fig 3j-k) and average precipitation 431 15 intensity (fig 3e-f), which is also true for extreme land-originating storms during autumn compared to their counterparts originating on the ocean. However, typical ocean storms in autumn and also winter (in terms of precipitation intensity) have slightly higher rain rates compared with storms originating on land. Therefore, typical ocean-originating storms are more spatially concentrated with higher rainrate and smaller areas compared to land-originating storms. In summer and spring, both types of storms (land and ocean) mostly move towards the east-southeast. However, in autumn and winter, they have different directions. Land storms still tend to move towards east-southeast, but ocean-storms usually move northward. 440 441 442 Fig. 4 PDFs of storm snapshot properties originating on land and transitioning to the ocean 443 (panels a-d) and originating on the ocean and hitting the land (panels e-l). Solid and dashed lines 444 Fig. 4 PDFs of storm snapshot properties originating on land and transitioning to the ocean 443 (panels a-d) and originating on the ocean and hitting the land (panels e-l). Solid and dashed lines 444 16 are related to the part of the storms that are over land and over the ocean, respectively. 419 420 421 422 423 424 425 426 427 428 429 430 431 St_no 445 refers to the number of storms that start from land and reach the ocean or vice-versa, and 446 St_snp_L and St_snp_O refer to the number of storm snapshots over the land and the ocean, 447 respectively. All differences between land and ocean distributions are statistically significant 448 based on the Kolmogorov–Smirnov test. 449 450 Next, we examine how storm properties change during the transition of storms between 451 land and ocean. Land-originating storms during summer/spring have higher max intensity over 452 land than later over the ocean (fig 4a, b). However, during the autumn and winter, these 453 differences are smaller (fig 4c, d). Summer and spring storms originating over the ocean and 454 reaching land tend to be smaller (fig 4e, f) without much change in rainfall intensity (fig 4i, j). In 455 addition, autumn and winter ocean-originating storms (that reach the land) are also more 456 spatially concentrated (with higher max intensity (fig 4k, l) and smaller sizes) when they are 457 raining over the ocean compared to when they are over land (fig 4g, h). 458 459 are related to the part of the storms that are over land and over the ocean, respectively. St_no 445 refers to the number of storms that start from land and reach the ocean or vice-versa, and 446 St_snp_L and St_snp_O refer to the number of storm snapshots over the land and the ocean, 447 respectively. All differences between land and ocean distributions are statistically significant 448 based on the Kolmogorov–Smirnov test. 449 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 are related to the part of the storms that are over land and over the ocean, respectively. St_no 445 refers to the number of storms that start from land and reach the ocean or vice-versa, and 446 St_snp_L and St_snp_O refer to the number of storm snapshots over the land and the ocean, 447 respectively. All differences between land and ocean distributions are statistically significant 448 based on the Kolmogorov–Smirnov test. 449 450 Next, we examine how storm properties change during the transition of storms between 451 land and ocean. Land-originating storms during summer/spring have higher max intensity over 452 land than later over the ocean (fig 4a, b). However, during the autumn and winter, these 453 differences are smaller (fig 4c, d). Summer and spring storms originating over the ocean and 454 reaching land tend to be smaller (fig 4e, f) without much change in rainfall intensity (fig 4i, j). In 455 addition, autumn and winter ocean-originating storms (that reach the land) are also more 456 spatially concentrated (with higher max intensity (fig 4k, l) and smaller sizes) when they are 457 raining over the ocean compared to when they are over land (fig 4g, h). 458 459 Next, we examine how storm properties change during the transition of storms between 451 land and ocean. Land-originating storms during summer/spring have higher max intensity over 452 land than later over the ocean (fig 4a, b). However, during the autumn and winter, these 453 differences are smaller (fig 4c, d). Summer and spring storms originating over the ocean and 454 reaching land tend to be smaller (fig 4e, f) without much change in rainfall intensity (fig 4i, j). In 455 addition, autumn and winter ocean-originating storms (that reach the land) are also more 456 spatially concentrated (with higher max intensity (fig 4k, l) and smaller sizes) when they are 457 raining over the ocean compared to when they are over land (fig 4g, h). 458 459 17 460 461 Fig. 5 Average rainfall intensity variation during the storm lifetimes (represented by seven bins) for storms originating on land (red) and ocean (blue). Only storms with more than seven timesteps have been selected in these plots. Note that all the storms with more than 7 timesteps are normalized (averaged) into seven bins (timestep). NL and NO refer to the number of selected storms over land and ocean, respectively. 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 The shaded areas show the 5 to 95 percentile range and the dashed/solid lines are the medians. Fig. 5 Average rainfall intensity variation during the storm lifetimes (represented by seven bins) for storms originating on land (red) and ocean (blue). Only storms with more than seven timesteps have been selected in these plots. Note that all the storms with more than 7 timesteps are normalized (averaged) into seven bins (timestep). NL and NO refer to the number of selected storms over land and ocean, respectively. The shaded areas show the 5 to 95 percentile range and the dashed/solid lines are the medians. Fig. 5 Average rainfall intensity variation during the storm lifetimes (represented by 2 seven bins) for storms originating on land (red) and ocean (blue). Only storms with more than 3 seven timesteps have been selected in these plots. Note that all the storms with more than 7 4 timesteps are normalized (averaged) into seven bins (timestep). NL and NO refer to the number 5 of selected storms over land and ocean, respectively. The shaded areas show the 5 to 95 6 percentile range and the dashed/solid lines are the medians. 7 Precipitation intensity varies during the lifetime of the storms, as shown in Fig. 5. Storm intensity generally peaks early in the storm lifetime during all seasons, though more clearly in the summer. In all seasons excluding winter, ocean-originating storms, on average, have lower average intensity during their lifetimes compared to their land counterparts. However, the 18 opposite is true in winter, when ocean-originating storms (on average) are more intense. In 473 addition, land-originating storms have a wider range of variability during their lifetimes 474 compared to ocean-originating storms and this range of variability is highest during summer and 475 lowest during the winter. 476 5.2. Diurnal cycle of storm/storm-object properties 477 5.2. Diurnal cycle of storm/storm-object properties 477 478 5.2. Diurnal cycle of storm/storm-object properties 7 8 9 0 Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for 1 different seasons (panels e-j). The results are also presented over land and ocean for different 2 seasons in panels k-z. NL and NO show the number of storm objects in each season over land 3 5.2. Diurnal cycle of storm/storm object properties Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for different seasons (panels e-j). 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 The results are also presented over land and ocean for different seasons in panels k-z. NL and NO show the number of storm objects in each season over land 80 Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for 481 different seasons (panels e-j). The results are also presented over land and ocean for different 482 seasons in panels k-z. NL and NO show the number of storm objects in each season over land 483 Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for 481 different seasons (panels e-j). The results are also presented over land and ocean for different 482 seasons in panels k-z. NL and NO show the number of storm objects in each season over land 483 19 and ocean, respectively. The shaded area shows the range from 5-95% and the dashed/solid lines 4 show the median. 5 show the median. 485 The diurnal cycle varies with season and with the location of storms (land/ocean). 486 Extreme summertime storm-objects have an afternoon peak around 5:00 UTC (15:00 Australian 487 Eastern Standard Time; AEST) in terms of size (fig 6e) and intensity (fig 6f, g). However, winter 488 storms don’t have such a peak during the day (fig 6e-f). Note that the afternoon peak intensity 489 (which exists for all seasons except winter) is mostly related to intense storm-objects over land. 490 In all seasons except winter, high-intensity storm-objects over the ocean are more intense around 491 10:00-23:00 UTC (20:00-09:00 AEST; fig 6l, 6p, 6t, 6m, 6n and 6q) compared to their land 492 counterparts, but in the afternoon and evening, the opposite is true. During spring, summer and 493 autumn, fast-moving ocean storm-objects move faster than land storm-objects during 0:00-10:00 494 UTC (10:00-20:00 AEST) with a peak around 5:00 UTC (15:00 AEST; fig 6n and 6r). However, 495 during the other times of the day, extreme land storm-objects have faster translation speeds. Note 496 that in winter, these peaks are less clear and land storm-objects are mostly faster than their ocean 497 counterparts (fig 6z). 498 521 5.3. Clustering analysis 499 Using clustering algorithms, we have grouped the storms with similar object-based 500 properties over the study area. Here, we employed the Agglomerative clustering technique over 501 the projected multi-dimensional storm data on a 2D map using t-SNE algorithm (more details in 502 section 4.2). The selected input storm properties in the clustering algorithm should not be highly 503 dependent on each other. Therefore, we have calculated the correlation between all pairs of the 504 studied storm-object properties to identify the dependent properties. Figure 7 shows the 505 correlated properties at the significance level of 0.01. By considering a threshold of 0.5 for 506 correlation coefficient, we have found that area vs. volume discharge (fig 7a) and maximum 507 intensity vs. average intensity (fig 7h) are highly dependent on each other and should not be used 508 together as the input in the clustering algorithm. Based on this analysis, the selected input 509 properties for the clustering algorithm are: 1) storm area, 2) storm translation speed, 3) storm 510 max intensity and 4) storm direction which is decomposed into x and y components and have 511 been considered as two independent input variables in the clustering algorithm. 512 The bi-variate histograms in figure 7 also show that storm-objects with higher intensities 513 are generally larger in size and volume (fig 7b, 7c, 7f and 7g). In addition, when the size of the 514 storm-objects increases, the shape of the storm-objects (on average) tends to be more linear (fig 515 7d, 7e). 516 20 Fig. 7 Bivariate histograms for storm-object properties. Tau (correlation coeffici in the figures are calculated based on Kendall’s tau method Fig. 7 Bivariate histograms for storm-object properties. Tau (correlation coefficient) and 519 P-value in the figures are calculated based on Kendall’s tau method 520 521 Fig. 7 Bivariate histograms for storm-object properties. Tau (correlation coefficient) and 519 P-value in the figures are calculated based on Kendall’s tau method 520 g g j p p ( ) P-value in the figures are calculated based on Kendall’s tau method 520 521 21 522 523 Fig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colour on 524 this map shows the density. Panel (b) shows the variation of CHI with the number of clusters. 525 Panels (c) represents the clustered groups of the 2d map (panel a) using the Agglomerative 526 method. 5.3. Clustering analysis 499 They are frequent during the whole year with a frequency peak in 540 spring 541  Type 2 (T2) storms often move south-eastward and include the fastest and largest storms 539 but with low rainfall intensity. They are frequent during the whole year with a frequency peak in 540 spring 541  Type 3 (T3) storms mostly occur during winter with a frequency peak in June, mostly 542 moving northward over the ocean, and include the very slow storms with the smallest size and 543 low intensity compared to the other types. 544  Type 4 (T4) includes the most extreme storm in terms of rainfall intensity and often 545 appears in large sizes moving eastward with low translation speed. They mostly occur during the 546 summer. 547  Type 5 (T5) storms mostly include very fast storms with small sizes and low rainfal 548 intensities that often occur during the winter, mostly moving northward (over the ocean). 549 To further demonstrate the characteristics of each cluster, a video representing the typica 550 storm for each cluster is provided in the supplemental material (Online Resources 2). 551 552 553 554 Fig. 9 Selected regression coefficients for the relationship between climate mode indices 555 and the object-based storm properties in different months. The detail of selection criteria is 556 explained in the manuscript. The coloured bars show the normalized coefficients that are 557 significantly different from zero. Note that the colours represent the storm groups and are 558 matched with the groups’ colours in figure 8. 559 560 We also investigated whether significant relationships exist between storm properties and 561 climate mode indices. Although no statistically significant relationships were found when 562 investigating all storms, we have found some significant relationships between climate indices 563 5  Type 3 (T3) storms mostly occur during winter with a frequency peak in June, mostly 542 moving northward over the ocean, and include the very slow storms with the smallest size and 543 low intensity compared to the other types. 544  Type 3 (T3) storms mostly occur during winter with a frequency peak in June, mostly 542 moving northward over the ocean, and include the very slow storms with the smallest size and 543 low intensity compared to the other types. 5.3. Clustering analysis 499 544  Type 4 (T4) includes the most extreme storm in terms of rainfall intensity and often 545 appears in large sizes moving eastward with low translation speed. They mostly occur during the 546 summer. 547  Type 5 (T5) storms mostly include very fast storms with small sizes and low rainfall 548 intensities that often occur during the winter, mostly moving northward (over the ocean). 549  Type 5 (T5) storms mostly include very fast storms with small sizes and low rainfall 548 intensities that often occur during the winter, mostly moving northward (over the ocean). 549 To further demonstrate the characteristics of each cluster, a video representing the typical 550 storm for each cluster is provided in the supplemental material (Online Resources 2). 551 553 554 Fig. 9 Selected regression coefficients for the relationship between climate mode indices 555 and the object-based storm properties in different months. The detail of selection criteria is 556 explained in the manuscript. The coloured bars show the normalized coefficients that are 557 significantly different from zero. Note that the colours represent the storm groups and are 558 matched with the groups’ colours in figure 8. 559 553 554 Fi 9 S l t d i ffi i t f th l ti hi b t li t d i di 555 Fig. 9 Selected regression coefficients for the relationship between climate mode indices and the object-based storm properties in different months. The detail of selection criteria is explained in the manuscript. The coloured bars show the normalized coefficients that are significantly different from zero. Note that the colours represent the storm groups and are matched with the groups’ colours in figure 8. Fig. 9 Selected regression coefficients for the relationship between climate mode indices and the object-based storm properties in different months. The detail of selection criteria is explained in the manuscript. The coloured bars show the normalized coefficients that are significantly different from zero. Note that the colours represent the storm groups and are matched with the groups’ colours in figure 8. Fig. 9 Selected regression coefficients for the relationship between climate mode indices and the object-based storm properties in different months. The detail of selection criteria is explained in the manuscript. The coloured bars show the normalized coefficients that are significantly different from zero. 5.3. Clustering analysis 499 Panels (d-h) show the PDFs of different properties for the clustered storms. The annual 527 cycle of each cluster is shown in panel (i). 528 529 ig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colo Fig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colour on this map shows the density. Panel (b) shows the variation of CHI with the number of clusters. Panels (c) represents the clustered groups of the 2d map (panel a) using the Agglomerative method. Panels (d-h) show the PDFs of different properties for the clustered storms. The annual cycle of each cluster is shown in panel (i). Fig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colour on 4 this map shows the density. Panel (b) shows the variation of CHI with the number of clusters. 5 Panels (c) represents the clustered groups of the 2d map (panel a) using the Agglomerative 6 method. Panels (d-h) show the PDFs of different properties for the clustered storms. The annual 7 cycle of each cluster is shown in panel (i). 8 The results show that there are five storm clusters (types) with similar object-based properties occurring over the study area. This number is based on the optimum value of CHI against the number of clusters (fig 9b; for more details see section 4.2.2). Figure 8d-h shows the PDFs of storm properties for different storm types with similar quantitative characteristics that have been identified using the Agglomerative and t-SNE algorithms over the study area. Based on these results, the detected storm types have the following characteristics:  Type 1 (T1) storms have a peak frequency in autumn and include mostly average size 36 storms with the lowest translation speeds but very high rainfall intensities compared to the other 37 groups. They often move towards the north (over the ocean) to the northwest (hit the land) 38 22  Type 2 (T2) storms often move south-eastward and include the fastest and largest storms 539 but with low rainfall intensity. They are frequent during the whole year with a frequency peak in 540 spring 541  Type 2 (T2) storms often move south-eastward and include the fastest and largest storms 539 but with low rainfall intensity. 5. Discussion 586 587 The results presented in Section 4.1 are broadly consistent with previous studies, but with 588 some notable exceptions. For example, during summer, storms are mostly larger, move faster 589 and are accompanied by higher rainfall intensities compared to the storms in winter (fig 2). This 590 is in agreement with the previous studies over Australia reporting that severe thunderstorms are 591 most prevalent in the warm season (Niall and Walsh 2005; Schuster et al. 2005; Davis and Walsh 592 2008; Warren et al. 2020). The extreme summer and spring storms in terms of rainfall intensity 593 are more intense when they are raining over land between 10:00 to 20:00 (AEST; peaking in the 594 afternoon) compared to when they are raining over the ocean, consistent with the diurnal cycle of 595 surface temperature and the maximum availability of heating over land. The diurnal peak of 596 severe thunderstorm over land during the warm season was also reported by Griffiths et al. 597 (1993) in studying the severe thunderstorms in New South Wales, Australia and Schuster et al 598 (2005) in studying the hail climatology of the Greater Sydney, Australia both using reported 599 data. 600 Although previous studies reported that thunderstorms are more prevalent during the 601 warm seasons, we have found that there are more storms during autumn and winter than spring 602 and summer. A reason behind this contradiction is that most previous studies employed ground 603 observations that provide information for storms only over land (Niall and Walsh 2005; Schuster 604 et al. 2005; Davis and Walsh 2008), and, based on our findings, autumn and winter storms 605 mostly initiate from the ocean and tend to move northward causing more precipitation over the 606 587 The results presented in Section 4.1 are broadly consistent with previous studies, but with 588 some notable exceptions. For example, during summer, storms are mostly larger, move faster 589 and are accompanied by higher rainfall intensities compared to the storms in winter (fig 2). This 590 is in agreement with the previous studies over Australia reporting that severe thunderstorms are 591 most prevalent in the warm season (Niall and Walsh 2005; Schuster et al. 2005; Davis and Walsh 592 2008; Warren et al. 2020). 𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡= 𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) 𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) (12) 581 Where σ is the standard deviation in this equation, “index coefficient” refers to any 583 calculated coefficient from equation 11 and “index normalized coefficient” is the partial 584 correlation between every index and storm property. 585 5.3. Clustering analysis 499 Note that the colours represent the storm groups and are matched with the groups’ colours in figure 8. We also investigated whether significant relationships exist between storm properties and 561 climate mode indices. Although no statistically significant relationships were found when 562 investigating all storms, we have found some significant relationships between climate indices 563 and storm properties in each cluster (identified in the previous section) using a multiple 564 regression model described in section 4.3. Based on this approach, 75 regression models have 565 been produced (3 indices × 5 storm properties × 5 clusters). In order to identify robust 566 associations, we have identified instances when at least five coefficients in a row have the same 567 sign (positive or negative), and among them at least three are significantly different from zero. 568 Since these climate indices often have impacts on weather for a period from 3-9 months, this 569 restriction helps us to better exclude those short periods in which precipitation has a statistically 570 acceptable link with climate indices but probably not in reality. Thus, from all of the results, only 571 23 five regressions passed this criterion and are shown in figure 9. The results show that during El- 572 Niño events in cold seasons, T3 and T5 storms have negative correlation with ENSO in cold 573 season with lower rainfall intensity in El Niño and higher rainfall intensity in La-Niña (fig 9a, 574 9b). ENSO also has an impact on T1 and T3 storms' translation speed during the warm season 575 with a positive correlation (faster in El Niño and slower in La-Niña; fig 9c, 9d). Finally, IOD has 576 also shown to have a positive correlation with T1 storms’ translation speed from mid-summer to 577 early winter (Feb, Apr and Jun; fig 9e). Note that in figure 9, all the coefficients have been 578 normalized to derive the partial correlation between every index and storm property as below: 579 580 𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡= 𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) 𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) (12) 581 582 Where σ is the standard deviation in this equation, “index coefficient” refers to any 583 calculated coefficient from equation 11 and “index normalized coefficient” is the partial 584 correlation between every index and storm property. 585 five regressions passed this criterion and are shown in figure 9. 5.3. Clustering analysis 499 The results show that during El- 572 Niño events in cold seasons, T3 and T5 storms have negative correlation with ENSO in cold 573 season with lower rainfall intensity in El Niño and higher rainfall intensity in La-Niña (fig 9a, 574 9b). ENSO also has an impact on T1 and T3 storms' translation speed during the warm season 575 with a positive correlation (faster in El Niño and slower in La-Niña; fig 9c, 9d). Finally, IOD has 576 also shown to have a positive correlation with T1 storms’ translation speed from mid-summer to 577 early winter (Feb, Apr and Jun; fig 9e). Note that in figure 9, all the coefficients have been 578 normalized to derive the partial correlation between every index and storm property as below: 579 580 𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡= 𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) 𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) (12) 581 582 Where σ is the standard deviation in this equation, “index coefficient” refers to any 583 calculated coefficient from equation 11 and “index normalized coefficient” is the partial 584 correlation between every index and storm property. 585 five regressions passed this criterion and are shown in figure 9. The results show that during El- 572 Niño events in cold seasons, T3 and T5 storms have negative correlation with ENSO in cold 573 season with lower rainfall intensity in El Niño and higher rainfall intensity in La-Niña (fig 9a, 574 9b). ENSO also has an impact on T1 and T3 storms' translation speed during the warm season 575 with a positive correlation (faster in El Niño and slower in La-Niña; fig 9c, 9d). Finally, IOD has 576 also shown to have a positive correlation with T1 storms’ translation speed from mid-summer to 577 early winter (Feb, Apr and Jun; fig 9e). Note that in figure 9, all the coefficients have been 578 normalized to derive the partial correlation between every index and storm property as below: 579 580 𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡= 𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) 𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ) (12) 581 582 Wh i th t d d d i ti i thi ti “i d ffi i t” f t 583 5. Discussion 586 616 We have calculated the average wind direction during the rainy days (based on radar 617 data) at 700 hPa and 850 hPa in ERA5 Reanalysis data (see fig S6 and S7; Online Resource 1) to 618 examine whether the storm directions are (on average) following the wind directions in different 619 seasons. The results show that typical storm direction in summer and spring seems to be in 620 agreement with climatological wind fields at 700 hPa in ERA5. This agreement seems to be even 621 better when compared with 850 hPa wind fields since the north westerly storm directions (fig 2e, 622 2l) during these seasons are better matched with the wind directions at this pressure level. 623 However, in autumn and winter, it doesn’t seem to agree well, especially over the ocean. Storms 624 in these seasons mostly move northward over the ocean (fig 2e, 2l). However, the average wind 625 directions in these seasons are south-westerly. A reason might be the climatological conditions during 626 the cold season are not supportive of isolated offshore showers/storms which are more frequent in these 627 seasons. However, further investigations are required by conducting a detailed comparison of wind 628 and storm directions. 629 In all seasons, land and ocean-originating storms tend to peak early in their lifetimes (fig 630 6) consistent with the findings of Ayat et. al. (2021b) and Prein et. al. (2017) over the United 631 States. Additionally, we have found that this peak is more prominent during the summer for 632 severe storms raining over land. Storm characteristics also change during the transition of storms 633 between land and ocean like the decrease in max intensity for summertime land-originating 634 storms when moving over the ocean and wintertime ocean-originating storms when moving over 635 land. These variations are probably related to a change in boundary layer instability in this 636 process, and shows the immediate effect of change in air mass characteristics (land/ocean) on the 637 storms. These characteristics can include surface temperature and humidity, sea/land breezes and 638 topographical interactions, the effects of elevated mixed layers advected over the coast, low-level 639 wind shear and convergence. 640 Using clustering analysis, we have found that there are five storm types with similar 641 object-based properties over this region and described in detail in section 5.3. 5. Discussion 586 The extreme summer and spring storms in terms of rainfall intensity 593 are more intense when they are raining over land between 10:00 to 20:00 (AEST; peaking in the 594 afternoon) compared to when they are raining over the ocean, consistent with the diurnal cycle of 595 surface temperature and the maximum availability of heating over land. The diurnal peak of 596 severe thunderstorm over land during the warm season was also reported by Griffiths et al. 597 (1993) in studying the severe thunderstorms in New South Wales, Australia and Schuster et al 598 (2005) in studying the hail climatology of the Greater Sydney, Australia both using reported 599 data. 600 Although previous studies reported that thunderstorms are more prevalent during the 601 warm seasons, we have found that there are more storms during autumn and winter than spring 602 and summer. A reason behind this contradiction is that most previous studies employed ground 603 observations that provide information for storms only over land (Niall and Walsh 2005; Schuster 604 et al. 2005; Davis and Walsh 2008), and, based on our findings, autumn and winter storms 605 mostly initiate from the ocean and tend to move northward, causing more precipitation over the 606 ocean than land. Thus, the previous studies have not captured the storms over the ocean in these 607 seasons. In addition, most of the previous studies were focused on the severe thunderstorms or 608 storms with deep convective clouds and high storm tops that are often accompanied by 609 24 electrification, which are less frequent during the cold seasons. Using lightning records, Dowdy 610 and Kuleshov (2014) also showed that a maximum in lightning activity during the cooler months 611 occurs over the ocean to the east of Australia, which is consistent with our results. However, they 612 reported a higher frequency of thunderstorms during the warm season. Since storms in cold 613 season are small-scale with low rainfall intensity, probably many of them are not accompanied 614 by lightning to be captured by the sensors. Therefore, a large number of storms over the ocean 615 during this season are probably missed in the mentioned study. 5. Discussion 586 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et 662 al. 2013; King et al. 2014; Ashcroft et al. 2019). While no relationship was found for all storms, 663 we have found that the rainfall intensity in T3 and T5 storm types (that are more frequent in 664 winter) decreases during these events (fig 9a, b). 665 We have found that there are relationships between some object-based storm properties 666 over the study area that have also been reported in previous studies over other regions. For 667 example, we find that storm objects with large volume and size tend to be more linear and are 668 accompanied by higher rainfall intensities (fig 7); this is consistent with the findings of Ayat et. 669 al. (2021b) in comparing a merged ground radar product and a merged satellite precipitation 670 product over the United States, and Prein et al. (2017) in simulating North American mesoscale 671 convective systems with a convection‑permitting climate model. Large-scale heavy storms over 672 the study area mostly fall in T2 and T4 storm categories, and typical T2 and T4 storms showed 673 that they are mostly frontal systems that elongated/oriented parallel to the front borders. 674 In summary, the results are showing that the storm intensity variations are consistent with 675 diurnal/seasonal cycles and are related to climate mode oscillations. However, other 676 characteristics of the storms like storm size and translation speed do not seem to always follow 677 the same relationship. This suggests that further investigations are required to find a more 678 definitive answer to the effect of atmospheric parameters variations (e.g. temperature, humidity, 679 etc.) on storm properties other than intensity. 680 5. Discussion 586 665 We have found that there are relationships between some object-based storm properties 666 over the study area that have also been reported in previous studies over other regions. For 667 example, we find that storm objects with large volume and size tend to be more linear and are 668 accompanied by higher rainfall intensities (fig 7); this is consistent with the findings of Ayat et. 669 al. (2021b) in comparing a merged ground radar product and a merged satellite precipitation 670 product over the United States, and Prein et al. (2017) in simulating North American mesoscale 671 convective systems with a convection‑permitting climate model. Large-scale heavy storms over 672 the study area mostly fall in T2 and T4 storm categories, and typical T2 and T4 storms showed 673 that they are mostly frontal systems that elongated/oriented parallel to the front borders. 674 In summary, the results are showing that the storm intensity variations are consistent with 675 diurnal/seasonal cycles and are related to climate mode oscillations. However, other 676 characteristics of the storms like storm size and translation speed do not seem to always follow 677 the same relationship. This suggests that further investigations are required to find a more 678 definitive answer to the effect of atmospheric parameters variations (e.g. temperature, humidity, 679 etc.) on storm properties other than intensity. 680 end of spring. This type of storm can be accompanied by severe winds. For instance, they 651 contributed to the storm that occurred on 8 January 2003, and brought a severe wind gust with a 652 maximum of 109 km/h. Finally, T4 storms mostly occur in summer and include the extreme 653 high-intensity storms that have very large sizes with slow translation speed. These characteristics 654 of the storms in this group can create devastating flash and riverine floods in this region (fig 8). 655 Their dominant contribution in the East Coast Low event on 1 and 2 February 2005 caused flash 656 flooding in Sydney with reports of 6 cm size hail. 657 Our results in investigating the relationships between climate mode indices and storm 658 properties are consistent with the previous studies over the eastern and southeastern parts of 659 Australia. Those studies have reported a reduction in rainfall intensity during El-Niño events in 660 the cold season (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008; 661 Nicholls et al. 5. Discussion 586 Among these 642 clusters, three storm types might be accompanied by natural disasters, due to their special 643 characteristics. The first storm type (T1) mostly includes the slowest storms that have high 644 rainfall intensities with small areas and mostly move towards the north-northwest with a peak 645 frequency in autumn. These storms can create flash floods if they hit the land like the high 646 impact event that occurred on 30 May 2011 with a dominant contribution of this type of storm 647 and led to flash flooding in some of Sydney's eastern suburbs (e.g., Zetland, Alexandria and 648 Kingsford). T2 storms mostly move towards the southeast and include the largest and fastest 649 storms but with very low rainfall intensity occurring in every season with a peak frequency at the 650 25 end of spring. This type of storm can be accompanied by severe winds. For instance, they 651 contributed to the storm that occurred on 8 January 2003, and brought a severe wind gust with a 652 maximum of 109 km/h. Finally, T4 storms mostly occur in summer and include the extreme 653 high-intensity storms that have very large sizes with slow translation speed. These characteristics 654 of the storms in this group can create devastating flash and riverine floods in this region (fig 8). 655 Their dominant contribution in the East Coast Low event on 1 and 2 February 2005 caused flash 656 flooding in Sydney with reports of 6 cm size hail. 657 Our results in investigating the relationships between climate mode indices and storm 658 properties are consistent with the previous studies over the eastern and southeastern parts of 659 Australia. Those studies have reported a reduction in rainfall intensity during El-Niño events in 660 the cold season (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008; 661 Nicholls et al. 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et 662 al. 2013; King et al. 2014; Ashcroft et al. 2019). While no relationship was found for all storms, 663 we have found that the rainfall intensity in T3 and T5 storm types (that are more frequent in 664 winter) decreases during these events (fig 9a, b). 6. Conclusion 681 Finally, T5 storms are wintertim 723 small scale storms over the ocean, moving northward with high translation speeds. We als 724 studied the connection between climate modes and storm properties for different clusters using 725 linear multivariate approach and the results show that during El-Niño events in cold seasons, T 726 and T5 storms have negative correlation with ENSO in cold season with lower rainfall intensit 727 in El Niño and higher rainfall intensity in La-Niña. In addition, ENSO has an impact on T1 an 728 T3 storms' translation speed during the warm season with a positive correlation (faster in El Niñ 729 The extreme storms in terms of size, intensity and translation speed are more frequent 690 during summer and spring. Storms in these seasons mostly originate on land, move towards the 691 east-southeast, are larger, faster and more intense compared to the storms originating on the 692 ocean. In these seasons, between ~10am and ~8pm (AEST), the extreme storms raining over land 693 (wherever they originate) are larger and have higher rain rate but slower compared to when they 694 are raining over the ocean (with a peak in the afternoon that is consistent with the diurnal 695 maximum of boundary layer instability). However, the opposite is true for storms later at night 696 into early morning. 697 Although severe storms are more frequent during summer and spring, typical storms in 698 autumn have higher rainfall intensity compared to the other seasons. In addition, the storms 699 (including non-severe ones) are more frequent in autumn and winter compared to summer and 700 spring. During the cold season, storms mostly initiate from the ocean and tend to move 701 northward, which causes more precipitation over the ocean than land. Ocean-originating storms 702 during these seasons like summer and spring are typically smaller and move slower but have 703 higher rainfall intensity than the land-originating storms which still tend to move east- 704 southeastward. 705 The results show that the change in the air mass characteristics (land/ocean) can 706 immediately affect the storm properties. For instance, the land-originating storms that cross to 707 the ocean in summer are more intense over land than ocean. However, in winter ocean 708 originating storms that can reach land are more intense over ocean than land. 6. Conclusion 681 In this study, we establish an object-based storm climatology using an S-band weather 682 radar located near Wollongong, NSW (34.26° S, 150.87° E, 471 m altitude) with more than 20 683 years of records (1996-2018). The study area is the radar coverage region (including land and 684 ocean), within 150 km of the radar. Here, we employed the Method of Object-based Diagnostic 685 Evaluation (MODE) Time Domain (MTD) to detect and track the storms. Using this object- 686 based approach helps us to better understand the climatology of storm properties (other than 687 rainfall intensity and frequency) that haven’t been explored in the previous studies over the study 688 area. 689 26 The extreme storms in terms of size, intensity and translation speed are more freque 690 during summer and spring. Storms in these seasons mostly originate on land, move towards th 691 east-southeast, are larger, faster and more intense compared to the storms originating on th 692 ocean. In these seasons, between ~10am and ~8pm (AEST), the extreme storms raining over lan 693 (wherever they originate) are larger and have higher rain rate but slower compared to when the 694 are raining over the ocean (with a peak in the afternoon that is consistent with the diurn 695 maximum of boundary layer instability). However, the opposite is true for storms later at nig 696 into early morning. 697 Although severe storms are more frequent during summer and spring, typical storms 698 autumn have higher rainfall intensity compared to the other seasons. In addition, the storm 699 (including non-severe ones) are more frequent in autumn and winter compared to summer an 700 spring. During the cold season, storms mostly initiate from the ocean and tend to mov 701 northward, which causes more precipitation over the ocean than land. Ocean-originating storm 702 during these seasons like summer and spring are typically smaller and move slower but hav 703 higher rainfall intensity than the land-originating storms which still tend to move eas 704 southeastward. 705 The results show that the change in the air mass characteristics (land/ocean) ca 706 immediately affect the storm properties. For instance, the land-originating storms that cross 707 the ocean in summer are more intense over land than ocean. However, in winter ocea 708 originating storms that can reach land are more intense over ocean than land. 6. Conclusion 681 These changes 709 storm properties during the storm lifetimes might be related to the differences in surfac 710 temperature and humidity, sea/land breezes and topographical interactions, the effects o 711 elevated mixed layers advected over the coast, low-level wind shear and convergence. Howeve 712 further research is needed to find a definitive answer. 713 We have found five types of storms with distinct object-based properties using clusterin 714 techniques. The first storm type (T1) peaks in autumn and mostly includes small-scale and slow 715 moving storms but with high rainfall intensities, often moving northward over the ocean. Th 716 type of storm has the potential to create flash floods when they move offshore. T2 storms are th 717 largest and fastest storms with low rainfall intensities, often moving southeastward with a pea 718 in spring. This storm type can be accompanied by severe gust winds. T3 storms, include th 719 smallest size storms moving northward with low intensities and translation speed and peak 720 winter. T4 storms include the most extreme storms in terms of rainfall intensity with large area 721 often moving slowly towards the east with a peak in summer, and can be a source of devastatin 722 flash and riverine floods over the study area in this season. Finally, T5 storms are wintertim 723 small scale storms over the ocean, moving northward with high translation speeds. We als 724 studied the connection between climate modes and storm properties for different clusters using 725 linear multivariate approach and the results show that during El-Niño events in cold seasons, T 726 and T5 storms have negative correlation with ENSO in cold season with lower rainfall intensi 727 in El Niño and higher rainfall intensity in La Niña In addition ENSO has an impact on T1 an 728 The extreme storms in terms of size, intensity and translation speed are more frequen 690 during summer and spring. Storms in these seasons mostly originate on land, move towards th 691 east-southeast, are larger, faster and more intense compared to the storms originating on th 692 ocean. In these seasons, between ~10am and ~8pm (AEST), the extreme storms raining over lan 693 (wherever they originate) are larger and have higher rain rate but slower compared to when the 694 are raining over the ocean (with a peak in the afternoon that is consistent with the diurna 695 maximum of boundary layer instability). 6. Conclusion 681 However, the opposite is true for storms later at nigh 696 into early morning. 697 Although severe storms are more frequent during summer and spring, typical storms i 698 autumn have higher rainfall intensity compared to the other seasons. In addition, the storm 699 (including non-severe ones) are more frequent in autumn and winter compared to summer an 700 spring. During the cold season, storms mostly initiate from the ocean and tend to mov 701 northward, which causes more precipitation over the ocean than land. Ocean-originating storm 702 during these seasons like summer and spring are typically smaller and move slower but hav 703 higher rainfall intensity than the land-originating storms which still tend to move east 704 southeastward. 705 The results show that the change in the air mass characteristics (land/ocean) ca 706 immediately affect the storm properties. For instance, the land-originating storms that cross t 707 the ocean in summer are more intense over land than ocean. However, in winter ocea 708 originating storms that can reach land are more intense over ocean than land. These changes i 709 storm properties during the storm lifetimes might be related to the differences in surfac 710 temperature and humidity, sea/land breezes and topographical interactions, the effects o 711 elevated mixed layers advected over the coast, low-level wind shear and convergence. Howeve 712 further research is needed to find a definitive answer. 713 We have found five types of storms with distinct object-based properties using clusterin 714 techniques. The first storm type (T1) peaks in autumn and mostly includes small-scale and slow 715 moving storms but with high rainfall intensities, often moving northward over the ocean. Thi 716 type of storm has the potential to create flash floods when they move offshore. T2 storms are th 717 largest and fastest storms with low rainfall intensities, often moving southeastward with a pea 718 in spring. This storm type can be accompanied by severe gust winds. T3 storms, include th 719 smallest size storms moving northward with low intensities and translation speed and peak i 720 winter. T4 storms include the most extreme storms in terms of rainfall intensity with large area 721 often moving slowly towards the east with a peak in summer, and can be a source of devastatin 722 flash and riverine floods over the study area in this season. 6. Conclusion 681 These changes in 709 storm properties during the storm lifetimes might be related to the differences in surface 710 temperature and humidity, sea/land breezes and topographical interactions, the effects of 711 elevated mixed layers advected over the coast, low-level wind shear and convergence. However, 712 further research is needed to find a definitive answer. 713 We have found five types of storms with distinct object-based properties using clustering 714 techniques. The first storm type (T1) peaks in autumn and mostly includes small-scale and slow- 715 moving storms but with high rainfall intensities, often moving northward over the ocean. This 716 type of storm has the potential to create flash floods when they move offshore. T2 storms are the 717 largest and fastest storms with low rainfall intensities, often moving southeastward with a peak 718 in spring. This storm type can be accompanied by severe gust winds. T3 storms, include the 719 smallest size storms moving northward with low intensities and translation speed and peak in 720 winter. T4 storms include the most extreme storms in terms of rainfall intensity with large areas 721 often moving slowly towards the east with a peak in summer, and can be a source of devastating 722 flash and riverine floods over the study area in this season. Finally, T5 storms are wintertime 723 small scale storms over the ocean, moving northward with high translation speeds. We also 724 studied the connection between climate modes and storm properties for different clusters using a 725 linear multivariate approach and the results show that during El-Niño events in cold seasons, T3 726 and T5 storms have negative correlation with ENSO in cold season with lower rainfall intensity 727 in El Niño and higher rainfall intensity in La-Niña. In addition, ENSO has an impact on T1 and 728 T3 storms' translation speed during the warm season with a positive correlation (faster in El Niño 729 27 and slower in La-Niña). Finally, IOD has also shown a positive correlation with T1 storms’ translation speed from mid-summer to early winter (Feb, Apr and Jun). Although previous studies using pixel-based approaches have provided us with valuable 2 information, the outcomes of this research show that the object-based approach can be a powerful means to better understand other aspects of the storms like storm size, translation 4 speed, direction, aspect ratio, lifetime, track length, etc. 6. Conclusion 681 which was not possible using pixel- 5 based approach. In this study, we have conducted a comprehensive study in understanding the 6 storm climatology. However, this study is limited to a specific region in Australia, and further 7 research is needed to do the same study over the other regions or over a larger scale using 8 regional/global datasets. In addition, there are lots of questions unanswered in understanding the 9 storm structures and the effects of parameters like temperature, humidity, topography, surface 0 type, etc. on the storm properties other than rainfall intensity, which might be better explored using an object-based approach. 2 Although previous studies using pixel-based approaches have provided us with valuable 732 information, the outcomes of this research show that the object-based approach can be a 733 powerful means to better understand other aspects of the storms like storm size, translation 734 speed, direction, aspect ratio, lifetime, track length, etc. which was not possible using pixel- 735 based approach. In this study, we have conducted a comprehensive study in understanding the 736 storm climatology. However, this study is limited to a specific region in Australia, and further 737 research is needed to do the same study over the other regions or over a larger scale using 738 regional/global datasets. In addition, there are lots of questions unanswered in understanding the 739 storm structures and the effects of parameters like temperature, humidity, topography, surface 740 type, etc. on the storm properties other than rainfall intensity, which might be better explored 741 using an object-based approach. 742 743 Declarations: 744 745 Funding: This work was supported by the Australian Research Council as part of the Center of 746 Excellence for Climate Extremes (CE170100023) for Jason P. Evans and Steven C. Sherwood. 747 748 Conflicts of interest/Competing interests: The authors declare no conflicts of 749 interest/competing interests. 750 751 Availability of data and material: Radar data used in this study are available at 752 https://my.nci.org.au/mancini/project/rq0. 753 754 Code availability: The codes that support the findings of this study are available upon request 755 from the corresponding author. 756 757 References: 758 759 Ahmed, R., M. Latif, S. Adnan, and M. K. Abuzar, 2019: Thunderstorm frequency distribution 760 and associated convective mechanisms over Pakistan. Theor. Appl. Climatol., 137, 755– 761 773, https://doi.org/10.1007/s00704-018-2619-x. 762 Allan, R., J. Lindesay, and D. Parker, 1996: El Niño southern oscillation &amp; climatic 763 variability. 6. Conclusion 681 CSIRO Publishing,. 764 Allen, J., D. Karoly, and G. Mills, 2011: A severe thunderstorm climatology for Australia and 765 associated thunderstorm environments. Aust. Meteorol. Oceanogr. 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Revealing the Radial Effect on Orientation Discrimination by Manual Reaction Time
Frontiers in neuroscience
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*Correspondence: Yujun Pan *Correspondence: Yujun Pan yujunpan@ems.hrbmu.edu.cn Revealing the Radial Effect on Orientation Discrimination by Manual Reaction Time Lixin Liang 1, Yang Zhou 2, 3, Mingsha Zhang 2 and Yujun Pan 1* 1 Department of Neurology, The First Clinical College of Harbin Medical University, Harbin, China, 2 State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China, 3 Department of Neurobiology, University of Chicago, Chicago, IL, United States It has been shown that the sensitivity and accuracy of orientation perception in the periphery is significantly better when the orientations are radial with respect to the fixation point than when they are tangential. However, since perception and action may be dissociated, it is unclear whether the perceptual radial effect has a counterpart in reaction time (RT) of motor responses. Furthermore, it is unknown whether or how stimulus-response-compatibility (SRC) effect interacts with the radial effect to determine RT. To address these questions, we measured subjects’ manual RT to grating stimuli that appeared across upper visual field (VF). We found that (1) RTs were significantly shorter when a grating was oriented closer to the radial direction than when it was oriented closer to the tangential direction even though the perceptual accuracies for the more radial and more tangential orientations were not significantly different under our experimental condition; (2) This RT version of the radial effect was larger in the left VF than in the right VF; (3) The radial effect and SRC effect interacted with each other to determine the overall RT. These results suggest that the RT radial effect reported here is not a passive reflection of the radial effect in perceptual accuracy, but instead, represents different processing time of radial and tangential orientations along the sensorimotor pathway. INTRODUCTION The radial effect refers to the observation that the sensitivity and accuracy of orientation perception in the periphery is significantly better when a stimulus is radially oriented with respect to the fixation point than when it is tangentially oriented (Rovamo et al., 1982; Bennett and Banks, 1991; Westheimer, 2003; Sasaki et al., 2006). The radial effect has also been used to explain the induced effect of vertical disparity (Matthews et al., 2003). Indeed, the anatomical and physiological basis of the radial effect has been revealed previously (Levick and Thibos, 1982; Leventhal and Schall, 1983; Schall et al., 1986; Sasaki et al., 2006). Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience Received: 20 April 2017 Accepted: 03 November 2017 Published: 24 November 2017 Received: 20 April 2017 Accepted: 03 November 2017 Published: 24 November 2017 Although the radial effect is well established, some questions remain open. First, while previous studies focused on comparing the perceptual sensitivity and accuracy between radial and tangential orientations, no study has investigated possible differences in reaction time (RT) of motor responses to these orientations. Since there are studies demonstrating dissociations between perceptual and motor measurements under certain conditions (Goodale and Milner, 1992; Goodale, 2014), a perceptual radial effect does not necessarily imply a RT radial effect. On the other hand, one of the main purposes of sensory processing is to guide motor response. It is therefore interesting to Keywords: orientation perception, grating stimuli, visual field, SRC, hemisphere asymmetry ORIGINAL RESEARCH published: 24 November 2017 doi: 10.3389/fnins.2017.00638 Reviewed by: Keywords: orientation perception, grating stimuli, visual field, SRC, hemisphere asymmetry Edited by: Sheng He, Edited by: Sheng He, Institute of Biophysics (CAS), China Institute of Biophysics (CAS), China Reviewed by: Dirk Kerzel, Université de Genève, Switzerland Pinglei Bao, California Institute of Technology, United States Edited by: Sheng He, Institute of Biophysics (CAS), China Reviewed by: Dirk Kerzel, Université de Genève, Switzerland Pinglei Bao, California Institute of Technology, United States Behavioral Tasks Orientation Discrimination Task (Figure 1A) A trial began with a red fixation point (16.0 ± 0.2 cd/cm2) appearing in the center of the screen (14.4 ± 0.2 cd/cm2). The subjects had to initiate fixation after the fixation onset, and were required to maintain fixation throughout each trial. A trial was excluded if the eye position left a circular window (3◦radius) centered at the fixation point. After a random delay (600–1,000 ms), a sine grating (10% contrast, 14.4 cd/m2 mean luminance, 2◦diameter) of either +45◦or −45◦diagonal grating appeared at one of nine possible locations, which were horizontally aligned with equal space (2◦) from 8◦left to 8◦ right of the vertical meridian and were 8◦above the fixation point (Figure 1B). Thus, the positive and negative diagonal lines from the fixation point went through the centers of the leftmost and rightmost locations, respectively. Consequently, the +45◦or −45◦gratings were, respectively, radial and tangential at the rightmost location, and tangential and radial at the leftmost location. When the gratings’ location changed from leftmost to rightmost, either of the two gratings will cover some intermediate orientations between radial and tangential. Subjects were instructed to report the self-judged grating orientation (left or right) as fast as possible by pressing a key accordingly (left or right) using the index or middle finger of the right hand, respectively. The inter-trial interval was 2 s. To address these questions, we designed an orientation discrimination task in which a grating stimulus (+45◦or −45◦) appeared randomly at one of nine equally spaced locations in the upper VF. Thus, when the grating appears at one of these nine locations, its direction with respect to the fixation point is radial, tangential or some intermediate orientations. We measured subjects’ manual RT for orientation discrimination across the range of radial/tangential conditions. Subjects showed significantly shorter RTs to gratings closer to radial orientation than closer to tangential orientation, and these differences were not attributable to the speed-accuracy tradeoff. Such RT differences decreased as the grating became less radially-oriented and tangentially-oriented. In addition, the magnitude of radial effect on RT was significantly larger in the left VF than in the right VF, which further supports the argument that the spatial perception is asymmetrically processed between left and right hemispheres (Corballis et al., 2002; Boulinguez et al., 2003; Corballis, 2003; Okubo and Nicholls, 2008). Behavioral Tasks Moreover, the radial effect and SRC effect strongly interact with each other to determine the overall RT. Citation: Liang L, Zhou Y, Zhang M and Pan Y (2017) Revealing the Radial Effect on Orientation Discrimination by Manual Reaction Time. Front. Neurosci. 11:638. doi: 10.3389/fnins.2017.00638 November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 1 Radial Effect on Orientation Discrimination Liang et al. investigate whether the perceptual radial effect has a motor counterpart. Second, previous studies only compared orientation sensitivity and accuracy between radial and tangential orientations at a given location in the VF. It is unclear how the radial effect on orientation perception changes as the orienting direction gradually changes from radial to tangential directions. Third, stimulus-response-compatibility (SRC) effect (also known as Simon effect) also affects RT (Fitts and Seeger, 1953; Simon and Rudell, 1967; Wallace, 1971; Whitaker, 1982; Hommel, 2011; Styrkowiec and Szczepanowski, 2013) and it has not been investigated whether and how it interacts with the radial effect. photometer. The subjects sat in front of the monitor at a viewing distance of 57.5 cm and with their head restrained on a chin rest. Their eye positions were monitored with an infrared image eye tracker (EyeLink 2000 Desktop Mount, SR Research). Two key buttons were placed in front of the subjects. We used MATLAB (Mathworks) with Psychtoolbox extension (Brainard, 1997; Pelli, 1997) on a PC to present stimuli and collect the manual RT data. Stimulus Parameters To reduce performance variations across sessions and subjects, we used a staircase procedure to determine stimulus parameters at threshold performance for each subject at the start of each session (day). For the orientation discrimination task, we varied the spatial frequency of gratings. For the color discrimination task, we varied the R or B index of color patches while keeping the remaining two indices equal to the RGB value of the gray background. For parameters determination, we fixed the duration of stimulus appearance (30 ms for gratings and 50 ms for color patches) and found thresholds at 78% correct performance by fitting cumulative Gaussian function to psychometric curves. Across all subjects and sessions, the Color Discrimination Task (Figure 1D) Eighteen right-handed subjects with normal vision (age 18–28, 17 naïve, 7 male and 11 female) participated in the present experiments. All subjects took part in the first experiment, named as orientation discrimination task (Figure 1A). Before the experiments started, each subject was examined about the self-judged orientation (left or right) to the two gratings (+45◦and −45◦), by pressing either a left key or a right key as response of his/her judgment. Then, the subjects were induced to remain such self-judged orientation throughout the orientation discrimination experiment. Twelve of eighteen subjects took part in the second experiment, named as color discrimination task (Figure 1D). All experiments followed the guideline of the Institutional Review Board (IRB) of the State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University. The protocol was approved by the same committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The aim of this experiment was to test whether there were intrinsic group differences in sensorimotor transformation. The task sequence was identical to the orientation discrimination task, except that the grating stimulus was replaced by a gray patch (0.8◦diameter, 14.3 ± 0.3 cd/m2 mean luminance) tinted either red or blue. Subjects were instructed to report the red and blue colors as fast as possible by pressing left and right key, respectively. Response Accuracy Calculation threshold of spatial frequency ranged from 3.8 cycle/degree to 5.6 cycle/degree, and the threshold of color- index ranged from 1 to 5%. We calculated each subject’s response accuracy (correct ratio) for each grating orientation (+45◦and −45◦) at each tested location. Experimental Setup All visual stimuli were presented on an LCD monitor (BENQ XL2720Z, 1,920 × 1,080 pixels, 100 Hz vertical refresh rate) calibrated and linearized with a Konica Minolta LS-110 November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 2 Radial Effect on Orientation Discrimination Liang et al. FIGURE 1 | Behavioral tasks and the two groups of subjects. (A) Orientation discrimination task. A trial started with a small red fixation point and subjects fixated on it. After a random delay, a grating appeared in upper visual field. Subjects had to press a left or right key to report the grating orientation (see Methods and Results for details). (B) Illustration of the grating orientations respect to the fixation point. The dashed circles indicate the grating possible locations across trials and green/red bar indicates grating with +45◦/−45◦. (C) Illustration of finger responses to the gratings in two groups. (D) Color discrimination task. A trial started with a small red fixation point and subjects fixated on it. After a random delay, a color patch (red or blue) appeared in upper visual field. Subjects had to press a left or right key to report the color of stimulus. FIGURE 1 | Behavioral tasks and the two groups of subjects. (A) Orientation discrimination task. A trial started with a small red fixation point and subjects fixated on it. After a random delay, a grating appeared in upper visual field. Subjects had to press a left or right key to report the grating orientation (see Methods and Results for details). (B) Illustration of the grating orientations respect to the fixation point. The dashed circles indicate the grating possible locations across trials and green/red bar indicates grating with +45◦/−45◦. (C) Illustration of finger responses to the gratings in two groups. (D) Color discrimination task. A trial started with a small red fixation point and subjects fixated on it. After a random delay, a color patch (red or blue) appeared in upper visual field. Subjects had to press a left or right key to report the color of stimulus SRC Effect Quantification Although the spatial locations of the grating were task- irrelevant, trials could be separated into SRC-compatible and SRC-incompatible conditions, based on the spatial relationship between the location of the grating and the direction of response key (finger). The SRC-compatible condition included trials in which the locations of grating and the response key were spatially compatible (e.g., gratings that appeared in the left VF were associated with left-key responses; gratings that appeared in the right VF were associated with right-key responses). In contrast, the SRC-incompatible condition included trials in which the grating and the response were spatially opposite (e.g., the gratings that appeared in the left VF were associated with right-key responses; gratings that appeared in the right VF were associated with left-key responses). The RT was calculated by using the same criteria as reported previously (Zhou et al., 2012, 2017). Briefly, we collected a total of 41,787 trials and excluded 6.18% of them in which subjects broke fixation or reported incorrectly, or the RTs differed from the mean by more than 3 standard deviations. For each subject, we calculated the mean of raw RT as well as the mean of normalized (differential) RT due to the fact of the existence of RT variation among subjects. Such RT variation may influence the results of statistical analysis. To calculate differential RT, we used both subtraction and division models. First, we set RTs responding to the grating (+45◦and −45◦) or patch (red and blue) appeared at the vertical meridian (straight ahead) as baseline condition, and then for each subject subtract/divide the mean RT at baseline condition from the mean RT at the other locations under same stimulus condition, respectively. Since the orientations of two gratings (+45◦and −45◦) at the vertical meridian location are in the middle between radial and tangential directions and there is no spatial SRC effect, the differential RTs predominately reflect the influence of radial effect on the temporal process of orientation discrimination. Frontiers in Neuroscience | www.frontiersin.org Reaction Times (RTs) for Orientation Discrimination Were Significantly Shorter When a Grating Was Oriented Closer to the Radial Direction than When It Was Oriented Closer to the Tangential Direction radial effect on RT, we need to exclude the influence of SRC. To do so, subjects were separated into two groups based on their subjective preference to the same grating as titled to left or right (Figure 1C). Group A contained 7 subjects who preferred the right-key response for the +45◦grating and the left-key response for the −45◦grating. Group B contained 11 subjects with the opposite preferences. Therefore, we created experimental conditions in which the SRC conditions were same for group A and group B subjects (same stimulus location and same key-press response), but the orientations of the gratings were opposite, with one being closer to the radial orientation and the other being closer to the tangential orientation (Figure 1B). The RT difference between two groups of subjects under the same SRC conditions predominately reflects the radial effect. To show more detailed results of radial effect on RTs, the data of differential RTs of two groups are arranged based on the key-response (manual response), i.e., pressing the same key but responding to the opposite gratings (+45◦vs. −45◦) in Figures 2A,B, respectively. Although the manual response is the same (Figure 2A, left-key response; Figure 2B, right-key response) and the spatial location of grating is the same (x-axis showing the horizontal eccentricities of grating), the distribution pattern of the differential RTs is very different between the two groups of subjects. Such difference could not be explained by the SRC effect, because the SRC conditions are identical between two groups of subjects in both Figures 2A,B. Thus, a reasonable explanation is that the differences in differential RT between two groups of subjects are mainly caused by the orientation of gratings (+45◦vs. −45◦). The differential RTs were shorter when the gratings were oriented closer to the radial direction than when they were oriented closer to the tangential direction in both left (solid line relative to dashed line) and right (dashed line relative to solid line) VFs. Magnitude of Radial Effect We defined the value of RT difference between two groups of subjects at each tested location as the magnitude of radial effect on orientation discrimination at this location. The magnitude of radial effect was calculated by comparing the absolute average RT difference between group A subjects and group B subjects. Frontiers in Neuroscience | www.frontiersin.org RESULTS The information of involvement of each subject in the experiments and the mean of raw RTs under different visual stimulation conditions (left VF vs. right VF) in orientation and color discrimination tasks are shown in Tables 1, 2, respectively. However, considering the fact that the large variation of RTs among individual subjects might affect the results of some statistical analyses, we calculated the differential RTs (see methods for details) for each subject and further used them in various statistical analyses. Since subtraction and division models showed consistent results, only data of differential RTs from subtraction model were shown thereafter. We further performed the post-hoc t-tests at each stimulated location to test the differential RT difference between two groups of subjects. For left-key response (Figure 2A, groups A subjects responding to the −45◦grating; group B subjects responding to +45◦grating), in the left VF the RTs of the group A subjects were significantly shorter at 3 of 4 locations [t-test, maximum p = 0.046, t(16) = −2.3] than that of the group B subjects; in TABLE 1 | The mean RTs under different conditions in orientation discrimination task. RT (ms) RT (ms) Subject Left-key Right-key Subject Left-key Right-key Group A LVF RVF LVF RVF Group B LVF RVF LVF RVF CY 567.3 594 571.5 563.5 GQ 622.3 585.4 648.3 614.4 DY 493 590.1 631.8 508.4 LLX 600 495.6 521.7 507 HZY 561.4 585 638 562.7 LD 620.8 579.6 636.2 592.9 LYY 469.6 506.6 560.5 500.2 LY 509.3 477.7 485.5 484 PY 555.7 568.3 617.1 495 LSK 487.7 458.7 498.3 491.9 YL 474.6 580.2 666.5 479.9 RSF 571.6 570.8 567.9 547.9 YYH 455.7 518.5 578.6 473.8 SJ 599.2 552.6 556.9 599.2 TRM 581.5 531.8 562.9 562.5 WYQ 460.2 446 455.3 426.5 WZY 424.2 421.1 442.9 407.8 XC 488.2 443.2 479.7 516.9 p g g g g p j p g +45◦grating), in the left VF the RTs of the group A subjects were significantly shorter at 3 of 4 locations [t-test, maximum p = 0.046, t(16) = −2.3] than that of the group B subjects; in TABLE 2 | The mean RTs under different conditions in color discrimination task. Radial Effect Quantification Considering the fact that subjects’ responses are highly influenced by the SRC effect (also known as the Simon effect) (Simon and Rudell, 1967; Wallace, 1971; Whitaker, 1982; Hommel, 2011; Styrkowiec and Szczepanowski, 2013), which results in the faster and more accurate responses in SRC compatible conditions than in SRC incompatible conditions. Thus, to quantify the November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 3 Liang et al. Radial Effect on Orientation Discrimination Reaction Times (RTs) for Orientation Discrimination Were Significantly Shorter When a Grating Was Oriented Closer to the Radial Direction than When It Was Oriented Closer to the Tangential Direction We employed the three-way ANOVA analysis [two key responses (left, right) × two gratings (+45◦, −45◦) × nine locations (−8◦, −6◦, −4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)] to test the significance of radial effect on RTs in the orientation discrimination task. The results of ANOVA analysis of the differential RTs showed a significant interaction between the grating orientations and grating locations [F(8, 216) = 31.8, p = 1.42e-35], and a significant interaction between the key-responses and grating locations [F(8, 216) = 11.5, p = 3.28e-14]. Reaction Times (RTs) for Orientation Discrimination Were Significantly Shorter When a Grating Was Oriented Closer to the Radial Direction than When It Was Oriented Closer to the Tangential Direction We employed the three-way ANOVA analysis [two key responses (left, right) × two gratings (+45◦, −45◦) × nine locations (−8◦, −6◦, −4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)] to test the significance of radial effect on RTs in the orientation discrimination task. The results of ANOVA analysis of the differential RTs showed a significant interaction between the grating orientations and grating locations [F(8, 216) = 31.8, p = 1.42e-35], and a significant interaction between the key-responses and grating locations [F(8, 216) = 11.5, p = 3.28e-14]. RESULTS RT (ms) RT (ms) Subject Left-key Right-key Subject Left-key Right-key Group A LVF RVF LVF RVF Group B LVF RVF LVF RVF CY 698.8 709 687.5 711.1 LLX 448.9 456.6 486.4 445.8 HZY 557 573.7 584 566.5 LD 637 691.2 695.4 657.2 LYY 495.9 529.2 541.2 533.3 LSK 472.6 476.6 520.4 495.4 YL 696.3 697.3 698.5 662.4 RSF 603.7 610.7 582.4 580.6 YYH 588.5 617.8 580.9 564.2 SJ 609.1 627.1 601.1 593.7 TRM 592.6 604.7 675.4 650.4 WZY 389.3 404.9 405.9 405.2 TABLE 1 | The mean RTs under different conditions in orientation discrimination task. RT (ms) RT (ms) Subject Left-key Right-key Subject Left-key Right-key Group A LVF RVF LVF RVF Group B LVF RVF LVF RVF CY 567.3 594 571.5 563.5 GQ 622.3 585.4 648.3 614.4 DY 493 590.1 631.8 508.4 LLX 600 495.6 521.7 507 HZY 561.4 585 638 562.7 LD 620.8 579.6 636.2 592.9 LYY 469.6 506.6 560.5 500.2 LY 509.3 477.7 485.5 484 PY 555.7 568.3 617.1 495 LSK 487.7 458.7 498.3 491.9 YL 474.6 580.2 666.5 479.9 RSF 571.6 570.8 567.9 547.9 YYH 455.7 518.5 578.6 473.8 SJ 599.2 552.6 556.9 599.2 TRM 581.5 531.8 562.9 562.5 WYQ 460.2 446 455.3 426.5 WZY 424.2 421.1 442.9 407.8 XC 488.2 443.2 479.7 516.9 TABLE 1 | The mean RTs under different conditions in orientation discrimination task. TABLE 2 | The mean RTs under different conditions in color discrimination task. RT (ms) RT (ms) Subject Left-key Right-key Subject Left-key Right-key Group A LVF RVF LVF RVF Group B LVF RVF LVF RVF CY 698.8 709 687.5 711.1 LLX 448.9 456.6 486.4 445.8 HZY 557 573.7 584 566.5 LD 637 691.2 695.4 657.2 LYY 495.9 529.2 541.2 533.3 LSK 472.6 476.6 520.4 495.4 YL 696.3 697.3 698.5 662.4 RSF 603.7 610.7 582.4 580.6 YYH 588.5 617.8 580.9 564.2 SJ 609.1 627.1 601.1 593.7 TRM 592.6 604.7 675.4 650.4 WZY 389.3 404.9 405.9 405.2 TABLE 2 | The mean RTs under different conditions in color discrimination task. November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 4 Liang et al. Radial Effect on Orientation Discrimination FIGURE 2 | The differential RTs and accuracies of two groups in orientation discrimination task. (A,B) The differential RTs are plotted against the grating horizontal eccentricity with left-key (A) and right-key (B) response. (C,D) The accuracies are plotted against the grating horizontal eccentricity with left-key (C) and right-key (D) response. RESULTS Similar results were observed from the RTs of right-key response (Figure 2B, groups A subjects responding to the +45◦ grating; group B subjects responding to −45◦grating). In the left VF, the RTs of the group B subjects were significantly shorter at 3 of 4 locations [t-test, maximum p = 0.042, t(16) = 2.2] than that of the group A subjects. In contrast, in the right VF, the RTs of the group A subjects were significantly shorter than that of the group B subjects at 1 of 4 locations [t-test, p = 0.010, t(16) = −2.3]. Overall, the differential RTs were significantly shorter when the grating orientation was closer to the radial direction than when it was closer to the tangential direction. RESULTS The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs/accuracies of group A and B, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs/accuracies of individual subject in group A and B, respectively. Asterisk denotes the results of post-hoc t-test between two groups: *P < 0.05, **P < 0.01, ***P < 0.001. FIGURE 2 | The differential RTs and accuracies of two groups in orientation discrimination task. (A,B) The differential RTs are plotted against the grating horizontal eccentricity with left-key (A) and right-key (B) response. (C,D) The accuracies are plotted against the grating horizontal eccentricity with left-key (C) and right-key (D) response. The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs/accuracies of group A and B, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs/accuracies of individual subject in group A and B, respectively. Asterisk denotes the results of post-hoc t-test between two groups: *P < 0.05, **P < 0.01, ***P < 0.001. [two key responses (left, right) × two gratings (+45◦, −45◦) × nine locations (−8◦, −6◦, −4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)] to analyze the variance of response accuracy between different experimental conditions. The results showed no significant interaction either between grating orientations and grating locations [F(8, 216) = 1.4, p = 0.18] or between key responses and grating locations [F(8, 216) = 1.4, p = 0.18]. Also, the post- hoc t-test at each location showed no significant difference in response accuracy between two groups of subjects in left-key response [Figure 2C, in left VF, minimum p = 0.17, t(16) = −1.5; in right VF, minimum p = 0.36, t(16) = −1.0]. Similar results were observed in right-key response condition [Figure 2D, in left VF, minimum p = 0.16, t(16) = −1.5; in right VF, minimum p = 0.25, t(16) = −1.2]. These results indicate that the radial effect on the temporal process of orientation discrimination is not due to SATO. contrast, in the right VF the RTs of the group B subjects were significantly shorter than that of the group A subjects at 2 of 4 locations [t-test, maximum p = 0.010, t(16) = 3.7]. The Radial Effect on RT in the Left VF Was Stronger than That in the Right VF It is commonly known that the speed of object discrimination could become faster by reducing the response accuracy, which is known as the SATO. To examine whether the radial effect on RT was due to SATO, we employed a three-way ANOVA To test whether the radial effect on RT is equally distributed in the left and right VFs, we performed a four-way ANOVA [two key responses (left, right) × two gratings (+45◦, −45◦) × four November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 5 Radial Effect on Orientation Discrimination Liang et al. eccentricities (2◦, 4◦, 6◦, 8◦) × two VFs (left, right)]. The results showed significant interaction among grating × eccentricity × VF for differential RTs [F(3, 192) = 38.1, p = 2.14e-20], which indicated the radial effect on RT is asymmetrically distributed between the left and right VFs. To further explore the spatial characteristics of radial effect on RT across VF, we estimated the magnitude of radial effect at each tested location by calculating the absolute RT difference between two groups of subjects under the same key response conditions. SRC effect on the unbalanced tuning curve, we mixed the radial effect of left-key (Figure 3A) and right-key (Figure 3B) response together correlated with the location of grating stimulation, and then the mean radial effect was calculated for each tested eccentric location. In average, the mixed radial effect was larger in the left VF than in the right VF (Figure 3C, the error bars will be described in next paragraph). The paired t-test of mixed radial effect between 4 tested locations in left VF and other 4 locations in right VF showed significant difference [t(3) = 3.5, p = 0.040]. However, considering the fact that the few number of samples (only four paired eccentric locations were tested between left and right VFs, respectively) might strongly bias the results of t-test, we used the following method to confirm the above result. We observed the “unbalanced U-shape” tuning curves of the magnitude of radial effect for both left-key and right-key response conditions (Figures 3A,B). The magnitude of radial effect was largest when the grating was either fully radial-oriented or fully tangential-oriented (the leftmost and rightmost locations) and became smaller as the grating became less radial-oriented and less tangential-oriented (closer to the vertical meridian). The RT Differences in Orientation Discrimination Task between Two Groups Were Not Caused by Subjects’ Difference in the Sensorimotor Transformation In the present study, the radial effect was quantified by calculating the RT differences between two groups. One intuitive argument is that the observed RT differences might be caused by intrinsic group differences in the sensorimotor process. Although this is very unlikely, we nevertheless performed a control experiment to test it. The Radial Effect on RT in the Left VF Was Stronger than That in the Right VF the mean radial effect of each subject (n = 11) at each tested eccentric location was calculated. Paired t-test of this mixed 9 × 11 matrix showed significant larger radial effect in the left VF than in the right VF [t(10) = 5.6, p = 2.25e-4]. Error bars in Figure 3C represents the mean standard deviation of these 11 subjects. differential RTs of group B show dramatically diminished SRC effect (Figure 4C). Two sample t-test confirmed that the differential RT differences in group A subjects were significantly greater than in group B subjects [t(16) = 5.0, p = 0.0010]. Thus, these results clearly showed that radial effect and SRC effect interact with each other to determine the overall RTs. Such asymmetric distribution of the radial effect was further confirmed by comparing the differential RT difference (radial effect) between mirror locations relative to the vertical meridian (Figure 3D), in which the mean radial effect with standard deviation at each tested eccentric location was calculated from the mixed 9 × 11 matrix. Paired t-test showed that the radial effect was stronger in the left VF than in the right VF at all tested off-middle locations [maximum p = 0.0035, t(10) = 3.8]. The RT Differences in Orientation Discrimination Task between Two Groups Were Not Caused by Subjects’ Difference in the Sensorimotor Transformation In the present study, the radial effect was quantified by calculating the RT differences between two groups. One intuitive argument is that the observed RT differences might be caused by intrinsic group differences in the sensorimotor process. Although this is very unlikely, we nevertheless performed a control experiment to test it. The Radial Effect on RT in the Left VF Was Stronger than That in the Right VF In addition, the radial effect tuning curve also differed between the left and right VFs in both left-key and right-key response conditions (Figures 3A,B). One possible reason is that the unbalanced tuning curve was, at least partially, due to the influence of SRC effect, i.e., shorter RTs in SRC compatible conditions and longer RTs in SRC incompatible conditions. To simply exclude the For each key-press condition, we first calculated the mean differential RT of group A subject (n = 7) at each tested location, and then calculated the absolute difference (radial effect) between this mean differential RT and differential RT of each group B subject (n = 11) at the same tested location. Thus, we created a 9 (locations) × 11 (subjects) matrix of data set for each key-press condition, respectively. To compare the radial effect between left and right VFs, these two 9 × 11 matrices were mixed together based on the eccentric location of grating stimulation, and then FIGURE 3 | The asymmetry of radial effect. (A,B) Tuning curve of the radial effect. The curve (through circles) represents the average of RT differences between two groups with left-key (A) and right-key (B) responses. (C) The radial effect between left and right VFs. (D) The radial effect between left and right VFs across each tested horizontal eccentricity. The gray curve (through gray circles) and black curve (through black circles), respectively represent the average of the radial effect in left and right VFs across each tested horizontal eccentricity. All the error bars denote ±SD. Asterisk denotes the results of paired t-test: **p < 0.01; ***P < 0.001. FIGURE 3 | The asymmetry of radial effect. (A,B) Tuning curve of the radial effect. The curve (through circles) represents the average of RT differences between two groups with left-key (A) and right-key (B) responses. (C) The radial effect between left and right VFs. (D) The radial effect between left and right VFs across each tested horizontal eccentricity. The gray curve (through gray circles) and black curve (through black circles), respectively represent the average of the radial effect in left and right VFs across each tested horizontal eccentricity. All the error bars denote ±SD. Asterisk denotes the results of paired t-test: **p < 0.01; ***P < 0.001. November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 6 Radial Effect on Orientation Discrimination Liang et al. The Radial Effect and SRC Effect Interact to Determine RT We employed a color discrimination task to test the similarity of sensorimotor process between two groups. In this task, the test stimuli and response strategy were exactly same for two groups of subjects (see details in the Methods). First, SRC effect was confirmed by a two-way ANOVA analysis [two key responses (left, right) × two VFs (left, right)]. The results showed a significant interaction between key response and VF for the differential RTs [F(1, 11) = 32.0, p =1.47e-04]. Second, the differential RTs were very similar between two groups for both left-key (Figure 5A) and right-key (Figure 5B) responses [left-key response: one-way ANOVA, 2 groups, F(1, 8) = 0.4, p = 0.53; right-key response: one-way ANOVA, 2 groups, F(1, 8) = 0.0, p = 0.89]. Also, the differences of differential RTs in all tested off-middle locations did not reach the significant level [t-test, minimum p = 0.25, t(10) = −1.2]. Thus, the difference in differential RTs between the two groups in orientation discrimination task was not due to the differences in sensorimotor process between subjects. A line of studies has found that the behavioral performance is faster and more accurate in SRC-compatible conditions than in SRC-incompatible conditions (Fitts and Seeger, 1953; Wallace, 1971; Whitaker, 1982), a finding also known as the Simon effect (Simon and Rudell, 1967; Hommel, 2011; Styrkowiec and Szczepanowski, 2013). We have shown earlier that the SRC effect strongly modulated the radial effect (Figures 2A,B). Here, we employed a three-way ANOVA analysis [two key responses (left, right) × two gratings (+45◦, −45◦) × nine locations (−8◦, −6◦, −4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)] to statistically test the interaction between radial effect and SRC effect. The results showed that the interaction between radial effect and SRC (key × grating × location) was significant [F(8, 216) = 2.7, p = 0.0069]. Furthermore, the influence of SRC to radial effect was very different between the two SRC conditions, as shown in Figure 4A (the averaged radial effect of left and right 4 off-middle locations in Figures 3A,B, respectively). The averaged radial effect was greater in the SRC-incompatible condition than in the SRC- compatible condition in both left and right key responses. Two sample t-test resulted the significant difference in radial effect between two SRC conditions [left key response, t(10) = −2.4, p = 0.037; right key response, t(10) = 6.6, p = 5.81e-05]. Frontiers in Neuroscience | www.frontiersin.org DISCUSSION consistent with the previously reported perceptual radial effect (Rovamo et al., 1982; Bennett and Banks, 1991; Westheimer, 2003; Sasaki et al., 2006), the former is not a passive reflection of the latter because under our experimental condition, there was no significant difference in perceptual accuracy between the more radial and more tangential orientations. locations in the upper VF, we produced more radial and more tangential orientations of various degrees. In this way, we were able to measure the radial effect by comparing the two equally oblique orientations and avoid the confound of the oblique effect. locations in the upper VF, we produced more radial and more tangential orientations of various degrees. In this way, we were able to measure the radial effect by comparing the two equally oblique orientations and avoid the confound of the oblique effect. A byproduct of the present study is that the RT radial effect is asymmetric between the left and right VFs: the effect is larger in the left VF than in the right VF (Figures 3C,D). Such results are consistent with findings of many previous studies (Heilman and Van Den Abell, 1979; Sturm et al., 1989; Corballis et al., 2002; Boulinguez et al., 2003; Corballis, 2003; Okubo and Nicholls, 2008; Zhou et al., 2012), which indicates that the right hemisphere of human brain plays a dominant role in processing spatial information than the left hemisphere does. It has been reported that the spatial information is dominantly processed in the left VF by the right hemisphere (e.g., orientation perception), whereas the non-spatial information (e.g., temporal perception) A byproduct of the present study is that the RT radial effect is asymmetric between the left and right VFs: the effect is larger in the left VF than in the right VF (Figures 3C,D). Such results are consistent with findings of many previous studies (Heilman and Van Den Abell, 1979; Sturm et al., 1989; Corballis et al., 2002; Boulinguez et al., 2003; Corballis, 2003; Okubo and Nicholls, 2008; Zhou et al., 2012), which indicates that the right hemisphere of human brain plays a dominant role in processing spatial information than the left hemisphere does. DISCUSSION The differential RT is plotted against the grating horizontal eccentricity in group A (B) and B (C). The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key responses, respectively. Asterisk denotes the results of post-hoc paired t-tests between left-key and right-key responses in each group: *P < 0.05, **P < 0.01, ***P < 0.001. FIGURE 4 | Mutual interaction between the radial effect and SRC effect. (A) The influence of SRC effect on the radial effect. The radial effect is shown in the SRC-compatible (gray) and SRC-incompatible (black) conditions with left-key and right-key response, respectively. The error bars denote SD. (B,C) The influence of the radial effect on SRC effect. The differential RT is plotted against the grating horizontal eccentricity in group A (B) and B (C). The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key responses, respectively. Asterisk denotes the results of post-hoc paired t-tests between left-key and right-key responses in each group: *P < 0.05, **P < 0.01, ***P < 0.001. FIGURE 5 | The differential RTs in color discrimination task. The differential RT is plotted against the color patch horizontal eccentricity with left-key (A) and right-key (B) response. The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key responses, respectively. FIGURE 5 | The differential RTs in color discrimination task. The differential RT is plotted against the color patch horizontal eccentricity with left-key (A) and right-key (B) response. The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key responses, respectively. DISCUSSION Orientation processing is a key element of visual perception. Despite the intuitive view that orientation processing is isotropic, it has been reported that human subjects exhibit perceptual differences for different orientations, including the oblique effect (Appelle, 1972; Berkley et al., 1975), the cardinal effect (Rijsdijk et al., 1980; Carrasco et al., 2001), and the radial effect (Rovamo et al., 1982; Bennett and Banks, 1991; Westheimer, 2003; Sasaki et al., 2006). Different from previous radial effect studies, which focused on comparing orientation sensitivity and accuracy between radial and tangential orientations, we systematically assessed the radial effect in term of the processing time along the sensorimotor pathway by measuring the manual RT to a +45◦or −45◦grating. The grating appeared randomly at one of nine equally spaced locations in the upper VF so that its orientation with respect to the fixation point is radial, tangential or somewhere in-between (Figure 1B). We found significantly shorter RTs to gratings closer to radial orientation than those closer to tangential orientation. Although this RT radial effect was On the other hand, the radial effect strongly affected the SRC effect too. Based on the relationship between the radial effect and SRC effect, trials could be separated into two types (Figure 1C): radial effect and SRC effect matched trials (group A: radially-oriented conditions were associated with SRC- compatible conditions and tangentially-oriented conditions were associated with SRC-incompatible conditions), and radial effect and SRC effect non-matched trials (group B: radial-oriented conditions were associated with SRC-incompatible conditions and tangential-oriented condition were associated with SRC- compatible condition). Although the SRC conditions were same, the distributions of the differential RTs were dramatically different between group A subjects (radial effect and SRC effect match, Figure 4B) and group B subjects (radial effect and SRC non-match, Figure 4C). While differential RTs of group A subjects showed clear SRC effect (circles are smaller than triangles in the left VF and vice versa in the right VF, Figure 4B), November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 7 Radial Effect on Orientation Discrimination Liang et al. FIGURE 4 | Mutual interaction between the radial effect and SRC effect. (A) The influence of SRC effect on the radial effect. The radial effect is shown in the SRC-compatible (gray) and SRC-incompatible (black) conditions with left-key and right-key response, respectively. The error bars denote SD. (B,C) The influence of the radial effect on SRC effect. Frontiers in Neuroscience | www.frontiersin.org DISCUSSION It has been reported that the spatial information is dominantly processed in the left VF by the right hemisphere (e.g., orientation perception), whereas the non-spatial information (e.g., temporal perception) When studying the radial effect, one must control for potential confound from another well-known phenomenon, the oblique effect, according to which the perception of cardinal orientations is superior to that of oblique orientations (Appelle, 1972; Berkley et al., 1975; Furmanski and Engel, 2000). Because of the oblique effect, we cannot assess the radial effect by comparing different orientations that include both more cardinal and more oblique orientations at a given location in the VF. Instead, we only used gratings of +45◦and −45◦orientations which are equally oblique. By presenting them at one of the nine equally spaced November 2017 | Volume 11 | Article 638 8 Radial Effect on Orientation Discrimination Liang et al. is dominantly processed in the right VF by the left hemisphere (Galati et al., 2010). In contrast, it has been also reported that the process of orientation perception was faster in the right VF than in the left VF when there were competing orienting stimuli appeared in the left and right VFs simultaneously (Carlei and Kerzel, 2017). Indeed, when we put all differential RTs of two groups of subjects together and then separate them to two groups based on the grating’s location either in the left or right VF, one-way ANOVA resulted that the effect of VF–RT was significant [F(1, 17) = 5.5, p = 0.020]. The mean differential RTs were significant faster in the right VF than in the left VF [post- hoc paired t-test, t(17) = 3.8, p = 0.0013], which was consistent with the above finding of shorter RT in the right VF than in the left VF. the RTs at several tested locations and in certain trial conditions (SRC-compatible condition and closer to radial orientation) are shorter than the RT in the baseline condition (grating in vertical meridian, with shortest eccentricity). Thus, it is reasonable to predict that the radial effect should be more obvious if the tested locations are distributed with an equal eccentricity. Thus, our results indeed reflect the influence of the radial effect and SRC effect on RT. Our results indicate that multiple factors interact to determine the overall RT. DISCUSSION We summarize our finding with the following simplified model: RT (stimulus, response, location) = Baseline + Radial effect (stimulus, response) + SRC effect (stimulus, response) + interaction (Radial and SRC effects) + VF (location). It would be interesting to investigate the neural mechanisms underlying these factors and their interactions. We found that the radial effect and SRC effect strongly interact with each other to determine RT. The radial effect was significantly larger in the SRC-incompatible condition than in the SRC-compatible condition, in both the left and right VFs (Figure 4A). Additionally, the RT difference between SRC- incompatible and SRC-compatible conditions was larger when the radial and SRC effects were matched than when they were unmatched (Figures 4B,C). Further studies are needed to explore the neural mechanisms underlying the interaction between the two effects. ACKNOWLEDGMENTS We thank Dr. Ning Qian of Columbia University, New York, USA, for his helpful suggestions and comments. FUNDING This study is supported by the following foundations: The Opening Foundation of State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (SKLN-2010A05 and SKLN-201203) awarded to YP; The Opening Foundation of State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University (CNLZD1303) awarded to YP. We measured the radial effect by comparing the RTs between two groups of subjects. It is thus important to rule out systematic differences in sensorimotor transformation between the two groups. To this end, we showed that the two groups of subjects had very similar RTs in a control task on color discrimination (Figure 5). One may argue that the RT difference among nine tested locations in the present study might be due to the different visual sensitivity, because the nine tested peripheral locations had different eccentricities (Figure 1B). However, our results cannot be explained by the variation of visual sensitivity with eccentricity. As shown in Figures 2A,B, 4B,C, for some subjects, AUTHOR CONTRIBUTIONS YZ designed the experiments. LL and YZ collected and analyzed the data. MZ and YP supervised the experiments. All authors are involved in writing the manuscript. REFERENCES M., and Van Den Abell, T. (1979). Right hemispheric dominance for mediating cerebral activation. Neuropsychologia 17, 315–321. doi: 10.1016/0028-3932(79)90077-0 Simon, J. R., and Rudell, A. P. (1967). Auditory S-R compatibility: the effect of an irrelevant cue on information processing. J. Appl. Psychol. 51, 300–304. doi: 10.1037/h0020586 Sturm, W., Reul, J., and Willmes, K. (1989). 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Psychol. 9, 202–215. doi: 10.5709/acp-0146-5 Wallace, R. J. (1971). S-R compatibility and the idea of a response code. J. Exp. Psychol. 88, 354–360. doi: 10.1037/h0030892 Matthews, N., Meng, X., Xu, P., and Qian, N. (2003). A physiological theory of depth perception from vertical disparity. Vis. Res. 43, 85–99. doi: 10.1016/S0042-6989(02)00401-7 Westheimer, G. (2003). The distribution of preferred orientations in the peripheral visual field. Vis. Res. 43, 53–57. doi: 10.1016/S0042-6989(02)00398-X Okubo, M., and Nicholls, M. E. (2008). Hemispheric asymmetries for temporal information processing: transient detection versus sustained monitoring. Brain Cogn. 66, 168–175. doi: 10.1016/j.bandc.2007. 07.002 Whitaker, L. A. (1982). Stimulus-response compatibility for left-right discriminations as a function of stimulus position. J. Exp. Psychol. Hum. Percept. Perform. 8, 865–874. doi: 10.1037/0096-1523.8.6.865 Zhou, Y., Liang, L., Pan, Y., Qian, N., and Zhang, M. (2017). Sites of overt and covert attention define simultaneous spatial reference centers for visuomotor response. Sci. Rep. 7:46556. doi: 10.1038/srep46556 Pelli, D. G. (1997). The VideoToolbox software for visual psychophysics: transforming numbers into movies. Spat. Vis. 10, 437–442. doi: 10.1163/156856897X00366 Zhou, Y., Liu, Y., Zhang, W., and Zhang, M. (2012). Asymmetric influence of egocentric representation onto allocentric perception. J. Neurosci. 32, 8354–8360. doi: 10.1523/JNEUROSCI.0829-12.2012 Rijsdijk, J. P., Kroon, J. N., and van der Wildt, G. J. (1980). REFERENCES frequency, eccentricity, task and set size. Spat. Vis. 15, 61–75. doi: 10.1163/15685680152692015 frequency, eccentricity, task and set size. Spat. Vis. 15, 61–75. doi: 10.1163/15685680152692015 Appelle, S. (1972). Perception and discrimination as a function of stimulus orientation: the “oblique effect” in man and animals. Psychol. Bull. 78, 266–278. doi: 10.1037/h0033117 Corballis, P. M. (2003). 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Grating visibility as a function of orientation and retinal eccentricity. Vis. Res. 15, 239–244. doi: 10.1016/0042-6989(75)90213-8 Furmanski, C. S., and Engel, S. A. (2000). An oblique effect in human primary visual cortex. Nat. Neurosci. 3, 535–536. doi: 10.1038/75702 Boulinguez, P., Ferrois, M., and Graumer, G. (2003). Hemispheric asymmetry for trajectory perception. Brain Res. Cogn. Brain Res. 16, 219–225. doi: 10.1016/S0926-6410(02)00276-8 Galati, G., Pelle, G., Berthoz, A., and Committeri, G. (2010). Multiple reference frames used by the human brain for spatial perception and memory. Exp. Brain Res. 206, 109–120. doi: 10.1007/s00221-010-2168-8 Brainard, D. H. (1997). The psychophysics toolbox. Spat. Vis. 10, 433–436. doi: 10.1163/156856897X00357 Goodale, M. A. (2014). How (and why) the visual control of action differs from visual perception. Proc. Biol. Sci. 281:20140337. doi: 10.1098/rspb.2014. 0337 Carlei, C., and Kerzel, D. (2017). Stronger interference from distractors in the right hemifield during visual search. Laterality. doi: 10.1080/1357650X.2017.1327539. [Epub ahead of print]. Goodale, M. A., and Milner, A. D. (1992). Separate visual pathways for perception and action. Trends Neurosci. 15, 20–25. doi: 10.1016/0166-2236(92)90 344-8 Carrasco, M., Talgar, C. P., and Cameron, E. L. (2001). Characterizing visual performance fields: effects of transient covert attention, spatial November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 9 Radial Effect on Orientation Discrimination Liang et al. Heilman, K. Frontiers in Neuroscience | www.frontiersin.org November 2017 | Volume 11 | Article 638 REFERENCES Contrast sensitivity as a function of position on the retina. Vis. Res. 20, 235–241. doi: 10.1016/0042-6989(80)90108-X Rovamo, J., Virsu, V., Laurinen, P., and Hyvarinen, L. (1982). Resolution of gratings oriented along and across meridians in peripheral vision. Invest. Ophthalmol. Vis. Sci. 23, 666–670. 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. Sasaki, Y., Rajimehr, R., Kim, B. W., Ekstrom, L. B., Vanduffel, W., and Tootell, R. B. (2006). The radial bias: a different slant on visual orientation sensitivity in human and nonhuman primates. Neuron 51, 661–670. doi: 10.1016/j.neuron.2006.07.021 Copyright © 2017 Liang, Zhou, Zhang and Pan. 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. Schall, J. D., Perry, V. H., and Leventhal, A. G. (1986). Retinal ganglion cell dendritic fields in old-world monkeys are oriented radially. Brain Res. 368, 18–23. doi: 10.1016/0006-8993(86)91037-1 November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 10
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ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA
Jurnal Ilmu Ekonomi
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Abstract The purpose of this study is to analyze what factors affect foreign exchange reserves in Indonesia. The focus in this study is the variables that affect changes in foreign exchange reserves in the 1999-2019 period are non-oil and gas exports, the rupiah exchange rate, foreign debt and inflation as independent variables. Using an open economy model and Keynes's balance of payments, the analytical tools used in this study are the PAM regression model (Partial Adjustment Model) and the adaptive expectation model to see the long-term and short-term effects of the independent variable on the dependent variable. The results of this study show that simultaneously non-oil and gas exports, the rupiah exchange rate, foreign debt and inflation affect Indonesia's foreign exchange reserves, while partially non-oil exports and the rupiah exchange rate significantly and positively affect Indonesia's long-term and short-term foreign exchange reserves, while debt foreign exchange and inflation are not significant and negatively affect long-term and short-term foreign exchange reserves. Non-oil exports, rupiah exchange rate, foreign debt and inflation can explain Indonesia's foreign exchange reserves of 97%, of which the remaining 3% is explained by other variables not included in this study. ANALISIS FAKTOR-FAKTOR YANG MEM CADANGAN DEVISA DI INDONESIA Norita Fitria*, Aris Soelistyo, Dwi Susilowati Program Studi Ekonomi Pembangunan, Fakultas Ekonomi dan Bissnis, Universitas Muhammadiyah Malang, Indonesia *Corresponding Author: noritafityaaa@gmail.com p g y Artikel Info Article history: Received 14 April 2021 Revised 18 August 2021 Accepted 30 August 2021 Available online 31 August 2021 Keywords: Cadangan Devisa, Ekspor Non Migas, Nilai Tukar Rupiah, Utang Luar Negeri, Inflasi Jurnal Ilmu Ekonomi (JIE) Vol. 5, No. 3, August 2021, pp. 451-460 PENDAHULUAN Indonesia merupakan negara berkembang dimana keadaan perekonomian masih di pengaruhi oleh adanya tekanan eksternal dari negara- negara maju yang berpengaruh terhadap keadaan perekonomian dunia hal ini di sebabkan karena Indonesia menganut sistem perekonomian terbuka. Keadaan ekonomi dunia yang determinasinya merupakan keadaan perekonomian negara-negara maju menyebabkan Indonesia sebagai negara berkembang yang merasakan dampak secara langsung atas kebijakan yang telah di tetapkan, salah satu dampak yang di rasakan yakni tidak stabilnya nilai mata uang, tingkat investasi asing yang menurun yang selanjutnya akan berdampak terhadap tingkat produksi hal ini juga dapat menghambat pembangunan di Indonesia yang dimana peranan ekonomi sangat penting untuk dapat melakukan pembangunan. (Asmanto and Suryandari 2009). Peranan Cadangan devisa sangat penting untuk menompang perekonomian negara karena Cadangan Devisa sendiri di gunakan untuk menjaga kestabilan negara saat adanya tekanan eksternal yang tidak terduga maupun tekanan dari dalam negeri yang berdampak pada ketahanan makroekonomi fungsinya untuk suatu Negara adalah sebagai tolak ukur kekayaan yang di miliki oleh Negara itu sendiri, memiliki cadangan devisa yang cukup sangat penting yang fungsinya untuk mendorong kegiatan perdagangan internasional kemudian fungsi untuk menjaga ketahanan nilai mata uang ketika adanya tekanan eksternal yang tidak terduga dan fungsi sebagai jenis kekayaan. Ketika neraca 451 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA pembayaran internasional mengalami defisit maka cadangan devisa akan di gunakan untuk melakukan pembayaran impor dan utang luar negeri sehingga neraca pembayaran kembali stabil. Untuk menjaga kestabilan moneter, cadangan devisa di gunakan untuk melakukan intervensi dalam pasar valuta asing untuk menjaga ketahanan nilai mata uang. Besaran penambahan cadangan devisa suatu Negara di tentukan oleh kemampuan Negara tersebut dalam melakukan kegiatan perdagangan internasional dan sistem nilai tukar yang di tetapkan. Di Indonesia sistem nilai tukar yang di tetapkan adalah sistem nilai tukar mengambang, jika nilai tukar berfluktuasi atau mengalami depresiasi maka cadangan devisa di gunakan untuk mengembalikan kestabilan nilai tukar dengan cara intervensi terhadap pasar valuta asing yang di lakukan oleh Bank Indonesia untuk mencapai nilai tukar yang di harapkan dan menjaga nilai tukar rupiah, tujuannya agar dapat menjaga laju inflasi dalam jangka menengah. Besaran cadangan devisa yang di miliki suatu Negara juga di pengaruhi oleh derajat keterbukaan ekonomi suatu Negara, sistem perekonomian yang terbuka akan mendorong transaksi perdagangan internasional, sehingga permintaan devisa akan semakin besar hal ini akan menyebabkan terdapat risiko ketimpangan neraca pembayaran internasional, untuk mencegah hal tersebut terjadi maka pengelolaan cadangan devisa, pembayaran impor dan utang luar negeri sangat penting untuk pencegahan krisis. PENDAHULUAN Di dalam perekonomian adanya utang luar negeri jangka pendek mempresentasikan tingkat kecukupan cadangan devisa Sehingga dalam hal ini ketersediaan Cadangan Devisa dapat membantu negara untuk mecapai kestabilan moneter dan ketahanan perekonomian negara. Sehingga pengelolaan terhadap cadangan devisa sangat penting untuk terus menjaga posisi cadangan devisa dengan cara melakukan pendorongan akan faktor- faktor yang dapat menambah cadangan devisa yang di miliki. Sehingga Indonesia dapat terus melakukan pembangunan dan menjaga kestabilan perekonomian, yang secara langsung yang akan berdampak terhadap ketahanan ekonomi ketika ada tekanan yang tidak terduga yang dapat berasal dari pasar keuangan luar negri maupun tekanan dari keuangan dalam negeri (Gandhi 2006; Tambunan 2000). Tabel 1. Cadangan Devisa Tahun 2010-2019 (US$) Tahun Cadangan Devisa Indonesia 2010 96,210,980,584.043 2011 110,136,597,662.435 2012 112,797,627,833.066 2013 99,386,826,239.083 2014 111,862,594,561.997 2015 105,928,847,088.719 2016 116,369,601,851.059 2017 130,215,330,382.957 2018 120,660,974,090.950 2019 129,186,464,020.187 Tabel 1. Cadangan Devisa Tahun 2010-2019 (US$) 452 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA Berdasarkan Tabel di atas dapat di lihat selama beberapa tahun Cadangan Devisa di Indonesia mengalami fluktuasi, hal tersebut dapat membahayakan kestabilan ekonomi di Indonesia apabila terjadi penurunan secara drastis dan terus menerus sehingga pengelolaan cadangan devisa sangat penting. Ketersediaan Cadangan Devisa yang di miliki tentunya di pengaruhi oleh beberapa faktor sehingga dalam hal ini untuk dapat menjaga ketahanan posisi cadangan devisa maka pendeteksian akan faktor-faktor apa saja yang dapat menambah cadangan devisa yang di miliki harus di lakukan. Penelitian yang di lakukan oleh (Kuswantoro 2017) Pengaruh Inflasi, Kurs Rupiah, Utang Luar Negri dan Ekspor Terhadap Cadangan Devisa. Adapun hasil penelitian Secara Simultan Kurs Rupiah, Utang Luar negri dan Ekspor berpengaruh Positif terhadap Cadangan Devisa. Sedangkan secara parsial Inflasi negatif tidak berpengaruh terhadap cadangan devisa dan secara parsial Kurs Rupiah, Utang Luar Negri dan Ekspor berpengaruh positif terhadap Cadangan Devisa. Selain itu hasil analisis koefisien determinasi menampilkan bahwa Cadangan Devisa dapat dipaparkan oleh variabel Inflasi, Kurs Rupiah, Utang Luar negri dan Ekspor sebesar 91,25%. Penelitian oleh(Rochman 2015b) Pengaruh Inflasi, Kurs, utang Luar Negri dan ekspor Terhadap Cadangan Devisa. Adapun perolehan hasil penelitian menunjukkan secara simultan Inflasi, Kurs, Utang Luar Negri dan Ekspor berpengaruh positif terhadap Cadangan Devisa. Sedangkan secara Parsial Inflasi negatif tidak berpengaruh terhadap Cadangan Devisa dan Kurs, Utang Luar Negri dan Ekspor berpengaruh positif terhadap Cadangan Devisa. Adapun hasil koefisien determinasi menunjukkan Cadangan Devisa dapat di paparkan oleh variabel Inflasi, Kurs, Utang Luar Negri dan Ekspor sebesar 91,25%. PENDAHULUAN Penelitian oleh(Sayoga and Tan 2017) Analisis Cadangan Devisa dan Faktor-faktor Yang Mempengaruhinya. Adapun hasil penelitian menggunakan teknik deskriptif menujukkan Cadangan Devisa tertinggi terletak pada tahun 2010. Sedangkan hasil teknik analisis kuantitafi menunjukkan secara simultan Ekspor, Nilai Tukar Rupiah, Utang Luar Negeri menunjukkan terdapat hubungan signifikan terhadap Cadangan Devisa. Secara Parsial Utang Luar Negri dan Ekspor terdapat hubungan positif terhadap Cadangan Devisa sedangkan untuk Nilai Tukar Rupiah terdapat hubungan negatif terhadap cadangan devisa. Relevansi penelitian terdahulu dengan penelitian ini adalah faktor yang mempengaruhi cadangan devisa sedangkan untuk gap penelitian ini adanya penggunaan model dan alat analisis yang berbeda serta keterbaruan tahun data. Sehingga dapat di lihat pengaruh jangka pendek dan jangka panjang ekspor non migas, nilai tukar rupiah, utang luar negeri dan inflasi terhadap cadangan devisa di Indonesia. METODE PENELITIAN Penelitian ini menggunakan pendekatan deskriptif kuantitatif dengan penentuan obyek penelitian di Indonesia ekspor non migas, nilai tukar rupiah, utang luar negri dan inflasi sebagai variabel bebas dan cadangan devisa sebagai variabel terikat. Sumber data yang di gunakan merupakan data sekunder yang dimana data tersebut di peroleh dari publikasi data Badan Pusat Statistik (BPS) Indonesia dan data World Bank (Bank Dunia) dengan teknik pengumpulan 453 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA data yang di gunakan yaitu dengan cara dokumentasi, yang kemudian akan di olah menjadi data Time Series dengan kurun waktu 21 tahun dari 1999-2019. Untuk mencapai tujuan penelitian di gunakan model ekonometrika dengan Analisis Regresi Partial Adjustment Model (PAM) untuk memperoleh persamaan jangka pendek dan Model ekspektasi adaptif untuk memperoleh persamaan jangka panjang selain itu untuk melihat kelayakan data maka akan di lakukan Uji Asumsi Klasik. data yang di gunakan yaitu dengan cara dokumentasi, yang kemudian akan di olah menjadi data Time Series dengan kurun waktu 21 tahun dari 1999-2019. Untuk mencapai tujuan penelitian di gunakan model ekonometrika dengan Analisis Regresi Partial Adjustment Model (PAM) untuk memperoleh persamaan jangka pendek dan Model ekspektasi adaptif untuk memperoleh persamaan jangka panjang selain itu untuk melihat kelayakan data maka akan di lakukan Uji Asumsi Klasik. Regresi Partial Adjustment Model (PAM) untuk estimasi jangka pendek dalam penelitian ini akan menggunakan formulasi sebagai berikut (Basuki 2014): 𝐷𝐸𝑉𝑡= 𝛽0 + 𝛽1 + 𝐿𝑂𝐺(𝐸𝐾𝑆)𝑡+ 𝛽2𝐿𝑂𝐺(𝐾𝑈𝑅𝑆)𝑡+ 𝛽3𝐿𝑂𝐺(𝐷𝐸𝐵𝑇)𝑡+ 𝛽4𝐿𝑂𝐺(𝐼𝑁𝐹)𝑡+ 𝛽5𝐿𝑂𝐺(𝐷𝐸𝑉)𝑡−1 + 𝜀𝑡 ………………… (1) 𝐷𝐸𝑉𝑡= 𝛽0 + 𝛽1 + 𝐿𝑂𝐺(𝐸𝐾𝑆)𝑡+ 𝛽2𝐿𝑂𝐺(𝐾𝑈𝑅𝑆)𝑡+ 𝛽3𝐿𝑂𝐺(𝐷𝐸𝐵𝑇)𝑡+ 𝛽4𝐿𝑂𝐺(𝐼𝑁𝐹)𝑡+ 𝛽5𝐿𝑂𝐺(𝐷𝐸𝑉)𝑡−1 + 𝜀𝑡 ………………… (1) 𝛽4 ( )𝑡 𝛽5 ( )𝑡1 𝑡 ( ) Model matematika untuk estimasi jangka panjang dengan model Ekspektasi adaptif(Damondaar N. Gujarati 2007): … (2) ) 𝑋𝑡 ∗−𝑋𝑡−1 ∗ = 𝛾(𝑋𝑡 ∗−𝑋𝑡−1 ∗ ) ……………… (2) HASIL DAN PEMBAHASAN Uji normalitas sendiri di butuhkan karena untuk melihat apakah data berdistribusi normal dan tidak bias. Uji normalitas tidak hanya di gunakan untuk melihat probabilitas selain itu dapat di gunakan untuk melakukan uji t, uji f dan r-square yang selanjutnya terdapat di regresi. Dalam penelitian ini Nilai probability menunjukkan 0,272453 dimana lebih besar dari 0,05 maka artinya error berdistribusi normal. Tabel 2 Hasil Uji Normaalitas Jarque-bera Probabilitas 0,272453 Tabel 2 Hasil Uji Normaalitas Jarque-bera Probabilitas 0,272453 Tabel 2 Hasil Uji Normaalitas Jarque-bera Probabilitas 0,272453 Uji Multikolinearitas pada dasarnya adalah adanya hubungan linear di dalam variabel penjelas. Di dalam kasus multikolinearitas yang terjadi di dalam time series biasanya di sebabkan oleh adannya common trends di dalam variabel bebas yang menyebabkan dapat dapat menurun ataupun meningkat dalam kurun waktu tertentu. Penelitian ini untuk mengetahui apakah adanya multikolinearitas maka di gunakan model koefisien determinasi regresi auxiliary dimana apabila nilai koefisien determinasi menunjukkan lebih dari 0,97 maka terdapat multikolinearitas. Dari hasil estimasi model koefisien determinasi regresi auxiliary dapat di ketahui bahwa koefisien determinasi auxiliary variabel bebas ekspor non migas, nilai tukar rupiah, utang luar negeri dan inflasi tidak ada yang lebih besar dari koefisien determinasi model utaman cadangan devisa (y) dengan demikian dapat di katakan tidak terjadi multikolinearitas di dalam data ini. Tabel 3 Hasil Uji Multikolinearitas Variabel Bebas Nilai Koefisien Determinasi Ekspor Non Migas 0,68 Nilai Tukar Rupiah 0,64 454 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA Variabel Bebas Utang Luar Negeri Inflasi Nilai Koefisien Determinasi Uji Heteroskedastisitas sendiri adalah masalah yang terdapat dalam regresi dimana terdapat sifat homo atau sama dalam variabel. Dalam penelitian ini untuk mendeteksi heteroskedastisitas maka di gunakan model glejser dimana apabila probanilitas chi-square yang di peroleh dari R-square auxiliary lebih dari 0,05 maka tidak terdapat heteroskedatisitas atau apabila nilai probabilitas chi-square kurang dari 0,05 maka terdapat heteroskedastisitas. Berdasarkan uji glejser di peroleh nilai prob chi sebesar 0,8858 dimana nilai tersebut lebih besar dari 0,05. Dengan demikian tidak terdapat heteroskedasitas. bel 4 Hasil Uji Heteroskedastisitas Probabilitas Chi-square 0,8858 Uji Autokorelasi sering terdapat di dalam data time series karena adanya korelasi antar variabel dalam penelitian sebelumnya dimana variabel tersebut tergantung dengan data tahun-tahun sebelumnya. Dalam penelitian ini di gunakan model LM Breusch-godfrey untuk mendeteksi autokorelasi, dalam penelitian ini menggunakan Lag untuk mendeteksi perbandingan nilai antar Akaike dengan Schwarz yang paling kecil. Berdasarkan output uji LM Breusch godfrey untuk berbagai panjang lag, lag optimum yang memiliki AIC dan SIC minimum adalah lag 1. Dari output di peroleh prob chi (1) = 30,37206. Nilai tersebut lebih besar dari 0,05 sehingga tidak terjadi autokorelasi pada data. Tabel 5 Hasil Uji Autokorelasi Lag 1 Lag 2 Lag 3 Lag 4 Lag 5 AIC 30,37206 30,41157 30,49847 30,56373 30,64933 SIC 30,67050 30,75974 30,89639 31,01139 31,14673 Hasil Uji Regresi Partial Adjutment Model (PAM) Tabel 6 Hasil Uji PAM Variable Coefficient t-statistic probability LOG(EKS) 0.559055 1233189 0.0000 LOG(KURS) 0.060897 2126704 0.0494 LOG(ULN) -0.003674 -0.650335 0.9380 LOG(INF) -0.142250 -1.831627 0.1119 LOG(DEV(-1)) 0.433503 2073461 0.0000 C 1.945.567 0.917698 0.3743 R-Squared 0.966371 Adjusted 0.957964 F-Statistic 114.9446 Tabel 5 Hasil Uji Autokorelasi Lag 1 Lag 2 Lag 3 Lag 4 Lag 5 AIC 30,37206 30,41157 30,49847 30,56373 30,64933 SIC 30,67050 30,75974 30,89639 31,01139 31,14673 Hasil Uji Regresi Partial Adjutment Model (PAM) Tabel 6 Hasil Uji PAM Variable Coefficient t-statistic probability LOG(EKS) 0.559055 1233189 0.0000 LOG(KURS) 0.060897 2126704 0.0494 LOG(ULN) -0.003674 -0.650335 0.9380 LOG(INF) -0.142250 -1.831627 0.1119 LOG(DEV(-1)) 0.433503 2073461 0.0000 C 1.945.567 0.917698 0.3743 R-Squared 0.966371 Adjusted 0.957964 F-Statistic 114.9446 Hasil Uji Regresi Partial Adjutment Model (PAM) Tabel 6 Hasil Uji PAM 455 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA Perolehan persamaan untuk jangka pendek sebagai berikut: LOG (DEV) = 1.945567 + 0.559055 LOG(EKS) + 0.060897 LOG(KURS) - 0.003674LOG(ULN) – 0.142250 LOG(INF) + 0.433503 LOG(DEV(-1)) + e Selanjutnya yaitu melakukan perhitunga koefisien penyesuaian untuk menentukan persamaan jangka panjang dimana hal tersebut di lakukan dengan cara membagi koefisien dalam jangka pendek dengan koefisien penyesuaian. Uji Koefisien Determinasi R-square sendiri menggambarkan seberapa besar kemampuan variabel Y di jelaskan oleh variabel X dalam hal ini semakin mendekati 1 maka kemampuan variabel X menjelaskan variabel X semakin besar karena 1 menggambarkan 100%. Dalam penelitian ini perolehan R-square sebesar 0.966371 atau sebesar 97%. Dimana artinya variabel X mampu menjelaskan variabel Y sebesar 97% sedangkan sisanya 3% di jelaskan oleh variabel lain yang tidak terdapat dalam penelitian ini. Nilai Koefisien Determinasi Perhitungan koefisen penyesuaian dengan perhitungan 1- 0.433503 =0.566497 artinya terdapat perbedaan antara cadangan devisa yang di harapkan dengan yang terjadi 57% di sesuaikan dalam periode tahunan. Sehingga dari koefisien penyesuaian dengan menggunakan ekspektasi adaptif maka perolehan persamaan untuk jangka panjang sebagai berikut: LOG (DEV) =0.566497 + 0.986863125 LOG(EKS) + 0.10749748 LOG(KURS) - 0.006485471 LOG(ULN) - 0.251104595 LOG(INF)+e LOG (DEV) =0.566497 + 0.986863125 LOG(EKS) + 0.10749748 LOG(KURS) - 0.006485471 LOG(ULN) - 0.251104595 LOG(INF)+e y g p p Uji Simultan (Uji F-Statistik) y g p p Uji Simultan (Uji F-Statistik) Pengujian F-Satatistik di gunakan untuk menguji secara serentak variabel bebas terhadap variabel terikat. Dalam penelitian di peroleh nilai F- statstik sebesar 114.9446 yang menunjukkan signifikan dimana nilai tersebut lebih besar dari nilai F-tabel yang sebesar 3.24 sehingga dapat di simpulkan menerima H1 yang artinya secara simultan Ekspor Non Migas, Nilai Tukar Rupiah, Utang Luar Negeri dan Inflasi mempengaruhi Cadangan Devisa di Indonesia. Tabel 7 Hasil Uji F F-hitung F-tabel (α = 5%) Kriteria 114.9446 3.24 Signifikan Uji t Statistik Pengaruh Ekspor Non Migas terhadap Cadangan Devisa g p g p g Berdasarkan hasil regresi PAM menunjukkan hasil secara jangka panjang dan jangka pendek ekspor non migas berpengaruh positif dan signifikan dimana hasil probabilitas uji t menunjukkan hasil sebesar 12.33189 menunjukkan terdapat pengaruh yang signifikan terhadap cadangan devisa. Hasil regresi PAM menunjukkan setiap kenaikan 1% ekspor non migas dapat meningkatkan cadangan devisa sebesar 56% dalam jangka pendek dan secara jangka panjang dapat meningkatkan cadangan devisa sebesar 99%. Hal tersebut sejalan dengan penelitian yang di lakukan oleh (Sukandar 2020) yang memperoleh hasil bahwa ekspor non migas berpengaruh signifikan terhadap cadangan devisa. Pada sistem perekonomian terbuka neraca perdagangan berperan penting hal ini di sebabkan neraca perdagangan yang surplus akan menabah ketersediaan devisa, laju pertumbuhan pendapatan yang di sebabkan oleh ekspor akan meyebabkan neraca pembayaran surplus sehingga peranan ekspor sangat penting bagi keseimbangan neraca pembayaran. Negara dapat memperoleh aset pembayaran dari ekspor yang berupa valuta asing yang akan menambah ketersediaan devisa. Selain itu dengan meningkatnya ekspor di sektor non migas dapat menstabilkan perekonomian karena laju pertumbuhannya yang lebih besar di bandingkan dengan sektor migas sehingga dapat meminimalisir risiko ekonomi Indonesia terhadap perubahan-perubahan yang terjadi di nilai tukar internasional. Uji t-Statistik j Pengujian t-statistik di gunakan untuk menguji satu persatu (individual) variabel bebas terhadap variabel terikat. Dalam penelitian ini di peroleh hasil uji t-statistik sebagai berikut: 456 j g Tabel 8 Hasil Uji t Variabel Koefisien t-hitung t-tabel (α = 5%) Keterangan EKS 0.559055 12.33189 2.120 Signifikan 456 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA Variabel Koefisien t-hitung t-tabel (α = 5%) Keterangan KURS 0.060897 2.126704 2.120 Signifikan ULN -0.003674 0.650335 2.120 Tidak Signifikan INF -0.142250 -1.831627 -2.120 Tidak Signifikan Pengaruh Utang Luar Negeri Terhadap Cadangan Devisa Berdasarkan hasil regresi PAM yang telah di lakuakan menunjukkan bahwa secara jangka pendek dan jangka panjang utang luar negeri terhadap cadangan devisa berpengaruh negatif dan tidak signifikan hal ini karena nilai uji t sebesar 0.650335 yang lebih kecil daripada nilai t-tabel sehingga di simpulkan tidak ada hubungan antara utang luar negeri dengan cadangan devisa. Hasil regresi PAM menunjukkan setiap kenaikan 1% utang luar negeri secara jangka pendek akan menurunkan cadangan devisa yang di miliki sebebsar 0,4% sedangkan secara jangka panjang utang luar negeri akan menurunkan sebesar 0,6% cadangan devisa yang di miliki. Hal tersebut menunjukkan hasil penelitian tidak sesuai dengan hipotesis yang telah di tentukan tetapi menurut Boediono apabila utang luar negeri yang di peroleh di gunakan untuk mengimpor barang-barang yang di gunakan oleh pemerintah maka tidak terdapat pengaruh terhadap perekonomian dalam negeri selain itu penelitian sebelumnya yang di lakukan oleh (Susanti 2019)perolehan dari hasil penelitian tersebut menunjukkan bahwa tidak terdapat pengaruh antara utang luar negeri terhadap cadangan devisa. Pengaruh Nilai Tukar Rupiah terhadap Cadangan Devisa. Syarat untuk mempertahankan kestabilan nikai tukar domestik adalah tersedianya cadangan devisa yang lebih dari cukup agar tercapainya kestabilan perdagangan internasional sehingga perolehan devisa dari ekspor terus mengalir(Syarifuddin 2015). Pengaruh Nilai Tukar Rupiah terhadap Cadangan Devisa. Berdasarkan hasil Regresi PAM yang telah di lakukan menunjukkan bahwa secara jangka pendek dan jangka panjang nilai tukar rupiah terhadap cadangan devisa berpengaruh positif dan signifikan dimana hasil uji t menunjukkan nilai probabilitas sebesar 2.126704 yang lebih besar dari nilai t- tabel terdapat pengaruh yang signifikan. Hasil regresi PAM menunjukkan setiap kenaikan 1% nilai tukar rupiah secara jangka pendek dapat meningkatkan cadangan devisa sebesar 6% sedangkan secara jangka panjang dapat meningkatkan cadangan devisa sebesar 11% sehingga setiap nilai tukar rupiah mengalami apresiasi akan meningkatkan cadangan devisa. Hasil penelitian ini sejalan dengan penelitian yang di lakukan oleh (Kuswantoro 2017; Rochman 2015a)hasil dari kedua penelitian tersebut menyatakan bahwa nilai tukar rupiah berpengaruh signifikan positif. Uang yang sifatnya tidak netral akan mempengaruhi kegiatan perekonomian Negara sehingga Nilai tukar sangat penting peranannya untuk menjaga kestabilan harga komoditas dimana nilai tukar berperan sebagai kunci dari kegiatan perdagangan internasional, apabila nilai tukar rupiah mengalami apresiasi menunjukkan ekspor sedang meningkat yang artinya akan menambah aset negara melalui pembayaran 457 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA ekspor yang akan menyebabkan neraca pembayaran internasional surplus dimana artinya cadangan devisa yang di miliki cukup. Sebaliknya ketika nilai tukar rupiah mengalami depresi maka harga komoditas akan mengalami penurunan hal ini akan menaikkan permintaan terhadap ekspor namun berdampak buruk terhadap impor bahan untuk melakukan produksi sehingga akan menghambat kegiatan ekonomi, untuk mestabilkan nilai tukar rupiah maka di butuhkan cadangan devisa untuk membeli valuta asing di pasar valas, yang akan menyebabkan defisit neraca pembayaran internasional, adanya peningkatan ekspor secara terus-menerus akan menyebabkan jangka panjang terhadap apresisasi nilai tukar domestik hal tersebut di sebabkan oleh terjualnya komoditas domestik secara terus-menerus. Syarat untuk mempertahankan kestabilan nikai tukar domestik adalah tersedianya cadangan devisa yang lebih dari cukup agar tercapainya kestabilan perdagangan internasional sehingga perolehan devisa dari ekspor terus mengalir(Syarifuddin 2015). ekspor yang akan menyebabkan neraca pembayaran internasional surplus dimana artinya cadangan devisa yang di miliki cukup. Sebaliknya ketika nilai tukar rupiah mengalami depresi maka harga komoditas akan mengalami penurunan hal ini akan menaikkan permintaan terhadap ekspor namun berdampak buruk terhadap impor bahan untuk melakukan produksi sehingga akan menghambat kegiatan ekonomi, untuk mestabilkan nilai tukar rupiah maka di butuhkan cadangan devisa untuk membeli valuta asing di pasar valas, yang akan menyebabkan defisit neraca pembayaran internasional, adanya peningkatan ekspor secara terus-menerus akan menyebabkan jangka panjang terhadap apresisasi nilai tukar domestik hal tersebut di sebabkan oleh terjualnya komoditas domestik secara terus-menerus. KESIMPULAN Perolehan dari penelitian ini secara simultan ekspor non migas, nilai tukar rupiah, utang luar negeri dan inflasi mempengaruhi cadangan devisa sedangkan secara parsial hasil uji regresi PAM jangka pendek dan jangka panjang menunjukkan ekspor non migas dan nilai tukar rupiah bahwa terdapat hubungan positif dan berpengaruh signifikan terhadap cadangan devisa di Indonesia, setiap kenaikan ekspor non migas akan menambah cadangan devisa yang di miliki hal ini karena terjadinya cash inflow kemudian untuk setiap nilai tukar rupiah terapresiasi akan mengakibatkan ekspor meningkat sehingga akan meningkatkan cadangan devisa yang di miliki. Sedangkan hasil untuk jangka pendek dan jangka panjang menunjukkan utang luar negeri dan inflasi hubungannya adalah negatif dan tidak ada pengaruh dengan cadangan devisa di Indonesia, sehingga adanya peningkatan atau penambahan dalam utang luar negeri dan inflasi hal ini akan menurunkan cadangan devisa di Indonesia. Ekspor non migas, nilai tukar rupiah, utang luar negeri dan inflasi dapat menjelaskan cadangan devisa sebesar 97% sedangkan untuk 3% di jelaskan oleh faktor lain yang tidak dimasukkan dalam penelitian ini. Pengaruh Inflasi terhadap Cadangan Devisa Berdasarkan hasil Regresi PAM yang telah di lakuakan menunjukkan bahwa secara jangka pendek dan jangka panjang inflasi terhadap cadangan devisa berpengaruh negatif dan tidak signifikan dimana hasil uji t menununjukkan sebesar -1.831627 yang lebih kecil dari nilai t-tabel sehingga dapat di simpulkan tidak terdapat pengaruh inflasi terhadap cadangan devisa. Hasil regresi PAM menunjukkan setiap kenaikann 1% inflasi akan menurunkan cadangan devisa yang di miliki secara jangka pendek sebanyak 14% sedangkan secara jangka panjang inflasi akan menurunkan cadangan devisa sebanyak 25%. Ketika terjadi inflasi hal ini akan berdampak terhadap kegiatan perekonomian yang dapat menurunkan tingkat produksi dan mempengaruhi tingkat harga sehingga menyebabkan menurunnya daya beli masyarakat yang akan menyebabkan jumlah uang beredar sangat banyak yang 458 Fitria Soelistyo Susilowati ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA akan menyebabkan depresiasi nilai mata uang. Ketika terjadi depresiasi pemerintah akan melakukan interfensi terhadap pasar valuta asing dengan melakukan pembelian valas maka akan ketersediaan cadangan devisa yang di miliki akan berkurang. Hasil penelitian ini di perkuat dengan sejalannya dengan penelitian terdahulu yang telah di lakukan (Kuswantoro 2017; Rochman 2015b)perolehan dari kedua penelitian tersebut menunjukkan bahwa pengaruh inflasi terhadap cadangan devisa tidak terdapat pengaruh. akan menyebabkan depresiasi nilai mata uang. Ketika terjadi depresiasi pemerintah akan melakukan interfensi terhadap pasar valuta asing dengan melakukan pembelian valas maka akan ketersediaan cadangan devisa yang di miliki akan berkurang. Hasil penelitian ini di perkuat dengan sejalannya dengan penelitian terdahulu yang telah di lakukan (Kuswantoro 2017; Rochman 2015b)perolehan dari kedua penelitian tersebut menunjukkan bahwa pengaruh inflasi terhadap cadangan devisa tidak terdapat pengaruh. Tambunan, T. and Wijanarko. 2000. Perdagangan Internasional Dan Neraca Pembayaran: Teori Dan Temuan Empiris. LP3ES. Sukandar. 2020. “Pengaruh Ekspor Migas Dan Non Migas Terhadap Cadangan Devisa Indonesia 2016-2019 Dalam Prespektif Islam.” : 6. g p Susanti, Tiyas. 2019. “Pengaruh Utang Luar Negeri, Ekspor,Kurs Dan Inflasi Terhadap Cadangan Devisa Indonesia.” 8(2): 2019. DAFTAR PUSTAKA Asmanto, Priadi, and Sekar Suryandari. 2009. “Cadangan Devisa, Financial Deepening Dan Stabilisasi Nilai Tukar Riil Rupiah Akibat Gejolak Nilai Tukar Perdagangan.” Buletin Ekonomi Moneter dan Perbankan 11(2). g g ( ) Basuki, Agus Tri. 2014. “Regresi Model PAM, ECM Dan Data Panel Dengan Eviews 7.” : 1–75. Damondaar N. Gujarati. 2007. Dasar-Dasar Ekonometrika. Edisi Keti. Jakarta: Erlangga. Gandhi, Dyah Virgoana. 2006. Seri Kebanksentralan Pengelolaan Cadangan Devisa Di Bank Indonesia. Kuswantoro, Muchammad. 2017. “Analisis Pengaruh Inflasi, Kurs, Utang Luar Negeri Dan Ekspor Terhadap Cadangan Devisa Indonesia.” Tirtayasa Ekonomika 12(1): 146. Rochman, Asep Maulana. 2015. “Pengaruh Inflasi, Kurs Rupiah, Utang Luar Negri Dan Ekspor Terhadap Cadangan Devisa Indonesia.” Sayoga, Pundy, and Syamsurijal Tan. 2017. “Analisis Cadangan Devisa Indonesia Dan Faktor-Faktor Yang Mempengaruhinya.” Jurnal Paradigma Ekonomika 12(1): 25–30. 459 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA Fitria Soelistyo Susilowati Sukandar. 2020. “Pengaruh Ekspor Migas Dan Non Migas Terhadap Cadangan Devisa Indonesia 2016-2019 Dalam Prespektif Islam.” : 6. Susanti, Tiyas. 2019. “Pengaruh Utang Luar Negeri, Ekspor,Kurs Dan Inflasi Terhadap Cadangan Devisa Indonesia.” 8(2): 2019. Syarifuddin, Ferry. 2015. “Konsep, Dinamika Dan Respon Kebijakan Nilai Tukar Di Indonesia.” Bank Indonesia (24): 1–101. Tambunan, T. and Wijanarko. 2000. Perdagangan Internasional Dan Neraca Pembayaran: Teori Dan Temuan Empiris. LP3ES. Fitria Soelistyo Susilowati ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA Susanti, Tiyas. 2019. “Pengaruh Utang Luar Negeri, Ekspor,Kurs Dan Inflasi Terhadap Cadangan Devisa Indonesia.” 8(2): 2019. p g ( ) Syarifuddin, Ferry. 2015. “Konsep, Dinamika Dan Respon Kebijakan Nilai Tukar Di Indonesia.” Bank Indonesia (24): 1–101. p g ( ) Syarifuddin, Ferry. 2015. “Konsep, Dinamika Dan Respon Kebijakan Nilai Tukar Di Indonesia.” Bank Indonesia (24): 1–101. ( ) Tambunan, T. and Wijanarko. 2000. Perdagangan Internasional Dan Neraca Pembayaran: Teori Dan Temuan Empiris. LP3ES. ( ) Tambunan, T. and Wijanarko. 2000. Perdagangan Internasional Dan Neraca Pembayaran: Teori Dan Temuan Empiris. LP3ES. 460 460
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Predicting attitudes toward ambiguity using natural language processing on free descriptions for open-ended question measurements
Scientific reports
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Predicting attitudes toward ambiguity using natural language processing on free descriptions for open‑ended question measurements OPEN Jimpei Hitsuwari 1,2, Hirohito Okano 1 & Michio Nomura 1* Individual traits and reactions to ambiguity differ and are conceptualized in terms of an individual’s attitudes toward ambiguity or ambiguity tolerance. The development of natural language processing technology has made it possible to measure mental states and reactions through open-ended questions, rather than predefined numerical rating scales, which have traditionally been the dominant method in psychological research. This study presented three ambiguity-related situations and responses collected online from 591 participants in an open-ended format. After the analysis with bidirectional encoder representations from transformers, correlations were calculated using scores from the numerical evaluation by conventional questionnaire, and a significant moderate positive correlation was found. Therefore, this study found that attitudes toward ambiguity can be measured using an open-ended response method of reporting everyday life states. It is a novel methodology that can be expanded to other scales in psychology and can potentially be used in educational and clinical situations where participants can be asked to respond with minimal burden. The ambiguous situations faced in the volatility, uncertainty, complexity, and ambiguity (VUCA) era are diverse, with individual differences in attitudes toward these ambiguous ­situations1. To measure individual differences, Lauriola et al.2 developed the Multidimensional Attitude toward Ambiguity Scale (MAAS) based on the Ambigu- ity Tolerance Scale, which measures individuals’ tolerance degree toward ambiguous situations. This scale has been validated for construct validity and internal ­reliability2. The MAAS is utilized globally, with ­Japanese3 and Swedish ­versions4 also being developed. It has been used in numerous behavioral experiments and psychologi- cal ­surveys5,6.i However, responding to a predefined numerical rating scale is not necessarily the optimal method to capture complex mental states and personality traits (People do not usually answer or express their states and emotions on a yes or no or 1–7 point scale, and most often use natural language.; for review, ­see7). Considering the recent popularity of ChatGPT, the development of large language models has made it possible to measure psychologi- cal states based on natural language, which was quite challenging in the past. For example, in Kjell et al.’s study, participants had to answer the question, “Overall, in your life, are you satisfied or not?”8. www.nature.com/scientificreports www.nature.com/scientificreports 1Graduate School of Education, Kyoto University, Kyoto, Japan. 2Japan Society for the Promotion of Science, Tokyo, Japan. *email: nomura.michio.8u@kyoto-u.ac.jp Methods Th d This study was approved by the Ethics Committee of the Graduate School of Education at Kyoto University (CPE- 571) and conducted in accordance with relevant guidelines and regulations. We obtained informed consent from the study participants before their participation. Participants p A total of 600 native English speakers of British nationality (the language used in most of the previous ­studies8 is English, so we targeted British nationals referring to those NLP studies) were recruited using an online survey platform Prolific (https://​www.​proli​fic.​com/). Nine were excluded because of duplicate IP addresses, extremely short response times (less than 255 s), and attention-checking errors, resulting in 591 participants ­(Mage = 43.35, SD = 14.44, 325 males, 255 females, 11 others) for the final analyses. A question for the attention check (For this question, select “5. I mildly agree”) was added to MSTAT II (detailed in Procedure section) to exclude participants who selected anything other than the required answer. They were paid£0.6 as a reward for their participation. Analysis Th d l The model for predicting the questionnaire scores was developed by fine-tuning the pre-trained BERT-base-cased model (https://​huggi​ngface.​co/​bert-​base-​cased). Closed models like ChatGPT raise scientific reproducibility and ethical concerns, as the precise architecture and training data are not disclosed, and updates are made without revealing the ­differences7. Therefore, for this study, a more open model, BERT, was used. Regarding hyperparam- eter selection during fine-tuning and final model evaluation, five-fold nested cross-validation (nested CV) was used. The nested CV has a low bias in estimation ­accuracy12 and is particularly effective for machine learning on small ­samples13. It allows obtaining an estimate of the model’s predictive accuracy, independent of the data used to build the model (see Supplementary Material for more information). Procedureh The participants provided open-ended responses to three ambiguous situations. The three situations correspond to the three factors of the MAAS: “How do you typically react when you are uncertain about the responsibilities of a job? (Discomfort with Ambiguity; DA),” “How do you typically react when ambiguous words like ‘probably,’ ‘approximately,’ or ‘perhaps’ are used? (Absolutism; AB),” and “How do you typically react when you are in situ- ations which can be interpreted in more than one way? (Need for Complexity and Novelty; NC)” The responses were required to have at least 100 characters (approximately 20 words), and at least 45 s had to pass before answering the next question. Subsequently, participants responded to a questionnaire containing the MAAS and the Multiple Stimulus Types Ambiguity Tolerance Scale-II (MSTAT-II)11. The MSTAT-II is a general measure of ambiguity tolerance and was employed to determine whether it could predict this scale score from the three situations created from the MAAS (usually, in MAAS, the average of each subscale score is calculated but not the overall score). Finally, respondents’ demographic data (sex, age, nationality, and education) were collected. Descriptive statistics from the MAAS and MSTAT-II and examples of open-ended responses obtained from the three texts are presented in Table 1. Predicting attitudes toward ambiguity using natural language processing on free descriptions for open‑ended question measurements OPEN They examined the correlation between the values calculated by bidirectional encoder representations from transformers (BERT), a large language model, and the scores of the Satisfaction with Life Scale (SWLS)9, which has been convention- ally used to measure life satisfaction. The BERT regression model transforms the participant’s free text into a multidimensional vector and uses that vector representation to predict the individual’s questionnaire score. The results indicated r = 0.74, implying that life satisfaction can be accurately measured using open-ended responses. In another ­study10, BERT was used to predict the Big Five personality traits based on user comments and posts comprising fiction (e.g., short stories) in a novel-writing community on Reddit (a bulletin board social site). The results indicated an average performance of r = 0.33, suggesting that personality can be predicted using free text. The present study asked participants to respond to open-ended questions in three situations (see below in the Method section) involving ambiguity (from the MAAS subscale), and the obtained texts were analyzed. The study aimed to determine the extent to which the survey methods consisting of free-text and natural language | https://doi.org/10.1038/s41598-024-59118-z Scientific Reports | (2024) 14:8276 www.nature.com/scientificreports/ processing (NLP) predicted ambiguity tolerance in comparison to conventional numerical scores. Additionally, this study examined whether the texts answered from the respective MAAS subscales could discriminate between the respective subscales answered with numerical values. processing (NLP) predicted ambiguity tolerance in comparison to conventional numerical scores. Additionally, this study examined whether the texts answered from the respective MAAS subscales could discriminate between the respective subscales answered with numerical values. Discussionhi The findings of this study are novel as they indicate that even free text can predict psychological states ­traits8,10 with regard to ambiguity.h Three questions were asked in this study; however, only one question from NC, “How do you typically react when you are in situations which can be interpreted in more than one way?” was moderately predictive. This question is more general than the other two questions and applies to various situations. This suggests that refin- ing situation settings and how questions are asked may allow attitudes toward ambiguity to be measurable, even with only one open-ended response. The DA, AB, and NC texts showed weak but significant correlations with their respective scores. Future studies should consider making it possible to discriminate between subscales, for example, by devising how the questions are asked.h This survey method consisting of free-text and NLP will allow for the measuring of an individual’s personal- ity in a more ecologically valid form; that is, an open-ended response method when expressing emotions and states in everyday ­life8,10,14,15. In Kjell et al.’s ­study8, questions aimed to examine overall life satisfaction, such as “Overall, in your life, are you satisfied or not?”; however, in this study, the question was constructed by specifying the situation and asking the respondent to imagine the situation, where “it can be interpreted in more than one way.” This allows the use of open-ended surveys that measure not only abstract concepts, such as life satisfaction, but also other personality traits and psychological states that are more specific.fi p y p y g pi While moderate correlation coefficients were observed, aligning with previous ­studies10, there is scope for further improvement in correlation by employing alternative language models (e.g., RoBERTa), a topic of inter- est for future studies. Consistent with previous studies, the results of this study are limited to English-language data. However, given the translation of the scale into various languages, efforts will be made to globally predict its scores in open-ended surveys in the future study. Both the MAAS and MSTAT-II used in this study were self- reported, and future research can attempt to predict a behavior (e.g., decision-making in ambiguous situations) based on participants’ open-ended responses and BERT scores. In conclusion, this study successfully predicted attitudes toward ambiguity by NLP of open-ended responses using BERT. Resultsh DA AB NC MSTAT​ Text DA 0.28** [0.10, 0.44] 0.12 [− 0.07, 0.29] 0.10 [− 0.08, 0.28] 0.20* [0.02, 0.37] Text AB 0.12 [− 0.06, 0.29] 0.23* [0.05, 0.40] 0.14 [− 0.04, 0.32] 0.06 [− 0.12, 0.24] Text NC 0.26** [0.09, 0.42] 0.16† [− 0.02, 0.33] 0.19* [0.01, 0.36] 0.38*** [0.22, 0.53] All texts 0.34*** [0.18, 0.49] 0.25** [0.07, 0.41] 0.28** [0.11, 0.44] 0.41*** [0.25, 0.55] DA AB NC MSTAT​ Text DA 0.28** [0.10, 0.44] 0.12 [− 0.07, 0.29] 0.10 [− 0.08, 0.28] 0.20* [0.02, 0.37] Text AB 0.12 [− 0.06, 0.29] 0.23* [0.05, 0.40] 0.14 [− 0.04, 0.32] 0.06 [− 0.12, 0.24] Text NC 0.26** [0.09, 0.42] 0.16† [− 0.02, 0.33] 0.19* [0.01, 0.36] 0.38*** [0.22, 0.53] All texts 0.34*** [0.18, 0.49] 0.25** [0.07, 0.41] 0.28** [0.11, 0.44] 0.41*** [0.25, 0.55] Table 2. Median correlation coefficient between each text and each questionnaire score. Text DA (Discomfort with Ambiguity), text AB (Absolutism), and text NC (Need for Complexity and Novelty) were calculated as free-text responses obtained from open-ended questions adopted from the DA, AB, and NC questions, respectively. The correlation coefficients in the table show the median correlation coefficients between the predicted values and true ones for the five test sets in the outer loop (n = 591/5 = 118 or 119). All texts were calculated by combining these three questions. *** p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.10. Table 2. Median correlation coefficient between each text and each questionnaire score. Text DA (Discomfort with Ambiguity), text AB (Absolutism), and text NC (Need for Complexity and Novelty) were calculated as free-text responses obtained from open-ended questions adopted from the DA, AB, and NC questions, respectively. The correlation coefficients in the table show the median correlation coefficients between the predicted values and true ones for the five test sets in the outer loop (n = 591/5 = 118 or 119). All texts were calculated by combining these three questions. *** p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.10. general ambiguity tolerance. Additionally, texts from the DA (r = 0.28, p = 0.002), AB (r = 0.23, p = 0.01), and NC (r = 0.19, p = 0.04) were weakly correlated with their respective MAAS subscale scores. Data availability Data availability All data and script are available online (https://​osf.​io/​jza53/?​view_​only=​dbdc4​b4c82​f9441​0aed7​e5ccb​b22a9​8d) Received: 14 February 2024; Accepted: 8 April 2024 Received: 14 February 2024; Accepted: 8 April 2024 Discussionhi Through the utilization of these technologies, complex human minds can be measured in a way that is natural to the participants, with little concern that the content of the questionnaire items will influence participants’ cognitions. Academically, as the scale is translated into other languages, attempts can be made to predict its scores in open-ended surveys globally to increase its accuracy and discrimination to apply it to social surveys, education, clinical situations, among other spheres. Resultsh The correlation coefficients between the BERT-predicted and true values of the questionnaire scores when using free-text responses to the three open-ended questions were calculated (Table 2 presents the medians; see Supple- mentary Table 1 for the minimum and maximum values). Results indicated that text NC (r = 0.38, p < 0.001) and the text combining all three texts (r = 0.41, p < 0.001) moderately predicted the MSTAT-II scores, which measure 2 4567890) Scientific Reports | (2024) 14:8276 | https://doi.org/10.1038/s41598-024-59118-z Table 1. MAAS and MSTAT descriptive statistics and examples of free-text responses from the three situations. M SD Examples DA 4.48 1.12 I get into a bit of a panic, try to work out how to do it on my own, and if I am still unable to, I go to someone with my tail between my legs Wait and see; I do not get stressed, I seek to clarify exactly what the responsibilities will be and, if needed, double- clarify MA 3.35 1.18 I do not mind them if it is just occasionally, but it can be annoying when they are used in every other sentence I would feel slightly distrustful and cautious. I have learned never to take assurances at face value. It depends on who is using such language NC 4.03 1.18 I always take my time, thinking through the options and how my actions could be interpreted by the people around me I react confused, I find it difficult to have to pick a certain path because I always question whether I have chosen correctly MSTAT​ 4.13 0.89 Scientific Reports | (2024) 14:8276 | www.nature.com/scientificreports/ Table 2. Median correlation coefficient between each text and each questionnaire score. Text DA (Discomfort with Ambiguity), text AB (Absolutism), and text NC (Need for Complexity and Novelty) were calculated as free-text responses obtained from open-ended questions adopted from the DA, AB, and NC questions, respectively. The correlation coefficients in the table show the median correlation coefficients between the predicted values and true ones for the five test sets in the outer loop (n = 591/5 = 118 or 119). All texts were calculated by combining these three questions. *** p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.10. References 1. Furnham, A. & Ribchester, T. Tolerance of ambiguity: A review of the concept, its measurement and applications. Curr. Psychol. 14, 179–199. https://​doi.​org/​10.​1007/​BF026​86907 (1995). p g 2. Lauriola, M., Foschi, R., Mosca, O. & Weller, J. Attitude toward ambiguity: Empirically robust factors in self-report personality scales. Assessment 23(3), 353–373. https://​doi.​org/​10.​1177/​10731​91115​577188 (2016). ( ) p g ( ) 3. Hitsuwari, J. & Nomura, M. Developing and validating a Japanese version of the multidimensional attitude toward ambiguity scale (MAAS). Psychology 12, 477–497. https://​doi.​org/​10.​4236/​psych.​2021.​124030 (2021).h ( ) p g ( ) 3. Hitsuwari, J. & Nomura, M. Developing and validating a Japanese version of the multidimensional attitude toward ambiguity scale (MAAS). Psychology 12, 477–497. https://​doi.​org/​10.​4236/​psych.​2021.​124030 (2021). p g 3. Hitsuwari, J. & Nomura, M. Developing and validating a Japanese version of the multidimensional attitude toward ambiguity scale (MAAS). Psychology 12, 477–497. https://​doi.​org/​10.​4236/​psych.​2021.​124030 (2021). ( ) y gy p g p y ( ) 4. Forsberg, E., Nilsson, A. & Jørgensen, Ø. Moral dichotomization at the heart of prejudice: The role of moral foundations and intolerance of ambiguity in generalized prejudice. Soc. Psychol. Personal. Sci. 10, 1002–1010. https://​doi.​org/​10.​1177/​19485​50618​ 817347 (2019). y gy p g p y 4. Forsberg, E., Nilsson, A. & Jørgensen, Ø. Moral dichotomization at the heart of prejudice: The role of moral foundations and intolerance of ambiguity in generalized prejudice. Soc. Psychol. Personal. Sci. 10, 1002–1010. https://​doi.​org/​10.​1177/​19485​50618​ 817347 (2019). y gy p g p y 4. Forsberg, E., Nilsson, A. & Jørgensen, Ø. Moral dichotomization at the heart of prejudice: The role of moral foundations and intolerance of ambiguity in generalized prejudice. Soc. Psychol. Personal. Sci. 10, 1002–1010. https://​doi.​org/​10.​1177/​19485​50618 817347 (2019). ( ) 5. Hitsuwari, J. & Nomura, M. Ambiguity tolerance can improve through poetry appreciation and creation. J. Creat. Behav. 57(2) 178–185. https://​doi.​org/​10.​1002/​jocb.​574 (2023). ( ) 5. Hitsuwari, J. & Nomura, M. Ambiguity tolerance can improve through poetry appreciation and creation. J. Creat. Behav. 57(2), 178–185. https://​doi.​org/​10.​1002/​jocb.​574 (2023). 6 S i lli C Ib hi M & Kh B C l i i bi i l h h i df l A i d i d i d ll d ( ) 5. Hitsuwari, J. & Nomura, M. Ambiguity tolerance can improve through poetry appreciation and creation. J. Creat. Behav. 57(2), 178–185. https://​doi.​org/​10.​1002/​jocb.​574 (2023). p g j ( ) 6. Spinelli, C., Ibrahim, M. & Khoury, B. Cultivating ambiguity tolerance through mindfulness: An induction randomized controlled trial. Curr. Psychol. www.nature.com/scientificreports/ PLOS One 18(2), e0267995. https://​doi.​org/​10.​1371/​journ​al.​pone.​02679​95 (2023). g y p p gh 15. Sikström, S., Pålsson Höök, A. & Kjell, O. Precise language responses versus easy rating scales: Comparing respondents’ views with clinicians’ belief of the respondent’s views. PLOS One 18(2), e0267995. https://​doi.​org/​10.​1371/​journ​al.​pone.​02679​95 (2023). Acknowledgementsh g This work was supported by Grant-in-Aid for JSPS Fellows Grant Number 22KJ1813, Leave a Nest Grant Incube Prize, and 3rd academist Prize. We also thank Katarina Woodman (Kyoto University) for the English proofread- ing of the questionnaire. Finally, this manuscript was reviewed in English by Editage (http://​www.​edita​ge.​com/) with the support of Global Education Office, Graduate School of Education, Kyoto University. g This work was supported by Grant-in-Aid for JSPS Fellows Grant Number 22KJ1813, Leave a Nest Grant Incube Prize, and 3rd academist Prize. We also thank Katarina Woodman (Kyoto University) for the English proofread- ing of the questionnaire. Finally, this manuscript was reviewed in English by Editage (http://​www.​edita​ge.​com/) with the support of Global Education Office, Graduate School of Education, Kyoto University. References https://​doi.​org/​10.​1007/​s12144-​021-​02597-4 (2022). https://doi.org/10.1038/s41598-024-59118-z Scientific Reports | (2024) 14:8276 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ 7. Kjell, O. N., Kjell, K. & Schwartz, H. A. Beyond rating scales: With targeted evaluation, language models are poised for psychologi- cal assessment. Psychiatry Res. 333, 115667. https://​doi.​org/​10.​1016/j.​psych​res.​2023.​115667 (2023). 7. Kjell, O. N., Kjell, K. & Schwartz, H. A. Beyond rating scales: With targeted evaluation, language models are poised for psychologi- cal assessment. Psychiatry Res. 333, 115667. https://​doi.​org/​10.​1016/j.​psych​res.​2023.​115667 (2023). 8. Kjell, O. N., Sikström, S., Kjell, K. & Schwartz, H. A. Natural language analyzed with AI-based transformers predict traditiona subjective well-being measures approaching the theoretical upper limits in accuracy. Sci. 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Evidence of the properties of an ambiguity tolerance measure: The multiple stimulus types ambiguity toler scale-II (MSTAT-II) Psychol Rep 105 975–988 https://doi org/10 2466/PR0 105 3 975-988 (2009) ( ) y p p g ( ) 12. Varma, S. & Simon, R. Bias in error estimation when using cross-validation for model selection. BMC Bioinform. 7(1), 1–8. https://​ doi.​org/​10.​1186/​1471-​2105-7-​91 (2006).f y p p g 2. Varma, S. & Simon, R. Bias in error estimation when using cross-validation for model selection. BMC Bioinform. 7(1), 1–8. https:// doi.​org/​10.​1186/​1471-​2105-7-​91 (2006).f g 3. Vabalas, A., Gowen, E., Poliakoff, E. & Casson, A. J. Machine learning algorithm validation with a limited sample size. PLOS One 14(11), e0224365. https://​doi.​org/​10.​1371/​journ​al.​pone.​02243​65 (2019). p g j p 4. Okano, H., Kawahara, D. & Nomura, M. Language model BERT can estimate trait self-compassion from people’s free texts with high accuracy. Open Science Framework. https://​doi.​org/​10.​31234/​osf.​io/​9zfh7 (2024). p g j p 14. Okano, H., Kawahara, D. & Nomura, M. Language model BERT can estimate trait self-compassion from people’s free texts with high accuracy. Open Science Framework. https://​doi.​org/​10.​31234/​osf.​io/​9zfh7 (2024). g y p p gh ( ) 15. Sikström, S., Pålsson Höök, A. & Kjell, O. Precise language responses versus easy rating scales: Comparing respondents’ views with clinicians’ belief of the respondent’s views. Competing interests h p g The authors declare no competing interests. Author contributions J.H.: Conceptualization, Methodology, Software, Writing—Original Draft, Visualization, Funding. H.O.: Methodology, Formal analysis, Visualization, Writing—Review & Editing. M.N.: Writing—Review & Editing, Supervision. © The Author(s) 2024 Additional informationh Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​024-​59118-z. Correspondence and requests for materials should be addressed to M.N. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2024 https://doi.org/10.1038/s41598-024-59118-z Scientific Reports | (2024) 14:8276 |
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Optimization of the Geometrical Parameters of a Solar Bubble Pump for Absorption-Diffusion Cooling Systems
American journal of engineering and applied sciences
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American J. of Engineering and Applied Sciences 3 (4): 693-698, 2010 ISSN 1941-7020 © 2010 Science Publications American J. of Engineering and Applied Sciences 3 (4): 693-698, 2010 ISSN 1941-7020 © 2010 Science Publications American J. of Engineering and Applied Sciences 3 (4): 693-698, 2010 ISSN 1941-7020 © 2010 Science Publications Corresponding Author: N. Dammak, Department of Chemical Engineering and Processes, National School of Engineers of Gabes (E.N.I.G), Omar Ibn El Khattab Street-6029 Gabes, Tunisia 693 Optimization of the Geometrical Parameters of a Solar Bubble Pump for Absorption-Diffusion Cooling Systems 1N. Dammak, 1B. Chaouachi, 1S. Gabsi and 2M. Bourouis 1Department of Chemical Engineering and Processes, National School of Engineers of Gabes (E.N.I.G), Omar Ibn El Khattab Street-6029 Gabes, Tunisia 2Department of Mechanical Engineering, University Rovira iVirgili, Avenida dels Països Catalans, 26, 43007 Tarragona, Spain Abstract: Problem statement: The objective of this study was to optimize the geometrical parameters of a bubble pump integrated in a solar flat plate collector. Approach: This solar bubble pump was part of an ammonia/water/helium (NH3/H2O/He) absorption-diffusion cooling system. Results: An empirical model was developed on the basis of momentum, mass, material equations and energy balances. The mathematical model was solved using the simulation tool “Engineering Equation Solver (EES)”. Conclusion/Recommendations: Using metrological data from Gabes (Tunisia) various parameters were geometrically optimized for maximum bubble pump efficiency which was best for a bubble pump tube diameter of 6 mm, a tube length of 1.5 m, an inclination to the horizontal between 30 and 50° of the solar flat plate collector and a submergence ratio between 0.2 and 0.3. Key words: Bubble pump, absorption-diffusion cycle, ammonia-water, solar cooling r: N. Dammak, Department of Chemical Engineering and Processes, National School of Engineers of Gabes (E.N.I.G), Omar Ibn El Khattab Street-6029 Gabes, Tunisia 693 INTRODUCTION A new Generator With Heat Exchanger (GWHX) was designed, built and tested. The test results of the DAR with GWHX were compared with baseline tests. The new generator design increased the cooling COP by as much as 50% more than the baseline tests while the cooling capacity remained constant. Srikhirin and Aphornratana (2002) reported an experimental study of a DAR cycle. The system was tested with heat input values between 1000 and 2500 W at a helium pressure of 6.1 bars. The system cooling capacities were found to be between 100 and 180 W with a COP between 0.09 and 0.15. Chaouachi and Gabsi (2007) designed and simulated an absorption diffusion refrigerator using solar energy for domestic use. They concluded that the COP would be best when the system was operated at a low generator temperature and high pressure. On the other hand, the numerical values of COP were still low and depended on the power of the solar bubble pump. Ammonia vapor is separated from the rich solution (1) in the separator. The vapor (2) then rises inside the bubble pump and flows through the rectifier where water vapor condenses (5) to join the weak solution (3). The pure ammonia (6) flows to the condenser, condenses (7) and flows to the evaporator. At the evaporator entrance the partial pressure of the ammonia liquid (7) drops because it mixes with the helium and ammonia vapor (13) from the absorber, which causes the refrigerant to evaporate. The ammonia-helium mixture (8) flowing in parallel through the shell side of the evaporator/gas heat exchanger enters the absorber and flows upward in counter flow to the weak solution entering the absorber at the top (10). In the absorber, the ammonia vapor is absorbed by the weak solution and the solution obtained flows down into the reservoir (11). Helium and ammonia residuals (12) flow toward the evaporator. INTRODUCTION numerically. The results showed that for the same heat input, the second configuration desorbed the highest amount of refrigerant and the first configuration desorbed the lowest amount. The third configuration proved to be less efficient in terms of COP than the second configuration. The first configuration gave the lowest performance, although heat was supplied directly to the rich solution. Corresponding Author: N. Dammak, Department of Chemica The influence of the cycle configuration on the performance of an absorption-diffusion cooling system has been investigated by various researchers. Zohar et al. (2007) compared the performance of two fundamental configurations of a Diffusion Absorption Refrigeration (DAR) cycle, with and without condensate sub-cooling prior to the evaporator entrance. They analyzed the performances of the two cycles by computer simulations and concluded that the DAR cycle without condensate sub-cooling, at higher evaporator temperature of about 15°C, shows higher COP of about 14-20% compared with the DAR cycle with condensate sub-cooling. Subsequently, they developed thermodynamic models for three generator and bubble pump configurations (Zohar et al., 2008): That is, (a) heat input into the rich solution without heat transfer to the poor solution; (b) heat input into the rich solution with heat transfer to the poor solution flowing in the annular space and (c) heat input into the rich solution through the poor solution, thus also desorbing refrigerant from the poor solution, with heat transfer to the poor solution in the annular space. The performance of three DAR systems, which differ in their generator and bubble pump configuration, was studied Shelton and Stewart (2002) developed a model for a bubble pump using two-phase flow correlations to establish the optimum design parameters. The experimental results showed that its performance was not sensitive to the length of the lift tube; however it was highly dependant on the submergence ratio. Koyfman et al. (2003) conducted experimental research on the bubble pump performance. A solution mixture of organic solvent and hydrochlorofluorocarbon refrigerant was used as the working fluid. It was found that the motive head is one of the parameters that most influences the bubble pump performance. Changing the motive head by 10% changed the mass flow rates by about 40%. The authors concluded that a low motive head provides higher refrigerant flow rates and, therefore, a higher cooling capacity. The maximum average generator temperature was below 90°C. Hence, they concluded that a complete diffusion absorption ( 693 Am. J. INTRODUCTION Engg. & Applied Sci., 3 (4): 693-698, 2010 Solar diffusion-absorption cycle: Figure 1 shows a schematic configuration of the solar heat driven ammonia/water/helium diffusion-absorption cooling system, consisting of a bubble pump, an absorber, a condenser, an evaporator, a rectifier, a heat exchanger and a reservoir. In this configuration the bubble pump tubes are integrated in the solar flat plate collector. Solar diffusion-absorption cycle: Figure 1 shows a schematic configuration of the solar heat driven ammonia/water/helium diffusion-absorption cooling system, consisting of a bubble pump, an absorber, a condenser, an evaporator, a rectifier, a heat exchanger and a reservoir. In this configuration the bubble pump tubes are integrated in the solar flat plate collector. refrigerating system that operates with organic solvents and hydrochlorofluorocarbon refrigerants may be driven by low temperature heat sources, such as solar thermal energy or waste heat. Chen et al. (1996) attempted to improve the current cycle performance of the Diffusion Absorption Refrigerator (DAR). They recommended using waste heat from the rectifier to preheat the weak solution leaving the absorber. A new Generator With Heat Exchanger (GWHX) was designed, built and tested. The test results of the DAR with GWHX were compared with baseline tests. The new generator design increased the cooling COP by as much as 50% more than the baseline tests while the cooling capacity remained constant. Srikhirin and Aphornratana (2002) reported an experimental study of a DAR cycle. The system was tested with heat input values between 1000 and 2500 W at a helium pressure of 6.1 bars. The system cooling capacities were found to be between 100 and 180 W with a COP between 0.09 and 0.15. Chaouachi and Gabsi (2007) designed and simulated an absorption diffusion refrigerator using solar energy for domestic use. They concluded that the COP would be best when the system was operated at a low generator temperature and high pressure. On the other hand, the numerical values of COP were still low and depended on the power of the solar bubble pump. refrigerating system that operates with organic solvents and hydrochlorofluorocarbon refrigerants may be driven by low temperature heat sources, such as solar thermal energy or waste heat. Chen et al. (1996) attempted to improve the current cycle performance of the Diffusion Absorption Refrigerator (DAR). They recommended using waste heat from the rectifier to preheat the weak solution leaving the absorber. MATERIALS AND METHODS & Applied Sci., 3 (4): 693-698, 2010 Momentum equation from Psys to “0”, from “0” to “1” and from state “1 to 2” yield (Shelton and Stewart, 2002): 2 2 t 0 2 sys l 2 l 2 G 1 d P P gz 2 d   − = ρ + −   ρ   (1) z sub H = (9) and: H Lsin( ) = β (10) Momentum equation from Psys to “0”, from “0” to “1” and from state “1 to 2” yield (Shelton and Stewart, 2002): z sub H = (9) Momentum equation from Psys to “0”, from “0” to “1” and from state “1 to 2” yield (Shelton and Stewart, 2002): z sub H = (9) (9) and: 2 2 t 0 2 sys l 2 l 2 G 1 d P P gz 2 d   − = ρ + −   ρ   (1) and: H Lsin( ) = β (10) 2 2 t 0 2 sys l 2 l 2 G 1 d P P gz 2 d   − = ρ + −   ρ   (1) and: H Lsin( ) = β 2 2 t 0 sys l 2 l 2 G 1 d P gz 2 d   − = ρ + −   ρ   (1) H Lsin( ) = β 2 0 2 2 1 d 2 d  −   (1) H Lsin( ) = β (10) H Lsin( ) = β (10) (10) For a bubble pump using nt tubes, the specific mass flow rate is given by: Where: t 0 l G v = ρ (2) (2) t 0 l G v = ρ t 0 l G v = ρ 2 1 tot 2 1 m ntG .d 4 • = π (11) (11) Momentum equation from Psys to “2” gives (vertical bubble pump) (Shelton and Stewart, 2002): Mass energy balance at the generator: Mass energy balance at the generator: 2 m 2 sys m tp 2 . L P P .g.L f . MATERIALS AND METHODS 2 d ρ ν = + ρ + (3) 1 2 3 m m m • • • = + (12) 1 1 2 2 3 3 m x m y m x • • • = + (13) 2 3 1 g 2 3 1 Q m h m h m h • • • = + − (14) (3) (12) For a bubble pump integrated in a solar flat plate collector, Eq. 3 gives: (13) 2 m 2 sys m tp 2 . L P P .g.Lsin( ) f . 2 d ρ ν = + ρ β + (4) Where: (14) 2 m 2 sys m tp 2 . L P P .g.Lsin( ) f . 2 d ρ ν = + ρ β + (4) (4) Useful solar heat (Sukhatme, 1997; Kalogirou, 2006): Where: u c L f a Q A ( U (T T ) = φ − − (15) (15) s poor poor 2 tp poor d f k   ρ ν =     µ   (5) (5) Liquid, gas and global efficiency are defined as: K and s are the BLASIUS factor (K = 0.316 and s = -0.25) (Shelton and Stewart, 2002) K and s are the BLASIUS factor (K = 0.316 and s = -0.25) (Shelton and Stewart, 2002) 2 G u m The gas efficiency : E Q • = (16) (16) Specific mass flow rate of the rich solution: tot m G . = ν ρ (6) tot m G . = ν ρ (6) (6) 3 L u m The liquid efficiency : E Q • = (17) gl G L The globalefficiency : E E .E = (18) 3 L u m The liquid efficiency : E Q • = (17) (17) Because of ρpoor >>> ρvapour, the velocity of the poor solution is given by: gl G L The globalefficiency : E E .E = (18) (18) tot poor poor G ν = ρ (7) Calculation procedure: The basic input data of the system shown schematically in Fig. 2 were: condenser temperature (Tc = 45°C), absorber temperature (Tab = 45°C), evaporator temperature (Tev = 15°C). MATERIALS AND METHODS In the present study, the new design of the bubble pump proposed by Chaouachi and Gabsi (2007) was optimized. A model for a system consisting of a bubble pump integrated in a solar flat plate collector was developed and used to optimize the geometrical parameters of the solar bubble pump of an ammonia/water/helium absorption-diffusion cooling system. An empirical model was developed on the basis of momentum, mass, material equations and energy balances. The mathematical model was solved using the simulation tool “Engineering Equation Solver (EES)”. Theoretical analyses: The following equations are based on Fig. 2. Frictional losses at the entrance to the tubes are considered insignificant and the steady state flow is established. Fig. 1: Schematic view of the absorption diffusion machine 694 Fig. 1: Schematic view of the absorption diffusion machine Fig. 2: Integrated solar bubble pump 4 Fig. 2: Integrated solar bubble pump Fig. 1: Schematic view of the absorption diffusion machine Fig. 2: Integrated solar bubble pump 694 Am. J. Engg. MATERIALS AND METHODS The calculation sequence was as follows: (7) Equation 1 and 4 gives 2 2 t 2 2 l 0 G 1 d neglecting 2 d     −         ρ     : • Calculate solar intensity (φ) (Liu and Jordan, 1960; 1961) ( ) 1.75 1 5 2 tot l m poor m 2 d 1 L G gz gLsin( ) K 2 d −           = ρ −ρ β         µ ρ         (8) • Calculate the Pressure of the system (Psys) using the condenser temperature (8) • Determine the rich solution concentration (x1) using the P-T-x relationship for the ammonia-water solution at equilibrium (Zohar et al., 2005) The submergence ratio is defined as: The submergence ratio is defined as: The submergence ratio is defined as: 695 Am. J. Engg. & Applied Sci., 3 (4): 693-698, 2010 , ( ) , Fig. 5: Global efficiency Vs submergence ratio (φav = 0.6 kW m−2, L = 1.5 m and d = 6 mm (month-8-)) Fig. 6: Liquid efficiency and gas efficiency Vs submergence ratio (φav = 0.6 kW m−2, L = 1.5 m and d = 6 mm (month-8- )) • Adjust the outlet temperature of the generator (Tg) until it satisfies the equality between the energy balance of the generator (Qg) and the useful solar heat (Qu) using Eq. 14 and 15 (since the useful heat available from the collector is transferred to the rich solution in the generator) • Determine the poor solution concentration (x3) using the P-T-x relationship (Zohar et al., 2005) • Determine the poor solution concentration (x3) using the P-T-x relationship (Zohar et al., 2005) • Calculate the refrigerant concentration (y2). The NH3H2O procedure called from EES provides the thermodynamic properties of ammonia water mixtures in saturated conditions • Calculate the mass flow rate of rich solution (m1), poor solution (m3) and the mass flow rate of refrigerant (m2), using Eq. 12 and 13 Fig. 5: Global efficiency Vs submergence ratio (φav = 0.6 kW m−2, L = 1.5 m and d = 6 mm (month-8-)) • Calculate the gas efficiency, the liquid efficiency and the global efficiency (Eq. 16-18) RESULTS AND DISCUSSION (L = 1.5 m, subav = 0.25, βav = 40° (month-8-)) p p Under the operating conditions Tab = 45°C, Tc = 45°C, Tev = 15°C, βav = 40°, subav = 0.25, φ = 0.83 kW m−2, nt = 6 and Ac = 3.8 m2, the cooling capacity of the diffusion absorption machine was equal to 450 W. In order to determine the minimal values of the submergence ratio, the variation in the mass flow rate of the generated vapor and pumped liquid vs. submergence ratio has been illustrated in Fig. 7. The intersection of the curve giving the mass flow rate of the generated vapor and the curve giving both the generated and pumped solution gives the minimal submersion ratio ( v l v l m m m givesm 0 • • • • + = = ). Figure 7 shows that the solar flat plate collector cannot be used with submersion values under 0.18 (the temperature required in the generator is too high). Figure 8 illustrates the variation of global efficiency vs. solar intensity for tube diameters between 4 and 6 mm at L = 1.5 m, subav = 0.25 and βav = 40°. Performance is most efficient when the bubble pump tube diameters are equal to 6 mm (corresponding to φav = 0.6 kW m−2). This can be explained as follows: If the bubble pump tube diameters are over 6 mm the flow rate of the rich solution increases when the heat input is constant. Thus, the poor solution flow rate increases and the refrigerant flow rate decreases, resulting in a decrease in the global efficiency. Fig. 9: Global efficiency Vs tube length (subav = 0.25, βav = 40° and d = 6 mm (month-8-)) Figure 3 and 4 illustrate the hourly variation of solar intensity obtained by the Liu and Jordan (1960; 1961) model for the region of Gabes in Tunisia. Solar intensity was maximum at 11.30 when the inclination angle of the solar flat plate collector varied between 30 and 50°. This can be explained by the fact that in winter, when the trajectory of the sun in the sky is low, the inclination angle must be high. However, it is recommended for the inclination angle to be low during the summer months when the sun in the sky is higher (Solar radiation is perpendicular to the collector). RESULTS AND DISCUSSION Figure 3-9 showed the most significant results obtained by simulation with the Equation Engineering Solver (EES) software. 696 Solver (EES) software. Fig. 3: Solar intensity Vs time (day 01/08) Fig. 4: Solar intensity Vs time (day 01/01) Fig. 6: Liquid efficiency and gas efficiency Vs submergence ratio (φav = 0.6 kW m−2, L = 1.5 m and d = 6 mm (month-8- )) Fig. 7: Mass flow rate of generated vapour and pumped liquid Vs submergence ratio (φmax = 0.83 kW m−2 (month-8- )) Fig. 3: Solar intensity Vs time (day 01/08) Fig. 4: Solar intensity Vs time (day 01/01) Fig. 6: Liquid efficiency and gas efficiency Vs submergence ratio (φav = 0.6 kW m−2, L = 1.5 m and d = 6 mm (month-8- )) 96 Fig. 7: Mass flow rate of generated vapour and pumped liquid Vs submergence ratio (φmax = 0.83 kW m−2 (month-8- )) Fig. 3: Solar intensity Vs time (day 01/08) Fig. 7: Mass flow rate of generated vapour and pumped liquid Vs submergence ratio (φmax = 0.83 kW m−2 (month-8- )) Fig. 4: Solar intensity Vs time (day 01/01) 696 Am. J. Engg. & Applied Sci., 3 (4): 693-698, 2010 Fig. 8: Global efficiency Vs solar intensity at different tube diameters. (L = 1.5 m, subav = 0.25, βav = 40° (month-8-)) Fig. 9: Global efficiency Vs tube length (subav = 0.25, βav = 40° and d = 6 mm (month-8-)) Fig. 8: Global efficiency Vs solar intensity at different tube diameters. (L = 1.5 m, subav = 0.25, βav = 40° (month-8-)) ratio ranges can be identified. In the first range global efficiency increases when the submergence ratio increases. This can be explained by the fact that any increase in the submergence ratio increases the mass flow rate of the rich solution. Hence, the mass flow of the pumped liquid increases and the mass flow of the generated vapor decreases (Fig. 6) so the global efficiency (defined as EG.EL) increases. At high submergence ratios (after reaching the maximum), however, the global efficiency decreases because the mass flow rate of the rich solution increases causing the mass flow rate of the refrigerant to drop sharply (Fig. 6) compared to the mass flow rate of the poor solution. Fig. 8: Global efficiency Vs solar intensity at different tube diameters. RESULTS AND DISCUSSION Figure 9 shows that the system pressure has considerable influence on the global efficiency. This is because the decrease in the system pressure (decrease in the absorber and condenser temperature) improves absorption and the mass flow rate of the rich solution increases which increases global efficiency. The variation of the global efficiency, gas efficiency and liquid efficiency of the solar bubble pump with submergence ratio at different solar flat plate inclination angles of the solar collector is shown in Fig. 5 and 6. For inclination angles of the solar flat plate collector between 30 and 50°, the optimal submergence ratio varied in the range 0.2-0.3. 7 Global efficiency values for a system pressure Psys = 17.82 bar (Tc = 45°C) were highest at a tube length of 1.5 m (Fig. 9). When the tube length increases, the collector area increases so more heat is supplied to the rich solution witch in turn increases the global efficiency. When the tubes are too long (over 2 m), the As can be seen in Fig. 5, global efficiency is sensitive to the submergence ratio. Two submergence 697 Am. J. Engg. & Applied Sci., 3 (4): 693-698, 2010 Liu, B.Y.H. and R.C. Jordan, 1960. The interrelationship and characteristic distribution of direct, diffuse and total solar radiation. Sol. Energy, 4: 1-19. DOI: 10.1016/0038- 092X(60)90062-1 submergence ratio (defined as z sub H = , where H Lsin = β ) decreases. Hence, the mass flow rate of the rich solution and the global efficiency decrease. submergence ratio (defined as z sub H = , where H Lsin = β ) decreases. Hence, the mass flow rate of the rich solution and the global efficiency decrease. H Lsin = β ) decreases. Hence, the mass flow rate of the rich solution and the global efficiency decrease. Liu, B. and R.C. Jordan, 1961. Daily insolation on surfaces tilted towards the equator. ASHRAE Trans., 10: 53-53. http://www.osti.gov/energycitations/product.biblio. jsp?osti_id=5047843 ACKNOWLEDGEMENT Zohar, A., M. Jelinek, A. Levy and I. Borde, 2005. Numerical investigation of a diffusion absorption refrigeration cycle. Int. J. Refrig., 28: 515-525. DOI: 10.1016/j.ijrefrig.2004.11.003 The researchers gratefully acknowledge the Spanish Ministry of Science and Innovation for its financial support (ENE2006-15250). Zohar, A., M. Jelinek, A. Levy and I. Borde, 2007. The influence of diffusion absorption refrigeration cycle configuration on the performance. Applied Therm. Eng., 27: 2213-2219. DOI: 10.1016/j.applthermaleng.2005.07.025 CONCLUSION A model of an integrated solar bubble pump was developed. Various parameters were geometrically optimized for maximum global efficiency by computer simulation. It was found that efficiency is best for a bubble pump tube diameter of 6 mm, a tube length of 1.5 m, an inclination angle of the solar flat plate collector between 30 and 50° and a submergence ratio in the range 0.2-0.3. In order to determine the minimal values of the submergence ratio, the mass flow rate variation of the generated vapor and pumped liquid vs. submergence ratio at different values of useful heat was studied. It was found that when the submersion ratio was too low, the solar flat plate collector could not be used. j p Srikhirin, P. and S. Aphornratana, 2002. Investigation of a diffusion absorption refrigerator. Applied Therm. Eng., 22: 1181-1193. http://www.thaiscience.info/Article%20for%20Tha iScience/Article/3/Ts- 3%20investigation%20of%20a%20diffusion%20ab sorption%20refrigerator.pdf Sukhatme, S.P., 1997. Solar Energy: Principles of Thermal Collection and Storage. 2nd Edn., McGraw-Hill Inc., USA., ISBN: 10: 0074624539, pp: 464. Shelton, S.V. and S.W. Stewart, 2002. Bubble pump design for single pressure absorption refrigeration cycles. ASHRAE Trans., 108: 1-10. REFERENCES Chaouachi, B. and S. Gabsi, 2007. Design and simulation of an absorption diffusion solar refrigeration unit. Am. J. Applied Sci., 4: 85-88. DOI: 10.3844/.2007.85.88 Zohar, A., M. Jelinek, A. Levy and I. Borde, 2008. The influence of the generator and bubble pump configuration on the performance of Diffusion Absorption Refrigeration (DAR) system. Int. J. Refrig. 30: 1-8. DOI: 10.1016/j.ijrefrig.2008.01.009 Chen, J., K.J. Kim and K.E. Herold, 1996. Performance enhancement of a diffusion-absorption refrigerator. Int. J. Refrig., 19: 208-218. DOI: 10.1016/0140- 7007(96)87215-X Kalogirou, S.A., 2004. Solar thermal collectors and applications. Prog. Energy Combust. Sci., 30: 231-295. DOI: 10.1016/j.pecs.2004.02.001 Koyfman, A., M. Jelinek, A. Levy and I. Borde, 2003. An experimental investigation of bubble pump performance for diffusion absorption refrigeration system with organic working fluids. Applied Therm. Eng., 23: 1881-1894. DOI: 10.1016/S1359- 4311(03)00162-5 698
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La Literatura Infantil y su Incidencia en el Desarrollo del Pensamiento Crítico en los Niños de Edad Preescolar
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LA LITERATURA INFANTIL Y SU INCIDENCIA EN EL DESARROLLO DEL PENSAMIENTO CRÍTICO EN LOS NIÑOS DE EDAD PREESCOLAR CHILDREN'S LITERATURE AND ITS IMPACT ON THE DEVELOPMENT OF CRITICAL THINKING IN PRESCHOOL CHILDREN Gloria Guadalupe Lozano López Universidad Politécnica Salesiana, Ecuador Lic.Ramiro Rubio.Msc Universidad Politécnica Salesiana, Ecuador DOI: https://doi.org/10.37811/cl_rcm.v8i1.10088 La Literatura Infantil y su Incidencia en el Desarrollo del Pensamiento Crítico en los Niños de Edad Preescolar Gloria Guadalupe Lozano López 1 Lozanogloria2000@gmail.com https://orcid.org/0000-0002-3847-0554 Universidad Politécnica Salesiana Ecuador Msc. Lic.Ramiro Rubio wrubio@ups.edu.ec https://orcid.org/0000-0002-8900-2664 Universidad Politécnica Salesiana Ecuador RESUMEN El desarrollo del pensamiento crítico en los estudiantes es un reto que asumen los docentes de todas las áreas de estudio, por eso es fundamental que se empleen estrategias efectivas en función de propiciar estas capacidades de evaluación y análisis, que en efecto pueden aportar una visión más holística acerca de su entorno. Teniendo en cuenta esta premisa, se planteó esta investigación que buscó analizar la influencia de la literatura infantil en el desarrollo del pensamiento crítico en niños preescolares de la Escuela de Educación Básica "1 de marzo" de la provincia de Morona Santiago en Ecuador. Se estableció un marco teórico que destaca la importancia de la lectura infantil en el desarrollo del pensamiento crítico. El estudio constó de dos fases: una revisión de la literatura científica sobre la relación entre la literatura infantil y el pensamiento crítico a través de la contextualización de las dos variables y, la segunda se trató de un diagnóstico de la población objetivo a través de encuestas a profesores, niños en edad preescolar y padres de familia. Los resultados revelaron que la literatura infantil desempeña un papel importante en el desarrollo del pensamiento crítico de los niños en edad preescolar. Sin embargo, se destacaron áreas susceptibles de mejora, como la selección de materiales literarios y estrategias didácticas específicas ya que los docentes en múltiples ocasiones recurren a las mismas lecturas de forma mecanizada y en otras ocasiones, los padres no indagan sobre textos pertinentes para el mismo fin. A través de esto se formularon sugerencias a todos los relacionados en esta problemática para potenciar este impacto positivo. En conclusión, el estudio ofrece un enfoque global al proponer medidas concretas para mejorar tanto la selección de la literatura como la aplicación de estrategias didácticas. Entre esto también se destacó que los recursos institucionales no deben ser una limitante para buscar estrategias de mejora. Palabras clave: literatura infantil, pensamiento crítico, estrategias. 1 Autor principal Correspondencia: Lozanogloria2000@gmail.com pág. 7580 Children's Literature and its Impact on the Development of Critical Thinking in Preschool Children ABSTRACT The development of critical thinking in students is a challenge assumed by teachers in all areas of study, that is why it is essential to use effective strategies in order to promote these evaluation and analysis skills, which can indeed provide a more holistic view of their environment. With this premise in mind, this research sought to analyze the influence of children's literature on the development of critical thinking in preschool children of the "1 de marzo" Basic Education School in the province of Morona Santiago in Ecuador. A theoretical framework was established highlighting the importance of children's reading in the development of critical thinking. The study consisted of two phases: a review of the scientific literature on the relationship between children's literature and critical thinking through the contextualization of the two variables, and a diagnosis of the target population through surveys of teachers, preschool children and parents. The results revealed that children's literature plays an important role in the development of critical thinking in preschool children. However, areas for improvement were highlighted, such as the selection of literary materials and specific didactic strategies, since teachers often resort to the same readings in a mechanized way and, on other occasions, parents do not inquire about relevant texts for the same purpose. Through this, suggestions were made to all those involved in this problem in order to enhance this positive impact. In conclusion, the study offers a global approach by proposing concrete measures to improve both the selection of literature and the application of didactic strategies. It was also emphasized that institutional resources should not be a limiting factor in the search for improvement strategies. Keywords: children's literature, critical thinking, strategies Artículo recibido 07 enero 2024 Aceptado para publicación: 15 febrero 2024 pág. 7581 INTRODUCCIÓN El presente documento describe la investigación realizada en el cantón Tiwintza, provincia Morona Santiago, en la Escuela de Educación Básica “1 de Marzo”, en la que se define una muestra poblacional de 91 padres de familia, 4 docentes y 91 niños de edad preescolar, a través de un enfoque mixto, según Hernández et al., (2010) y un método de investigación tipo exploratoria. Para lograrlo, se parte del diagnóstico que identifique la importancia de la Literatura Infantil y que facilite la formulación de estrategias tomando como base la literatura infantil para el desarrollo del pensamiento crítico en los niños de preescolar. Los cimientos serán los referentes teóricos; en la propuesta se procede a formular las estrategias que permitan que el pensamiento crítico sea fortalecido para los niños en edad preescolar de la Institución de Educación Básica “1 de Marzo”. Este trabajo halla su justificación, primero que todo en el deber docente de mejorar la calidad de la enseñanza para sus dicentes, como también en los principios la legislación ecuatoriana y el Ministerio de Educación (2016), en la forja de los hombres del mañana que planteen soluciones, sean analíticos, responsables, participativos en sus realidades. Es por ello también que los autores citados, se enfocan en la necesidad latente de mejoramiento de la educación frente a los nuevos paradigmas que se plantea la sociedad actual. La falta de énfasis y comprensión sobre la relevancia de la literatura infantil se convierte en un desafío significativo para los niños en edad preescolar de la Institución Educativa Básica "1 de Marzo" en el cantón Tiwintza, provincia de Morona Santiago. Esta problemática se manifiesta en la dificultad de los estudiantes para identificar personajes, realizar lecturas comprensivas, carecer de motivación, así como la incapacidad para hacer evaluaciones y críticas sobre los textos literarios. Considerando este panorama, la falta de enfoque y apreciación hacia la literatura infantil emerge como un obstáculo relevante para el desarrollo del pensamiento crítico en los niños de preescolar. Esta dificultad se refleja en la incapacidad de los estudiantes para comprender los textos, identificar personajes y expresar valoraciones y críticas sobre los contenidos literarios. Además, se nota una carencia de motivación en el proceso de aprendizaje. El razonamiento crítico es una habilidad cognitiva que permite a los estudiantes analizar, evaluar y reflexionar sobre la información de forma objetiva, teniendo en cuenta su edad y nivel de desarrollo. pág. 7582 Canchihuaman et al. (2018), sostienen que es importante que los docentes promuevan el desarrollo del pensamiento crítico desde la primera infancia, para que los estudiantes puedan desarrollar su creatividad y solucionar problemas de forma efectiva a medida que crecen. De acuerdo al autor, es primordial que los docentes desarrollen el pensamiento crítico desde los niveles preescolares, con la finalidad de que, al ir avanzando en sus estudios, éstos sean unos referentes para que puedan desarrollar y dar solución a problemáticas presentadas de acuerdo a su nivel. En ese sentido Vargas (2019) afirma que la literatura infantil puede ayudar a promover el pensamiento crítico, ya que expone a los estudiantes a diferentes perspectivas y puntos de vista. En el contexto de este estudio, la población objeto de estudio es una comunidad rural en Ecuador. La mayoría de los habitantes se dedican a la agricultura, la ganadería o el comercio. La lectura no es una práctica común en esta comunidad, por lo que es importante encontrar estrategias para promover el pensamiento crítico a través de la literatura infantil. El contexto sociocultural de la comunidad en estudio explica la baja prevalencia de la lectura en el hogar y la escuela. La mayoría de los habitantes se dedican a actividades agrícolas, ganaderas o comerciales, lo que les deja poco tiempo para la lectura. Además, las costumbres y tradiciones de la comunidad no alientan el hábito de la lectura. Es crucial presentar y detallar las estrategias específicas para fomentar el pensamiento crítico a partir de la literatura infantil, considerando la diversidad lingüística y cultural de los estudiantes en la Escuela "1 de Marzo". Además, es importante enfatizar la relación directa entre el desarrollo del lenguaje y el pensamiento crítico, respaldándolo con estudios y ejemplos de Jean Piaget, lo que podría enriquecer y sustentar de manera más efectiva el apartado relacionado con el lenguaje y su papel en el desarrollo del pensamiento crítico. La importancia del pensamiento crítico radica en su estrecha relación con la comprensión lectora, permitiendo al lector adentrarse en el texto y obtener una comprensión profunda y reflexiva, especialmente en un mundo cada vez más globalizado. Según Serrano (2008) la lectura trasciende la simple repetición de palabras; implica una comprensión profunda del contenido, facilitando la adquisición de conocimiento y la aplicación práctica de los mensajes en la vida cotidiana. El pensamiento crítico no solo es crucial en el ámbito educativo, sino que también desempeña un papel esencial en la esfera profesional. Esta perspectiva busca educar para la vida, promoviendo la pág. 7583 creatividad, la transformación y el cuestionamiento de lo establecido, tanto en el ámbito personal como profesional. Los cuestionamientos críticos incitan a las personas a generar conocimiento, provocar cambios y explorar nuevas alternativas, abriendo caminos hacia respuestas innovadoras y desafiando la complacencia. Un lector competente distingue sus habilidades al analizar un texto desde múltiples perspectivas, desglosando sus elementos significativos, evaluando su estructura narrativa, la solidez de sus argumentos y la credibilidad de las ideas presentadas. Identificar las ideas principales y secundarias es parte esencial de esta competencia lectora. Por otro lado, según las reflexiones de Lizarazo (2022), el pensamiento crítico ha sido analizado desde diversas disciplinas como la filosofía, la psicología y la ciencia, destacando su influencia en el desarrollo educativo. Desde edades tempranas, se considera crucial que los estudiantes aprendan a discernir y emitir juicios críticos frente a aspectos positivos y negativos de su entorno. Existen varios estudios orientados a abordar esta temática, con el propósito de no solo resolver esta problemática, sino también de fomentar habilidades que permitan a los estudiantes evaluar la literatura infantil desde una perspectiva crítica. Destacan los trabajos llevados a cabo en instituciones como la Universidad UNAE, la Universidad UNIANDES, entre otras, las cuales están promoviendo la escritura para niños y el desarrollo del pensamiento crítico entre los educandos. Esto motiva a los estudiantes a gozar la lectura de un texto de literatura de cualquier género o tipo, por lo que la institución educativa debe proveer estos insumos para que los estudiantes enriquezcan sus conocimientos, desarrollen habilidades críticas y estén prestos a resolver problemáticas de su contexto. Acordes con los lineamientos curriculares, el Ministerio de Educación del Ecuador (2021), menciona que: Los elementos del Currículo se evidencian en el diseño de destrezas básicas que se tornan imprescindibles y deseables. Las habilidades con criterios de desarrollo necesarios se analizarán en el transcurso del subnivel para impedir que el estudiante entre en el peligro de exclusión social y rezago en sus estudios; mientras que, las destrezas con criterios de desempeño deseables pueden desarrollarse en este subnivel o en el siguiente. (p. 13) pág. 7584 Esto conlleva la existencia de una comisión encargada de proporcionar los recursos virtuales y digitales a las Instituciones educativas, las mismas que son elaboradas tomando en cuenta los años de básica en la que cursan, como también mediante actividades de trabajo que ayuden en el impulso del raciocinio crítico mediado por la literatura infantil. En la literatura infantil, se destacan estudios con autores que promueven la didáctica para que los niños, comprendan lo que su docente les transmite, a lo cual Escalante et al. (2008) afirman que, desde la enseñanza inicial y preescolar, los niños señalan interés por indagar diferentes materiales de escritura, que a su vez les induce a expresar ideas, vivencias, emociones, sentimientos, experiencias reales externalizando su mundo interior. La literatura infantil comprende entonces varios géneros, destacando: Cuentos de hadas: Son historias fantásticas que involucran momentos mágicos, con personajes imaginarios y heroicos, lugares atraen a los niños a formar parte de ellos. Fábulas: Cuentos cortos, en que se personifican a los animales, humanizándolos, lo fascinante es que dejan una enseñanza o moraleja. Cuentos populares y leyendas: Son narrativas que pasan de generación en generación y reflejando la cultura autóctona de los diferentes pueblos y países. Poesía infantil: Son colecciones de poemas presentados en un estilo lúdico, propio para los niños, suelen usar la rima y el ritmo para entretener y educar a los niños. Cuentos de aventuras: Historias llenas de emoción en que los protagonistas se sumergen en viajes y desafíos, estimulando la imaginación y la exploración de nuevos mundos. Libros informativos: Proporcionan una amplia gama de temas, como animales, ciencia, historia y geografía, para que los niños comprendan fácilmente. Literatura de aprendizaje: Son libros interactivos que enseñan habilidades básicas, como contar, reconocer colores, letras, a desarrollar la memoria y la percepción. Cuentos humorísticos: Hacen reír a los niños con situaciones cómicas, juegos de palabras y personajes risibles. Literatura multicultural: Exponen diversidad cultural y étnica, con historias y personajes de diferentes procedencias pág. 7585 Las interrogantes planteadas apuntan a solucionar esta problemática: ¿Qué estrategias metodológicas pueden aplicarse para fomentar el pensamiento crítico desde la literatura infantil en niños de edad preescolar? ¿De qué manera la literatura infantil contribuye al desarrollo del pensamiento crítico en los niños preescolares de la Escuela "1 de Marzo"? ¿Qué parámetros metodológicos pueden emplearse para impulsar la literatura infantil y promover el desarrollo del pensamiento crítico en la institución educativa? ¿Es viable implementar estrategias metodológicas para fortalecer el pensamiento crítico desde la literatura infantil en los niños de preescolar en la Escuela de Educación Básica "1 de Marzo"? Estas preguntas buscan identificar y desarrollar estrategias efectivas que puedan superar la falta de énfasis en la literatura infantil, mejorando así la comprensión crítica entre los estudiantes de preescolar en la institución educativa mencionada. METODOLOGÍA El trabajo investigativo partió de un enfoque cuantitativo-cualitativo. Cuantitativo pues la información numérica se trató con estadística descriptiva, la muestra fue determinada a través de fórmula, la graficación se realizó a través de Excel y enfoque cualitativo porque estos datos obtenidos fueron sometidos a revisión analítica basada en el marco teórico, donde se realizan descripciones y de hecho las premisas o preguntas de los cuestionarios, son de tipo descriptivo, hacen referencia a situaciones referidas precisamente a la literatura infantil y pensamiento crítico. En el análisis se utilizó el método mixto, que, según Hernández et al., (2010) expresan un grupo de eventos “empíricos, sistemáticos y críticos de investigación. Como modalidad del presente estudio, se acudió a la de campo, cuyo análisis de casos se realizó en el aula de la institución educativa 1 de Marzo. Esta modalidad permitió a la investigadora contactar directamente con los sujetos investigados, concordando con los objetivos investigativos. Así también se recurrió a la bibliográfica – documental, misma permitió conocer y profundizar los enfoques, conceptualizaciones, teorías de autores sobre literatura infantil y el pensamiento crítico, cuyos documentos han sido citados debidamente para una mejor comprensión y enfoque del estudio. pág. 7586 Se utilizó la investigación de tipo exploratoria, factible de utilizar en el método cualitativo y cuantitativo; aquí el estudio se aplica a hechos no indagados con anterioridad, que se quieren examinar sus características, (Ramos, 2020); el enfoque cualitativo se basa en los estudios de la literatura infantil y su incidencia para desencadenar pensamiento crítico en los niños. Se usó además el tipo descriptivo, por detallar al grupo estudiado, como mencionan Guevara et al., (2020) señalan que el objeto de esta investigación es caracterizar conjuntos homogéneos de fenómenos, con criterios sistemáticos dando una estructura o el comportamiento de la situación en estudio, para dar información ordenada, comparable con otras fuentes. La investigación se centra en la influencia de los textos infantiles en el desarrollo del pensamiento crítico en la población preescolar de la Escuela de Educación Básica "1 de Marzo". Se dispuso de una población total 120 estudiantes de preescolar. Además, se incluyeron 120 padres de familia y 4 docentes correspondientes al nivel de preescolar. Se tomarán las 4 docentes del grado de preescolar para establecer la muestra de los niños, siendo 120 niños de preescolar y 120 padres de familia de la escuela “1 de Marzo”, los resultados se obtuvieron aplicando la siguiente formula: 𝑛= 𝑍 2 𝑃𝑄𝑁 𝑍 2 𝑃𝑄 + 𝑒 2 (𝑁 − 1) 𝑍 2 𝑃𝑄𝑁 𝑛= 2 𝑍 𝑃𝑄 + 𝑒 2 (𝑁 − 1) 𝑛= 1.962 ∗ 0.5 ∗ 0.5 ∗ 120 1.962 ∗ 0.5 ∗ 0.5 + 0.052 ∗ (120 − 1) 𝑛= 3.84 ∗ 0.5 ∗ 0.5 ∗ 120 3.84 ∗ 0.5 ∗ 0.5 + 0.0025 ∗ (120 − 1) 𝑛= 115.2 1.2575 𝑛 = 91 Dando como resultado las siguientes muestras. Tabla 1. Resultado de la población Muestra estudiantes Muestra padres de familia Muestras docentes 91 91 4 pág. 7587 Así, se reúne un total de encuestados de 186, cada uno de los tres grupos contará con una encuesta dirigida a su grupo correspondiente (padres, docentes, estudiantes). El instrumento para utilizar es la encuesta, la cual es un método de recopilación de datos para obtener información y opiniones de un grupo específico de personas; se elaboraron tres formatos para cada grupo respectivo de encuestados. Por lo general, se utilizan preguntas estandarizadas para recopilar datos referentes a comportamientos, preferencias, opiniones, actitudes, o características demográficas de la muestra poblacional (Monserrat, 2015). El cuestionario es un conjunto de preguntas donde los encuestados expresan su respuesta al requerimiento. La técnica para analizar los datos recolectados se basa en el uso de la herramienta ofimática de Excel, misma que permita el conteo o totalización de las preguntas, de acuerdo a las opciones de respuesta, permite a la vez su traficación y el porcentaje obtenido, de acuerdo con las respuestas. RESULTADOS Y DISCUSIÓN Los resultados obtenidos se relacionan con un conjunto común de temas planteados en las preguntas, los cuales se organizaron considerando los objetivos establecidos en la investigación. La información proporcionada por los docentes en las preguntas abiertas se analiza para determinar el porcentaje basado en la frecuencia de repeticiones, lo cual es significativo. Al consultar sobre su experiencia laboral, los docentes destacaron los beneficios de los textos infantiles en el mejoramiento del pensamiento crítico. El 100% de los docentes afirmaron que estos textos fomentan el disfrute, la creatividad y la absorción de la lectura, permitiendo así potenciar la imaginación. Esta práctica genera interés en los estudiantes por explorar los aspectos narrativos, especialmente durante la etapa evolutiva, en la que los niños son más susceptibles a asimilar cualidades y adoptar estados de ánimo, deseos y sentimientos como modelos a seguir (Romero & Silveira, 2019), Esto da lugar a lo que se conoce como Zona de Desarrollo Próximo, evidenciando que las estrategias metodológicas basadas en la literatura infantil promueven el pensamiento crítico desde una edad temprana. Además, al solicitar a los docentes que identifiquen los tipos de literatura infantil que más les gustan a los estudiantes en base a su experiencia, se evidencia un especial interés en los cuentos infantiles, con un 75%, en comparación con los libros ilustrados, que representan un 25%. Esta aportación es valiosa, ya que los niños muestran mayor atención cuando alguien más les lee, especialmente si la literatura está pág. 7588 acompañada de ilustraciones, ya que estas captan su interés y motivan la exploración continua (Figura 1). Se propone un listado de textos de literatura infantil y se solicita que se clasifiquen en orden de importancia, según el criterio personal y profesional de las maestras, considerando cuatro grupos de textos: libros con imágenes, poesía infantil, fábulas y cuentos de hadas, con una distribución del 25% para cada categoría. El objetivo es descubrir y aprender mediante la heurística del descubrimiento, practicando la resolución de problemas y esforzándose por descubrir; cuanta más práctica, mayor capacidad de generalización. Aunque el gráfico resalta la relevancia de las ilustraciones en el proceso de lectura, la trama, los personajes y el contenido de los cuentos en sí mismos prevalecen. Esta idea es respaldada por Soto (2017) quien señala que los cuentos desarrollan micro destrezas del pensamiento crítico, como la anticipación, las competencias comunicativas, la inferencia, la realización de juicios, la interpretación y la predicción. Imaginar los diversos escenarios narrados en el cuento potencia la creatividad y la imaginación; las ilustraciones funcionan mejor cuando es el niño quien lee. Al analizar los parámetros metodológicos que ayudan al docente a promover la literatura infantil, se formuló la pregunta sobre cómo se apoya para estimular el pensamiento crítico en los niños. El 75% indicó un apoyo significativo, mientras que el 25% lo calificó como poco. Esto indica que fomentar el trabajo en equipo desde una edad temprana, promoviendo círculos de estudio que fomenten la competitividad académica, refuerza el interés por la literatura infantil y enriquece el lenguaje visual, promoviendo la imaginación y la fantasía. Respecto a la percepción sobre la importancia de la literatura infantil y su influencia en el desarrollo del pensamiento crítico en los niños en edad preescolar, el 75% considera que es muy importante y el 25% la considera importante. Ambos porcentajes reflejan una percepción positiva, lo que indica la necesidad de aplicar y seguir fomentando la literatura infantil desde edades tempranas. En este punto, se alinean tanto la motivación intrínseca como la extrínseca, ya que el aprendizaje por descubrimiento conduce a una tendencia hacia la independencia y la auto gratificación por los hallazgos propios. En relación con el tema de estudio, se planteó la pregunta de si es factible establecer estrategias metodológicas, y el 100% de los docentes respondieron afirmativamente. Este resultado subraya la necesidad de impulsar el pensamiento crítico en la educación, ya que, en el contexto actual de las pág. 7589 competencias según la Constitución Ecuatoriana de 2008, el pensamiento crítico debe ser considerado como una destreza esencial. Además, se evidencia que, dentro del plantel educativo, en la sección de educación inicial y preparatoria, no existen estrategias metodológicas para mejorar el pensamiento crítico de los estudiantes, por lo que sería recomendable desarrollar estrategias con el fin de mejorar este aspecto educativo. En esta misma dinámica se indagó, si la literatura infantil ha apoyado en distintos ámbitos al desarrollo de destrezas de los infantes, el 50% señalan que ha posibilitado a mejorar las relaciones interpersonales y un 50% indican que los niños son más reflexivos; de hecho, esto se corrobora cuando se consultó si en los libros han encontrado desafíos cognitivos o conceptos complejos, y señalaron con el 75% que sí y el 25% no, este apunte es relevante, puesto que, el uso de la literatura infantil impulse un esfuerzo cognitivo; nace entonces la inquietud si los juegos con expresiones verbales , pueden potenciar el pensamiento crítico de los niños, los encuestados se ratifican con el 100% que ayuda a la expresión verbal en el desarrollo del pensamiento crítico, bien lo ratifica González, et al. (2014) que manifiesta que la actividad lúdica permite al niño eliminar sus fronteras y le posibilita a desarrollar su personalidad. Los resultados muestran que los docentes consideran la factibilidad de establecer estrategias metodológicas, como se mencionó con anterioridad al no tener establecidas las metodologías respectivas, no se está cumpliendo al 100% el desarrollo del pensamiento crítico en los estudiantes de educación inicial. Con relación a los padres de familia, pues forman parte del conjunto de educación, con lo cual los representantes conviven más tiempo con los estudiantes, por tal motivo su obligación es realizar el seguimiento y cumplimiento de las tareas escolares. En esa sintonía y con el fin de analizar cómo la literatura infantil promociona el desarrollo del pensamiento crítico en los niños, y consecuentes del rol de los padres en la enseñanza, se solicitó señale tres actitudes y/o comportamientos que ayuden a desarrollar habilidades de análisis y pensamiento crítico, de los encuestados el 60% manifiestan que han desarrollado la creatividad, saber pensar antes de contestar y aprender nuevas palabras, el 20% han encontrado niños motivados, animados y críticos; el 20%, señaló que han desarrollado la curiosidad, el pensamiento y las emociones; por tanto, los padres son el eje de educación de sus hijos y el interés por la enseñanza lo asume desde su concepción, lo que honra un compromiso con la escuela, he ahí la pág. 7590 importancia de forjar una colaboración mutua entre docentes y representantes; sin ser ambiguo o arbitrario para que relacionen las ideas pertinentes que se hallen en la capacidad de aprendizaje del estudiante. En la figura 2, sobre el impulso de la lectura de la familia en el niño, se observa que el 70% ha respondido como aspecto relevante a la motivación, el 20% señala por instrucciones, el 7,5% lúdicamente, y el 2,5% de forma obligada, lo que se pone de manifiesto que el padre de familia colabora en la educación de sus hijos, apoyándose en las tareas en casa, según Martínez et al. (2020) afirman que los padres de familia son “modelos del desarrollo” (p. 4) del niño al promover su socialización, formación y crecimiento, asumiendo características específicas; bien se dice que la familia es la base de la sociedad pues en ella se desarrollan dimensiones del ambiente familiar, la forma de estudio y de comportamiento en la comunidad; por tanto, a la pregunta del tipo de textos prefiere que su hijo lea para promover su pensamiento crítico, manifiestan los encuestados que el 84% tiene preferencia por los cuentos, el 4,9% por los textos de secuencia lógica, el 4,9% la poesía y otros (3,7), y el 2,5% textos lúdicos, de ahí la importancia de los roles que tienen los involucrados en la educación, ya se dijo que uno son los padres, el segundo son los docentes y su clima organizacional, y por último el estudiante. De acuerdo a los resultados obtenido los cuentos son una parte esencial para promover el desarrollo del pensamiento crítico de los estudiantes de acuerdo a los padres de familia, siendo que dentro de la institución en el bloque de educación inicial tiene escaso material lector sobre todo cuentos, debido a esto no se puede reforzar el pensamiento crítico de los estudiantes dentro del establecimiento educativo. Al evaluar la importancia de la literatura infantil en el desarrollo del pensamiento crítico de los niños y en coherencia con los parámetros metodológicos, se planteó la pregunta sobre los tipos de textos que los padres leen con sus hijos. El 43,8% indicó las fábulas, el 36,3% mencionó las historias, el 15% se refirió a otro tipo de texto y el 4,9% eligió la poesía. Estos datos se respaldan con la pregunta sobre la frecuencia de lectura de cuentos, donde el 56,8% respondió que a veces, el 22,2% casi siempre, el 17,3% siempre y el 3,7% casi nunca. Estos resultados generan preocupación, ya que al asociar el 56,8% que lee a veces con el 3,7% que casi nunca lee, revela un alto índice de falta o escaso acompañamiento en la lectura con sus hijos. Tal como se mencionó anteriormente, la carencia de material adecuado para fomentar la lectura en los estudiantes de preescolar pág. 7591 es evidente, y los resultados de la Figura 3 sugieren que en los hogares también existe una falta de fomento de la lectura. Esto posiblemente incide en que el estudiante no desarrolle de manera efectiva su pensamiento crítico. En esencia la lectura es la base del crecimiento intelectual del niño, Delgado (2022) sostiene que es un proceso intelectual forjado desde el nivel preescolar, por ser la fase preoperacional del desarrollo del niño y las actividades deben ser coherentes con las características del niño así, mientras más temprano se inicia, generará un mejor rendimiento del potencial que se está formando; desde la perspectiva de las emociones que se manifiesta con la lectura se aprecia que el 96,3% declara alegría, el 2,5% desconocen y el 1,2% demuestran confusión, sin embargo, el gusto es un buen indicador de la importancia de ser consecuentes y continuar con el trabajo gestado en el aula y que debe complementarse en casa, con los padres de familia. La tercer meta u objetivo es el diseño de tácticas metodológicas factibles para el desarrollo del pensamiento crítico de los niños, se ha establecido preguntar si su hijo demuestra interés por lo que lee, los encuestados señalan que el 48,7% a veces demuestran interés, el 46,3% siempre y el 5% nunca, esta pregunta tiene concordancia. Al plantear si su hijo puede diferenciar entre realidad y ficción al leer libros infantiles, se establece en los siguientes resultados (figura 4). En cuanto a la capacidad de diferenciar la realidad y ficción, el 55% señalan poco, el 37,5% mucho y el 7,5% nada, por ello hay que recurrir al rol del docente en incentivar en los niños y así como en los padres de familia a asumir su rol; lo importante está en hacer reflexionar que ser padre no solo es el hecho de matricular a su hijo y despreocuparse, pues es obligación de los padres “propiciar un ambiente de aprendizaje adecuado en su hogar, organizando espacios dedicados a las obligaciones y responsabilidades escolares…” (Ministerio de Educación de Ecuador, 2017, Artículo 13, literal f.), por ello la corresponsabilidad es importante, para generar un desarrollo holístico de sus hijos. Empero al preguntar sobre en qué medida cree que los libros infantiles pueden estimular la imaginación y la creatividad del niño el 78,8% señalaron que mucho, el 17,5% medianamente y el 3,7% poco, lo que corrobora la importancia de promover la participación activa del estudiante, dada la importancia del aprendizaje por descubrimiento que requiere una situación del contexto como reto permanente a la inteligencia del aprendiz y conseguir que resuelva problemas e incluso que comparta su aprendizaje. pág. 7592 Para dar respuesta a las preguntas del antecedente, en ilación con los objetivos, se hace necesario examinar el tercer grupo encuestado, los estudiantes para analizar cómo la literatura infantil influye en la potenciación del desarrollo del pensamiento crítico en los niños de preescolar, por ello fue preciso conocer el lugar mágico el 72% señalan que estarían en un río, el 15% se verían en el bosque y el 13% en su casa, lo que se acercó a determinar cómo sería el cuento ideal estableciendo que el 80% sería la familia y el 20% de animales; en los niños de preescolar se manifiestan la curiosidad y es un indicador que debe ser aprovechado por el docente, según Correa (2009) la narrativa de cuentos permite satisfacer profundas necesidades, contrastando al niño con su entorno para que lo reinvente desde su perspectiva, se trata de sumergir al educando en el contexto del cuento, de la trama de la lectura. Dado los resultados es importante tener un área de lectura donde el niño pueda simular o sentirse a gusto al momento de leer, ya que esto ayudara a mejor la concentración, al igual que su pensamiento crítico, al ser una institución con pocos recursos, no es posible tener este tipo de área o ambiente, por lo cual el estudiante no va a sentirse cómodo y no podrá concentrarse, debido a esto no podrá desarrollar correctamente su pensamiento crítico. En el propósito de explorar los fundamentos teóricos que sustentan la literatura infantil y determinar el aporte al crecimiento del intelecto crítico en los estudiantes de preescolar, se planteó la pregunta del gusto por los cuentos, el 98.9% respondieron que sí y el 1.1% que no, lo señalado tiene consecuencia con la pregunta sobre si lee cuentos en su casa ya que el 92,1% dice que sí, el 6,7% que no y el 1,1% rara vez; por lo visto se determina que los estudiantes gustan del cuento e inclusive se corrobora con la pregunta le gustaría escribir cuentos donde el 95.5% dijeron que sí, y apenas el 4.5% se pronunciaron que no, entonces, corresponde a los docentes a seguir utilizando y trabajando en este tipo de literatura infantil, pues a decir de Oviedo & Páez (2020) señalan que el pensamiento crítico es como piensa en situaciones educativas que lo favorecen, por ejemplo si se alientan por la investigación educativa. Con el objeto de diagnosticar la relevancia de la literatura infantil en el desarrollo del pensamiento crítico se estableció el cuestionamiento de qué emociones siente al leer cuentos, se evidenció que el 83,1% alegría, el 12,4% emoción, el 3,4% responden tristeza y el 1,1% enfado, lo propio cuando se le preguntó los sentimientos que tiene cuando dibuja alguna escena de un cuento donde el 92.1% respondió alegría, el 3,4% entre el miedo y la tristeza, en tanto que el 1.1% siente enojo, lo que motiva a escoger pág. 7593 con diligencia la literatura infantil para motivar el interés en los niños, bien lo señala Oviedo & Páez, son las situaciones áulicas donde se generan las motivaciones y que los maestros tendrían que estar preparados y capacitados, en consideración que son niños que absorbe como esponja todo lo que ve, escucha y hace. Ahora, en cuanto a diseñar estrategias metodológicas que sean factibles hacia el desarrollo del pensamiento crítico de los niños, se estableció la pregunta de qué le motiva para crear un cuento (figura 5); De lo señalado, fue oportuna la consulta sobre si le gustaría que le cuenten cuentos donde puede observar dibujos, y el 98.9% respondió que sí y el 1.1% dijo no, lo que ratifica la importancia de seguir incentivando la literatura infantil acompañada de gráficas atractivas que llamen la atención de los niños, donde los espacios de aprendizaje son sitios relevantes y atractivos para los niños de preescolar porque desarrollan hábitos, independencia, siguen reglas y estimulan la capacidad de tomar decisiones; el aprendizaje se construye y produce a partir de las situaciones que cambian los esquemas mentales. Las comunidades construyen ideas sobre su entorno y éstas se replantean y evolucionan para cambiar. Sin lugar a dudas y desde diferentes perspectivas, es la literatura infantil el pilar sobre el cual se construye el pensamiento crítico, con la enorme posibilidad de sus diferentes manifestaciones desde lo poético, histórico, ficción, hasta las novedosas posibilidades que cada estudiante pueda dar en su pensamiento mientras desarrolla y proyecta su raciocinio. Así entonces, desde las perspectivas del docente, del padre de familia y del mismo educando, la tierra está abonada para sembrar la semilla de la lectura como catapulta al pensamiento crítico, bastión del desarrollo humano. Figura 1. Tipos de Literatura infantil les agrada más a sus estudiantes Libros ilustrados 25% Literatura tradicional Adaptaciones clásicas Literatura popular. 75% Ficción Cuento. pág. 7594 Figura 2. Manera de promover la lectura en su hijo Figura 3. Frecuencia de lectura en casa 4% 17% Siempre Casi siempre 22% 57% A veces Casi nunca Figura 4. Capacidad del niño de discernir entre la realidad y la ficción al leer libros infantiles 8% 38% Mucho Poco Nada 54% Figura 5. Que le motiva para crear un cuento pág. 7595 CONCLUSIONES El ejercicio integral permitió obtener los siguientes resultados; primero que todo, con relación al diagnóstico se conoció que los docentes tienen conocimiento de cómo promover el pensamiento crítico a través de la literatura infantil, sin embargo, se hace necesario ahondar o capacitar en el tema para que los que desconocen cómo proceder, tengan un mayor campo de posibilidades en el desarrollo de su labor. El diagnóstico facilitó una importante información de los docentes y padres de familia con respecto a su experiencia y conocimiento acerca de la literatura infantil en el desarrollo del pensamiento crítico, permitiendo afirmar que hay mucho camino por recorrer, sin embargo la actitud tanto de padres de familia como de los docentes es activa y alentadora en la materia; hay mucho potencial para trabajar en el objetivo general; de hecho la aplicación de las encuestas despertó inquietud y ganas de trabajar en el tema, por parte de los encuestados. Los padres y docentes tuvieron en común en algunas de sus respuestas que la literatura infantil mejora en los discentes, el vocabulario, la pronunciación, la imaginación, la resolución de conflictos, la creatividad, la rapidez de pensamiento, sus acciones en sus hogares, las relaciones interpersonales, genera motivación, disfrute, actitud positiva, sonrisas, mejora su comprensión de los hechos y de las palabras. Si bien es cierto, como se estableció a comienzos de la investigación, en la escuela no se cuenta con suficientes recursos y materiales para trabajar es posible generar espacios e ideas que contribuyan al aporte de la construcción de conocimiento y como propende la presente investigación, del fortalecimiento del raciocinio crítico por medio de la Literatura Infantil, lo que implica la consecución de un número mayor de textos, ya sea en físico, o electrónico con la posibilidad de hacerlo visible por televisor o computador a los niños de preescolar, ya que como también fue establecido en el trabajo, no se cuenta con materiales de este tipo dentro de la escuela. En lo que refiere a la exploración de fundamentos teóricos, permitieron no solamente ampliar los conocimientos en la materia de estudio, sino que fortalecen la estructura del trabajo, ampliaron los horizontes, y dieron una orientación con base en la información plasmada. Haciendo referencia al tercer objetivo, las posibles estrategias requieren una dinámica primero de planeación y orientadora, para que pág. 7596 las mismas circunstancias y entorno en que se dan, faciliten el ejercicio que sea el vehículo esperado para conseguir un fortalecimiento del pensamiento crítico. REFERENCIAS BIBLIOGRAFICAS Canchuhuaman, M., & Calero, E. (2018). Pensamiento crítico y habilidades sociales en los niños de 5 años de las instituciones educativas del nivel inicial de San Juan Pampa-Yanacancha. http://repositorio.undac.edu.pe/bitstream/undac/335/1/T026_71990891_T.pdf,Ed. Universidad Nacional "Daniel Alcídes Carrión". Correa, M. (2009). El cuento, la lectura y la convivencia como valor fundamental en la educación inicial. Educere, 44, 89-98. Delgado, V. (2022). La lectura recreativa y su aporte en el aprendiza<je de los niños y niñas en la etapa pre-operacional. Polo del conocimiento, 7(7), 462-473. Escalante, D., & Caldera, R. (2008). Literatura para niños: una forma natural de aprender a leer. Educere, 12(43), 669-678. Obtenido de https://www.redalyc.org/pdf/356/35614570002.pdf González, C., Solovieva, Y., & Quintanar, L. (2014). El juego de roles sociales: aportes al desarrollo en la edad preescolar. Avances en Psicología Latinoamericana, 32(2), 287 - 308. Guevara, G., Verdesoto, A., & Nelly, C. (2020). Metodologías de investigación educativa (descriptivas, experimentales, participativas, y de investigación-acción). Recimundo, 4(3), 163-173. Obtenido de https://recimundo.com/index.php/es/article/view/860 Hernández, C., Fernández, R., & Baptista, P. (2010). Metodología de la investigación. Quinta edición. Ciudad de México: Mac Graw Hill. Obtenido de https://www.icmujeres.gob.mx/wpcontent/uploads/2020/05/Sampieri.Met.Inv.pdf Lizarazo, N. (2022). Aprender a pensar: Propuesta para la estimulación del pensamiento crítico en niños de preescolar del colegio Luz de la Esperanza, sede B, del corregimiento de Berlín, municipio de Tona. Universidad Autónoma de Bucaramanga. Martínez, G., Torres, M., & Ríos, V. (2020). El contexto familiar y su vinculación con el rendimiento académico. Investigación educativa de la Rediech, 11, 1-17. Ministerio de Educación de Ecuador. (19 de mayo de 2017). Ley Orgánica de Educación Intercultural. Quito: Ministerio de Educación . pág. 7597 Ministerio de Educación del Ecuador. (2021). Lineamientos curriculares para instituciones educativas multigrado. Subsecretaría de Fundamentos Educativos. Monserrat, P. (2015). Aplicación de la metodología de la investigación para identificar las emociones. 6(11), 35-42. Obtenido de https://www.redalyc.org/pdf/4981/498150319048.pdf Oviedo, P., & Páez, R. (2020). Pensamiento crítico en la educación. En R. Páez, & P. Oviedo, Problemas y posibilidades del pensamiento crítico en la educación (págs. 11-34). Universidad La Salle. Ramos, C. (2020). Los alcances de una investigación. CienciAmérica, 9(3), 2. Obtenido de Los alcances de una investigación: https://dialnet.unirioja.es/descarga/articulo/7746475.pdf Romero, C., & Silveira, D. (2019). Importancia de la literatura infantil en el desarrollo del niño de 4 años. Institución de formación docente "Ercilia Guidali de Pisano". Serrano, M. (2008). El desarrollo de la comprensión crítica en los estudiantes universitarios: hacia una propuesta didáctica. Educere, 12(42), 505-514. Obtenido de https://www.redalyc.org/pdf/356/35614569011.pdf Soto, M. (2017). El cuento como mediación pedagógica para el fortalecimiento de la lectoescritura. Zona Próxima(27), 51-65. Vargas, N. (2019). Experiencias didácticas mediante la Literatura infantil latinoamericada. Repertorio Americano(29), 328-352. pág. 7598
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WaterSwap Analysis, a Computation-based Method for the Discovery of Effective and Stable Binding Compounds for Mutant EGFR Inhibition
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WaterSwap Analysis, a Computation-based Method for the Discovery of Effective and Stable Binding Compounds for Mutant EGFR Inhibition Kshipra S. Karnik Dr Babasaheb Ambedkar Marathwada University Aniket P. Sarkate Dr Babasaheb Ambedkar Marathwada University Vaishanavi S. Jambhorkar Dr Babasaheb Ambedkar Marathwada University Pravin Wakte (  pswkshipra16@gmail.com ) Dr Babasaheb Ambedkar Marathwada University https://orcid.org/0000-0002-8118-2963 Research Article Keywords: EGFR, Waterswap, Virtual screening, Molecular docking, ADMET, Styrylquinoline Posted Date: October 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-981656/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/10 Abstract The epidermal growth factor receptor of the tyrosine kinase family has been largely targeted in mutations associated with nonsmall cell lung cancer. EGFR inhibitors have been produced that bind allosterically to the C797S mutant EGFR enzyme. Here, the Waterswap tool has been used for the interpretation and visualization of trajectories of mutant EGFR-ligand complexes. Virtual screening of the generated compounds has been carried out along with its molecular docking and ADMET analysis. Out of the generated library of compounds, the top 15 have been selected. Waterswap calculated the binding free energies of the compounds and thermodynamic properties of the enumerated compounds were compared with that of standard EAI045. It was observed that styrylquinoline stabilized better than EAI045. The results show that Waterswap analysis offers a promising new path in the hunt for improved tools for analyzing and visualizing molecular driving forces in protein-ligand complex simulations. Introduction Cancer is the second biggest cause of death in the world, and it is one of the primary causes of morbidity and mortality. In 2015, cancer claimed the lives of 8.8 million people. Cancer is responsible for about one-sixth of all fatalities worldwide [1]. Despite decades of attempts to find and develop small molecule anticancer medications, the demand for novel antitumor medicines with enhanced tumour efficiency, selectivity, and safety remains critical [2, 3]. One of the most significant enzymes in the tyrosine kinase family is the Epidermal Growth Factor Receptor. EGFR is a transmembrane receptor that is found on the cell surface. The EGFR enzyme is classified as (ErbB1, HER1), ErbB2 (HER2, neu in rodents), ErbB3 (HER3), and ErbB4 (HER4) based on structural similarities [4]. EGFR and its members have become a popular target for anticancer therapy, particularly in the treatment of non-small cell lung cancer [5]. Activating mutations in the EGFR gene had a significant impact on non-small cell lung cancer therapy approaches (NSCLC). The recent identification of novel EGFR inhibitors was critical in overcoming such mutations [6]. Drugs' affinity for their targets is described by thermodynamic characteristics. As a result, accurate thermodynamic property calculations have long been a goal of computational chemistry. Hence, accurate thermodynamic property calculations have long been a computational chemistry goal. In the industry, study of solvation patterns surrounding pharmaceuticals and binding sites has become a major goal for molecular modelling [7–9]. According to an estimate, hydrophobic surfaces cover 75% of surface drug sites [10], implying that the hydrophobic effect is one of the most energetic contributions to ligand binding and recognition. Individual water molecules that serve a crucial stabilising role, for example, by building bridges between the ligand and the receptor, have been shown by high-resolution co-crystallized structures of protein-ligand complexes [11, 12]. Improved binding affinity has also been linked to a network of H-bond-enriched water molecules around the ligand [13–15]. The water-swap reaction coordinate was described by Woods et al. as a new reaction coordinate (WSRC) [16]. The ligand and water are exchanged from being bound to the protein to being solvated by a periodic box of bulk water in this approach. Because no molecules are moved into a vacuum, the enormous decoupling free energy associated with double decoupling procedures is avoided. Furthermore, a new approach for restricting the identity of solvent clusters within bulk has been established, allowing the swap to be accomplished without the requirement for any restrictions on the ligand or swapped water, and this strategy applies to both buried and solvent-exposed binding sites [16]. The concept behind the water-swap reaction coordinate is that it permits the identity of water clusters to be limited during simulation. During a simulation, this identity constraint [17] works by modifying how water molecules are labeled. We used computation-based methods such as virtual screening, molecular docking, and WaterSwap analysis to find an effective compound with stable binding and mutant EGFR inhibition effect in this study. Materials And Methods Insilico methodology ChemT tool was used for library generation and enumeration. The molecular docking tool, AutoDock Vina, was used for docking studies of all compounds on the EGFR enzyme. SwissADME tool was used to determine ADMET studies. Waterswap study was performed on trial version of Cresset’s Flare software. Page 2/10 ChemT and Library Enumeration ChemT, a Biochemcore-based software, was used to enumerate the library. In Marvin Sketch, the R-group position on the template styrylquinoline. The styrylquinoline was then assigned to the identified R-groups through the enumeration model of the software. EAI045 input determined the standard template. Additionally, few available pharmacokinetic properties such as molecular weight, H-bond donors, and Lipinski rule of five was set for designing drug-like compounds [18]. Molecular Docking To investigate the binding pocket, residue interactions, and predicted binding affinity of the enumerated compounds, molecular docking protocol was carried out using AutoDock tools. Through Chimera modeler, the protein was fetched from the open-source PDB libraries. Explicit hydrogen bonds were added followed by the removal of water molecules. Using Gastegier charges, ions were removed and finally, the protein was minimized using the Amber94 force field. The total number of loops was set to 1000 to ensure maximum minimization of the protein. The minimization was terminated when the energy converged or the root mean square deviation (RMSD) reached a maximum cut-off of 0.30 Å. Ramachandran plot was set to check the total number of disallowed residues. Through AutoDock 4.0 GUI, the minimized PDB was converted to a PDBQT file followed by generation of the grid. To perform flexible docking, torsions were chosen in the selected residues and saved as flex.PDBQT. The configuration file, which was used to outline the grid area as well as the protein and ligand input, was defined and saved as a text document. All the ligands were prepared using Gastegier charges and saved as a PDBQT file. The files saved in the fold were called through the command line under the vina environment to start the docking process. The results for each input ligand were saved as a text file consisting of docking scores and RMSD values of each ligand conformation which was used for the analysis of data. The output file was then converted into Maestro readable extension for image saving [19–22]. WaterSwap methodology The PDB: 5D41 was downloaded initially and full preparation on proteins and ligands was carried out. Under flare protein preparation wizard, at pH 7.0 small side chains movement were allowed, atoms were removed from residues with incomplete backbone, along with filling of 1 and 2 residue gaps at 6.00 A° active site size. Then the protein was minimized under the normal calculation method with a gradient cutoff of 0.200 kcal/mol/A with 1300 maximum iterations. The ligands were loaded in the sdf format for docking. Docking was performed under the normal calculation method under Autodock flexible docking protocol. The ligands to dock were already selected, the template ligand was defined, the co-crystallized ligand was selected for docking grid preparation, the minimized protein was selected and docking calculation was started [16, 23]. For WaterSwap calculation, the protein along with docked ligand was prepared again and minimized as per the above method. The calculation method was kept custom and no changes were made. The small molecule force field was kept as AMBER with GAFF version with AM1-BCC as the charge method. The solvate box buffer was kept at 10.0 A. Open MM platform was GPU, minimized energy tolerance as 0.25 (kcal/mol), the system was equilibrated for 100 ps. For Waterswap, the iterations were kept as 1000 -1300 (1300 for docked ligand), maximum threads were kept on Auto, with Water monitor distance as 7.00 A, the protein was selected, the ligand was selected along with the proper chain, and the calculation was started. Result And Discussion Computational Methods Database building and Library Screening The most potent substituted styrylquinoline with the highest antitumour activity was taken from the literature [24], and by modification, at R1 position a library of around 2000 compounds was generated virtually. Based on the obtained fitness score using ChemT, the top 1000 hits were screened for further pre-ADMET analysis. All 1000 hits were screened through Lipinski’s Rule of 5 and around 100 hits were chosen for further study fitting with the criteria. In the virtual screening workflow protocol, all 100 hits were subjected to a flexible docking approach using AutoDock. A score cutoff of -9.20 kcal/mol for docking discarded lowerPage 3/10 scoring compounds selecting 15 highest scoring compounds. Top 15 highest scoring, best pocket fitting, and possessing similar binding interactions with that of standard EAI045, novel and potent compounds were obtained through virtual screening and were selected for further studies. Molecular Docking Molecular docking was carried out on mutant EGFR enzyme (PDB ID: 5D41) to check the binding pattern and interactions. It was seen that all 15 molecules docked correctly into the allosteric binding pocket of the protein with common interacting amino acid residues LYS745, LEU788, ASP855 and PHE856. From Figures 1a and 1b, it was observed that all compounds fit into the hydrophobic pocket of the protein interacting with the common amino acid residues. The combined docking scores along with structures are shown in Table 1. Figure 1b Docked images of top 15 compounds over T790M/C797S mutant EGFR (PDB: 5D41). Blue lines in the figures indicate π-π stacking interaction and pink lines indicate hydrogen bonding interaction. ADMET studies SwissADME website (http://www.swissadme.ch/index.php) was used to quantify the physicochemical and pharmacokinetic properties of the synthesized derivatives with respect to EAI045. Table 2 shows the analysis of the pharmacokinetic parameters needed for ADMET study of compounds KSK (1-15). Table 2 ADMET study of top 15 compounds with EAI045. Compound #HB acceptor #HB donors TPSAa MLOGPb GI absorption BBB permeant Violation of Lipinski’s Rule Bio availability Score Synthetic Accessibility EAI045 5 2 110.77 1.93 High No 0 0.55 3.28 KSK-1 2 2 70.83 3.65 High No 0 0.55 3.37 KSK-2 5 2 88.68 0.81 High No 0 0.55 3.99 KSK-3 1 1 42.15 3.75 High No 0 0.55 3.33 KSK-4 2 1 56.4 3.15 High No 0 0.55 4.16 KSK-5 2 1 36.36 3.85 High Yes 0 0.55 3.05 KSK-6 2 0 58.5 3.78 High No 0 0.55 3.94 KSK-7 3 2 75.27 3.38 High No 0 0.55 3.34 KSK-8 3 1 100.88 3.91 Low No 0 0.56 4.39 KSK-9 3 1 70.89 3.41 High No 0 0.55 4.47 KSK-10 2 1 41.05 3.54 High Yes 0 0.55 3.54 KSK-11 2 0 29.02 3.18 High No 0 0.55 3.25 KSK-12 3 1 45.59 3.41 High Yes 0 0.55 4.19 KSK-13 2 0 67.82 3.98 High No 0 0.55 3.28 KSK-14 3 1 36.36 3.75 Low No 0 0.55 3.24 KSK-15 2 1 55.04 3.33 High Yes 0 0.55 3.41 a TPSA 40Å - 160Å bMLOGP must be less than 4.15 Page 4/10 The drug must pass Lipinski’s rule to show good oral absorption in humans [25, 26]. All 15 compounds do not show Lipinski’s rule violation, passing the criteria. The MLOGP values were also within the acceptable range, showing the compounds' correct drug absorption and distribution within the body. Almost all the compounds show good GI absorption and some of them were bloodbrain barrier permeant. Through bioavailability scores, it was indicated that a potent compound is available at the site of action. Overall, the compounds showed satisfactory pharmacokinetic parameters range defined for human use along with good docking scores [27–29]. Waterswap When dealing with the cavitation and large-value problem of double decoupling, we used Waterswap, an absolute binding free energy method. Continuum solvent methods do not include the molecular detail of protein–water, ligand–water, and protein– water–ligand interaction interactions because they use an explicit model of water [23]. It uses a single simulation to replace the ligand-bound protein with bulk water molecules of the same shape and volume as shown in Figure 2. An increase in protein-ligand interactions occurs when the protein's resulting cavity is filled with a cluster of water molecules. With only one reaction coordinate, it's possible to get a more accurate free energy value because it's not the product of two huge numbers [30]. Moreover, the beneficial waters around the protein-ligand complex are also identified to obtain a stable pocket. These beneficial waters also become a potential bridge in creating interactions between likely residues and ligand. Both proteins favourable and ligand favourable waters are important to identify to assess the pocket interactions and their stability [31]. Two different types of colours are given for the protein depending on its preference i.e., the protein present in green colour states that it prefers ligand, and protein present in red colour states that it prefers water as indicated in Figure 3. The water box identified by the system further provides the required binding free energy for the protein and ligand [16]. To analyze Waterswap studies, binding free energy (∆G), shown in Figure 4 comes into play describing the stability of the waters around the pocket. If ∆Gprotein in the complete simulation decreases, it does not stabilize with the set number of iterations, the possibility of the existence of unfavourable water distorting the pocket intensity is high. In such cases, the ligand would exit the simulation box resulting in an inactive compound [32]. The similar identity of ∆Gwater is identified through drastic increase or instability of the waters resulting in distortions in the protein. This could also result in terminating the protein-ligand interactions. EGFR protein becomes the perfect example of identifying and comparing the potential waters for the binding affinity and checking the potency of the complexed ligand. A mutant EGFR protein with known standard EAI045 is initially run for 1300 iterations using Waterswap to identify all the favourable waters and their binding free energy. Then the ligand is swapped to check the potential binding affinity and stability of the designed compounds concerning the identified favourable waters. If the binding free energy results obtained during the complete iterative simulations are similar or better than the results for standard drug EAI045, the compounds designed are the potential to withstand the waters and show potency. Figure 5 describes the relative binding free energy graph during the 1300 iterations performed for EAI045 compared to the styrylquinoline compound identified from the literature. The blue line in the graph shows the average binding free energy change for the number of iterations. The thermodynamic properties compared in the Figure showed that the styrylquinoline stabilized better than the standard compound EAI045. Towards the end of the iterations, the graph showed stability after a drop in relative binding energy in styrylquinoline. Overall, all the attributes taken into account concludes the potential of styrylquinoline compound as an EGFR inhibitor. The results show that Waterswap analysis offers a promising new path in the hunt for improved tools for analyzing and visualizing molecular driving forces in protein-ligand complex simulations. Conclusion The binding free energy calculations using the Waterswap analysis for interpreting and visualizing trajectories of EGFR-ligand complexes have been demonstrated. These visualizations provide precise molecular-level information that should help medicinal chemists construct better-binding ligands. Although we tested these decompositions on mutant EGFR, we believe they should Page 5/10 apply to other protein-ligand systems as well. The thermodynamic properties of the enumerated compounds were compared with that of standard EAI045, and it was observed that styrylquinoline stabilized better than EAI045. The results show that Waterswap analysis offers a promising new path in the hunt for improved tools for analyzing and visualizing molecular driving forces in protein-ligand complex simulations. Declarations Acknowledgement The authors K. S. K and P. S. W. are thankful to Indian Council of Medical Research (ICMR) for providing funding in support of Senior Research Fellowship with Ref No: 3/2/2/33/2018/Online OncoFship/NCD-III. 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Woods CJ, Malaisree M, Pattarapongdilok N, Sompornpisut P, Hannongbua S, Mulholland AJ (2012) Long Time Scale GPU Dynamics Reveal the Mechanism of Drug Resistance of the Dual Mutant I223R/H275Y Neuraminidase H1N1-2009 Influenza Page 7/10 Virus. Biochemistry 51:4364–4375 31. Kurdekar V, Giridharan S, Subbarao J, Nijaguna MB, Periasamy J, Boggaram S, Bharatham K, Potluri V, Shivange AV, Sadasivam G, Padigaru M, Venkitaraman AR Structural features underlying the activity of benzimidazole derivatives that target phosphopeptide recognition by the tandem BRCT domain of the BRCA1 protein. bioRxiv preprint. https://doi.org/10.1101/555623 32. Liu J, Liu L, Tian Z, Li Y, Shi C, Shi J, Wei S, Zhao Y, Zhang C, Bai B, Chen Z, Zhang, H In silico discovery of a small molecule suppressing lung carcinoma A549 cells proliferation and inducing autophagy via mTOR pathway inhibition. Mol Pharm. https://doi.org/10.1021/acs.molpharmaceut.8b00996 Tables Due to technical limitations, table 1 is only available as a download in the Supplemental Files section. Figures Figure 1 Page 8/10 a Docked images of top 15 compounds over T790M/C797S mutant EGFR (PDB: 5D41). Blue lines in the figures indicate π-π stacking interaction and pink lines indicate hydrogen bonding interaction. b Docked images of top 15 compounds over T790M/C797S mutant EGFR (PDB: 5D41). Blue lines in the figures indicate π-π stacking interaction and pink lines indicate hydrogen bonding interaction. Figure 2 (a) Protein pocket filled with ligand. (b) Protein pocket filled with waters. Figure 3 Interpretation of which protein residues prefer binding to water or ligand. Green represents ligand favourable and red represents water favourable. Figure 4 Page 9/10 Swapping of Protein:Water to Protein:Ligand. Figure 5 The relative binding free energy graph of standard EAI045 and Styrylquinoline. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table1Structures.docx Graphicalabstract.docx Page 10/10
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Use of apple genetic resources in apple breeding programmes at INRA: Present status and perspectives
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Use of apple genetic resources in apple breeding programmes at Inra: present status and perspectives Francois Laurens Francois Laurens To cite this version: Francois Laurens. Use of apple genetic resources in apple breeding programmes at Inra: present status and perspectives. European Malus Germplasm, European cooperative programme for crop genetic resources networks., Jun 1995, London, United Kingdom. ￿hal-01603372￿ Use of apple genetic resources in apple breeding programmes at Inra: present status and perspectives HAL Id: hal-01603372 https://hal.science/hal-01603372v1 Submitted on 4 Jun 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 - ShareAlike 4.0 International License
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Whole-Genome Sequence of the Ancestral Animal-Borne ST398 Staphylococcus aureus Strain S123
Genome announcements
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To cite this version: Nathalie van Der Mee-Marquet, David Hernandez, Xavier Bertrand, Roland Quentin, Anna-Rita Corvaglia, et al.. Whole-Genome Sequence of the Ancestral Animal-Borne ST398 Staphylococcus aureus Strain S123.. Genome Announcements, 2013, 1 (5), pp.e00692-13. ￿10.1128/genomeA.00692- 13￿. ￿hal-00926042￿ Whole-Genome Sequence of the Ancestral Animal-Borne ST398 Staphylococcus aureus Strain S123 Nathalie van der Mee-Marquet,a,b David Hernandez,c Xavier Bertrand,d Roland Quentin,a Anna-Rita Corvaglia,c Patrice Françoisc Service de Bactériologie et Hygiène, Centre Hospitalier Régional Universitaire, Tours, Francea; Réseau des Hygiénistes du Centre, Centre Hospitalier Universitaire, Tours, Franceb; UMR 6249 Chrono-environnement Université de Franche-Comté Besançon Franced; Genomic Research Laboratory University of Geneva Hospitals Geneva Marquet,a,b David Hernandez,c Xavier Bertrand,d Roland Quentin,a Anna-Rita Corvaglia,c Patrice Françoisc Nathalie van der Mee-Marquet,a,b David Hernandez,c Xavier Bertrand,d Roland Quentin,a Anna-Rita Corvaglia,c Patrice Françoisc Service de Bactériologie et Hygiène, Centre Hospitalier Régional Universitaire, Tours, Francea; Réseau des Hygiénistes du Centre, Centre Hospitalier Universitaire, Tours, Franceb; UMR 6249 Chrono-environnement, Université de Franche-Comté, Besançon, Franced; Genomic Research Laboratory, University of Geneva Hospitals, Geneva, Switzerlandc Sequence type 398 (ST398) Staphylococcus aureus was originally reported in livestock. We announce the complete genome se- quence of an ST398 methicillin-susceptible S. aureus strain of animal origin, S123. The genome sequence reveals a wild-type ge- nome that probably corresponds to an ancestral clone. Received 5 August 2013 Accepted 5 August 2013 Published 29 August 2013 Citation van der Mee-Marquet N, Hernandez D, Bertrand X, Quentin R, Corvaglia A-R, François P. 2013. Whole-genome sequence of the ancestral animal-borne ST398 Staphylococcus aureus strain S123. Genome Announc. 1(5):e00692-13. doi:10.1128/genomeA.00692-13. Received 5 August 2013 Accepted 5 August 2013 Published 29 August 2013 Copyright © 2013 van der Mee-Marquet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Patrice François, patrice.francois@genomic.ch. S tem. Similar to many other sequenced ST398 strains, S123 is de- void of DNA from phagic elements despite the presence of a functional type I restriction system. S equence type 398 (ST398) Staphylococcus aureus was originally described in livestock (1) but was recently reported as the causative agent of severe infections in humans (2). An evolution- ary hypothesis suggests that an ancestral wild-type S. aureus strain acquired mobile genetic elements triggering host adaptation and evolution to the methicillin-resistant genotype after insertion of a staphylococcal cassette chromosome mec (SCCmec) element (3). We report here the genome sequence of a carriage methicillin- susceptible S. aureus isolate showing sequence type 398 and de- void of any phage element. We conclude that S123 is potentially an ancestral clone still missing a phagic element that contributes to host adaptation. Nucleotide sequence accession number. The whole-genome sequence of S. Whole-Genome Sequence of the Ancestral Animal-Borne ST398 Staphylococcus aureus Strain S123 aureus S123 was deposited in the DDBJ/EMBL/ GenBank databases under the accession number AUPU00000000. ACKNOWLEDGMENTS This study was supported in part by the University Hos We thank the technicians for their dedicated help. This study was supported in part by the University Hospi h k h h f h d d d h l This study was supported in part by the University Ho We thank the technicians for their dedicated help. S. aureus strain S123 was recovered from a pig during a screen- ing campaign in a Dutch farming area in 2010. Purified genomic DNA was subjected to whole-genome shotgun sequencing by us- ing an HiSeq2000 system (Illumina, Inc.). Following fragmenta- tion, end reparation, and sample tagging, the sequencer produced 21.6 million 100-bp paired-end reads, yielding appreciable cover- age of around 800. Assembly was performed using Edena 3.0 (4) and resulted in 49 contigs. The larger contigs showed sizes of 498,000 bp. Overall assembly values were satisfactory, showing a total length of 2.70 Mbp and an N50 of 152,000 bp. A total of 2,475 predicted coding sequences (CDS) were detected by rapid anno- tations using subsystems technology (RAST) (5). More than 55% of the genes were assigned to specific subsystem categories by RAST (5). In addition to CDS, RAST revealed 77 structural genes, including 59 tRNA and 18 rRNA genes. Note that S123 contains two circular plasmids of 15 kb and 4.5 kb at apparent copy num- bers of 2 and 10, respectively. y pp p y y We thank the technicians for their dedicated help. September/October 2013 Volume 1 Issue 5 e00692-13 HAL Id: hal-00926042 https://hal.science/hal-00926042v1 Submitted on 29 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. REFERENCES 1. Witte W, Strommenger B, Stanek C, Cuny C. 2007. Methicillin-resistant Staphylococcus aureus ST398 in humans and animals, Central Europe. Emerg. Infect. Dis. 13:255–258. doi:10.3201/eid1302.060924. 2. van der Mee-Marquet N, François P, Domelier-Valentin AS, Coulomb F, Decreux C, Hombrock-Allet C, Lehiani O, Neveu C, Ratovohery D, Schrenzel J, Quentin R, Bloodstream Infection Study Group of the Réseau des Hygiénistes du Centre (RHC). 2011. Emergence of unusual bloodstream infections associated with pig-borne–like Staphylococcus au- reus ST398 in France. Clin. Infect. Dis. 52:152–153. doi:10.1093/cid/ciq053. 3. van der Mee-Marquet N, Corvaglia AR, Valentin AS, Hernandez D, Bertrand X, Girard M, Kluytmans J, Donnio PY, Quentin R, François P. 2013. Analysis of prophages harbored by the human-adapted subpopula- tion of Staphylococcus aureus CC398. Infect. Genet. Evol. 18:299–308. 4. Hernandez D, François P, Farinelli L, Osterås M, Schrenzel J. 2008. De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Res. 18:802–809. doi:10.1101/gr.072033. 107. The genome of strain S123 encodes known virulence factors, such as pore-forming toxins (only hemolysins) and bacterial ad- hesins. Annotation confirmed the absence of important resistance determinant genes, except those for tetracycline and erythromy- cin. Interestingly, strain S123 is missing a type IV restriction sys- 5. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: rapid annotations using subsystems technology. BMC Genomics. 9:75. doi:10.1186/1471-2164-9-75. genomea.asm.org 1 Genome Announcements genomea.asm.org 1 genomea.asm.org
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Social Support for People with Morbid Obesity in a Bariatric Surgery Programme: A Qualitative Descriptive Study
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  Keywords: social support; morbid obesity; bariatric surgery; qualitative study Academic Editor: Paul B. Tchounwou Received: 31 May 2021 Accepted: 15 June 2021 Published: 17 June 2021 International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2021, 18, 6530. https://doi.org/10.3390/ijerph18126530 Article Social Support for People with Morbid Obesity in a Bariatric Surgery Programme: A Qualitative Descriptive Study í é á 1 á 1 2 é 2 Public Health 2021, 18, 6530. https://doi.org/10.3390/ ijerph18126530 Academic Editor: Paul B. Tchounwou Received: 31 May 2021 Accepted: 15 June 2021 Published: 17 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// ti /li /b /   Citation: Torrente-Sánchez, M.J.; Ferrer-Márquez, M.; Estébanez-Ferrero, B.; Jiménez-Lasserrotte, M.d.M.; Ruiz-Muelle, A.; Ventura-Miranda, M.I.; Dobarrio-Sanz, I.; Granero-Molina, J. Social Support for People with Morbid Obesity in a Bariatric Surgery Programme: A Qualitative Descriptive Study. Int. J. Environ. Res. Public Health 2021, 18, 6530. https://doi.org/10.3390/ ijerph18126530 Article Social Support for People with Morbid Obesity in a Bariatric Surgery Programme: A Qualitative Descriptive Study í é á 1 á 1 2 é 2 María José Torrente-Sánchez 1, Manuel Ferrer-Márquez 1,2, Beatriz Estébanez-Ferrero 2, María del Mar Jiménez-Lasserrotte 3, Alicia Ruiz-Muelle 3, María Isabel Ventura-Miranda Iria Dobarrio-Sanz 3 and José Granero-Molina 3,4,* María José Torrente-Sánchez 1, Manuel Ferrer-Márquez 1,2, Beatriz Estébanez-Ferrero 2, María del Mar Jiménez-Lasserrotte 3, Alicia Ruiz-Muelle 3, María Isabel Ventura-Miranda 3 , Iria Dobarrio-Sanz 3 and José Granero-Molina 3,4,* 1 Hospital HLA Mediterráneo, 04007 Almería, Spain; mjtorrentes@gmail.com (M.J.T.-S.); Manuferrer78@hotmail.com (M.F.-M.) 1 Hospital HLA Mediterráneo, 04007 Almería, Spain; mjtorrentes@gmail.com (M.J.T.-S.); Manuferrer78@hotmail.com (M.F.-M.) 2 Hospital Universitario Torrecárdenas, 04009 Almería, Spain; beatriz.estebanez.sspa@juntadeandalucia.es 3 Department of Nursing, Physiotherapy and Medicine, University of Almeria, 04120 Almería, Spain; mjl095@ual.es (M.d.M.J.-L.); arm297@ual.es (A.R.-M.); mvm737@ual.es (M.I.V.-M.); ids135@ual.es (I.D.-S.) 4 Faculty of Health Sciences, Universidad Autónoma de Chile, Santiago 7500000, Chile * Correspondence: jgranero@ual.es 2 Hospital Universitario Torrecárdenas, 04009 Almería, Spain; beatriz.estebanez.sspa@juntadeandalucia.es 3 Department of Nursing, Physiotherapy and Medicine, University of Almeria, 04120 Almería, Spain; mjl095@ual.es (M.d.M.J.-L.); arm297@ual.es (A.R.-M.); mvm737@ual.es (M.I.V.-M.); ids135@ual.es (I.D.-S.) 4 Faculty of Health Sciences, Universidad Autónoma de Chile, Santiago 7500000, Chile * Correspondence: jgranero@ual.es * Correspondence: jgranero@ual.es Abstract: Background—Morbid obesity (MO) is a chronic metabolic disease affecting physical, psychological and social wellbeing. Bariatric surgery is a reliable method for losing weight in the long term, improving the quality of life, body image and social life of people with MO. Current literature recognises the importance of social support in controlling weight and coping with MO. The objective of this study was to describe and understand experiences related to social support for patients with MO included in a bariatric surgery programme. Methods—A qualitative descriptive study, where data collection included thirty-one interviews with people diagnosed with MO involved in a bariatric surgery programme. Results—Three main themes emerged from the analysis: (1) accepting the problem in order to ask for help, (2) the need for close support and (3) professional support: opposing feelings. Conclusions—A partner, family and friends are the key pillars of social support for those with MO included in a bariatric surgery programme. Healthcare professionals gave formal support; the bariatric surgery team provided information, trust and assurance. Nurses provided healthcare 24 h a day, making them the main formal support for people in the bariatric surgery programme.   Citation: Torrente-Sánchez, M.J.; Ferrer-Márquez, M.; Estébanez-Ferrero, B.; Jiménez-Lasserrotte, M.d.M.; Ruiz-Muelle, A.; Ventura-Miranda, M.I.; Dobarrio-Sanz, I.; Granero-Molina, J. Social Support for People with Morbid Obesity in a Bariatric Surgery Programme: A Qualitative Descriptive Study. Int. J. Environ. Res. 1. Introduction Obesity is a chronic disease that is characterised by an accumulation of fat or hyper- trophy of fatty tissue. Obesity is a public health issue, although the prevalence varies depending on the country [1]. It affects approximately 13% of the global adult popula- tion [2]. The prevalence of obesity in Spain is 22.8% of men and 20.5% of women [3]. Obesity is defined as having a body mass index (BMI) > 30 kg/m2, and morbid obesity (MO) as having a BMI between 40–49.9 kg/m2 [4,5]. Obesity is a multifactorial problem that is associated with metabolic, endocrine [6] and vascular problems, as well as diabetes and hypertension [7]. Added to this are external factors, such as poverty [8], unhealthy diet, lifestyle and physical exercise, as well as socio-cultural factors [9]. MO is a health risk factor, as it is related to anxiety, depression, body image disorders and sexual dysfunction [10,11]. MO can be prevented with a multidisciplinary approach, including health education, diet, physical exercise and pharmacological treatment [12–14]. However, therapies or treatments are not always effective and therefore surgical treatment is required [15]. Bariatric surgery is a reliable method for losing weight in the long term, improving the quality of life, body image and social life of people with MO. The criteria for inclusion for a person with MO in a bariatric surgery programme are: to be an adult with a BMI ≥40 kg/m2 or an adult with a BMI between 35–39.9 kg/m2 and a serious associated comorbidity, such as type Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Int. J. Environ. Res. Public Health 2021, 18, 6530. https://doi.org/10.3390/ijerph18126530 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 6530 2 of 14 2 diabetes, high blood pressure or hyperlipidemia [16]. Bariatric surgery involves a set of surgical procedures to treat obesity, prevent the associated morbidity and mortality and improve the quality of life of obese patients following weight loss. Bariatric surgery includes restriction techniques that reduce gastric volume [17], malabsorptive techniques and mixed techniques [18]. q Physical, psychological, social and cultural factors make living with MO difficult. 2.1. Design A qualitative descriptive study was used. This methodology allows for little-known experiences to be described from a naturalist perspective without losing their essence in the analysis process. The study was developed and reported in accordance with the Consolidated Criteria for Reporting Qualitative Research (COREQ) [42]. 1. Introduction People with MO face physical impediments, difficulty in moving [19], low self-esteem or self-control [20], disorders associated with negative body image [21], shame and psychoso- cial stress, all of which can lead to limited social relations, stigma and the loss of quality of life [22,23]. Facing MO not only requires weight loss but also dealing with psychological and social problems [24]. Social support refers to the social resources that are available for people facing a health issue, which are provided by formal and/or informal support groups. Social support is key in preventing and overcoming chronic pathologies, such as diabetes, cardiovascular disease, osteoarticular disease or MO [25,26]. Various studies have demonstrated the importance of social support in different phases of obesity [27]. Social support is associated with making the decision to undergo bariatric surgery [28], post-surgery results [29–32], stress levels [33], information prior to treatment [34] and coping strategies [35]. Other studies have explored the experiences of patients in MO support groups after undergoing bariatric surgery [36,37] or patients who are expecting bariatric surgery alongside their partners [38,39]. Although the liter- ature recognises the importance of social support in controlling weight and overcoming MO, the experiences of people with MO in a bariatric surgery programme have scarcely been studied. In line with Leahy-Warren’s social support theory [40], social support has a structural and functional dimension. The structural dimension refers to the social networks of people with MO, which are divided into informal (family, friends and acquaintances) and formal (essentially healthcare professionals). The functional dimension is related to the exchange of activities (informative, instrumental and emotional support), types of social support received and the perception/satisfaction of them [41]. The objective of this study was to describe and understand the experiences regarding the social support of people with MO that were included in a bariatric surgery programme. 2.2. Participants and Setting The researchers used purposive sampling to select people with MO that were included in a bariatric surgery programme. The participants were recruited by surgeons and nurses associated with the bariatric surgery programme for people with MO. The criteria for inclusion were: be a person diagnosed with MO at least 10 years prior to the study, in the phase prior to surgical intervention, a Spanish speaker, over 18 and having signed an informed consent form. The exclusion criteria were: rejected participating in the study, not be included in a bariatric surgery programme or be in a different phase of treatment. Out of 44 people with MO selected for telephone contact and recruitment, two did not answer the phone, nine refused to discuss the topic and two did not have time to be interviewed. The interviews took place with a total of 31 participants, of which, 21 were women and 10 were men (Table 1). The study was carried out in two hospitals in the south of Spain between April 2019 and January 2020. Int. J. Environ. Res. Public Health 2021, 18, 6530 3 of 14 Table 1. Sociodemographic data. Table 1. Sociodemographic data. Participant Sex Onset of MO Marital Status Job IDI1 F Adulthood Married Housewife IDI2 F Adolescence Married Farmer IDI3 F Adulthood Relationship Housewife IDI4 F Adolescence Single Unemployed IDI5 F Adulthood Separated Seamstress IDI6 F Pregnancy Separated Chef IDI7 F Adolescence Relationship Administrative assistant IDI8 F Adulthood Married Healthcare assistant IDI9 F Pregnancy Married Administrative assistant IDI10 F Pregnancy Married Farmer IDI11 F Pregnancy Married Teacher IDI12 F Childhood Married Administrative assistant IDI13 F Adolescence Married Housewife IDI14 F Adolescence Single Unemployed IDI15 F Adolescence Single Shop assistant IDI16 F Pregnancy Married Teacher IDI17 F Adolescence Single Civil servant IDI18 F Childhood Single Student IDI19 F Adolescence Married Teacher IDI20 F Childhood Single Beautician IDI21 F Childhood Relationship Waitress IDI22 F Adolescence Single Unemployed IDI23 M Adolescence Married Engineer IDI24 M Adulthood Married Salesperson IDI25 M Childhood Single Farmer IDI26 M Adulthood Married Salesperson IDI27 M Adulthood Married Petrol station worker IDI28 M Adulthood Married Hospitality worker IDI29 M Adulthood Married Farmer IDI30 M Adulthood Married Farmer IDI31 M Childhood Married Unemployed MO: morbid obesity. IDI: in-depth interview. 2.3. Data Collection Data Analysis Thematic analysis was carried out using Braun and Clarke’s [43] method with the following steps: (1) familiarisation with the data—data transcription, reading and re- reading and annotation of initial ideas; (2) generate initial codes—systematic codification of data groups; (3) find themes—convert codes into themes; (4) revise themes—verify codes with themes; (5) define themes—analyse the details of each theme; (6) elaborate the report—select examples of themes and subthemes, associate the analysis with the research question and generate the final report. 2.5. Rigor Credibility: participants and researchers were familiarised with the context of the study to ensure trust and obtain rich data. Various researchers participated in the codifica- tion, analysis and interpretation of the data. Transferability: the researchers described the experiences and context of the study in detail. Reliability and confirmability: the nurses did the transcriptions and then other members of the research group revised them, verified them and accepted the participants. Reflexivity: the researchers examined their own values and preconceptions about the issue at hand before describing the results. 2.6. Ethical Issues The study was carried out in accordance with the ethical standards established by the Helsinki Declaration. It was approved by the Ethical Committee of the University of Almería’s Nursing, Physiotherapy and Medicine Department (protocol number: 45/2018). Prior to the study, informed written consent and permission to record the interview were obtained. The participants were informed that their participation in the study was inde- pendent of the bariatric surgery programme. 3. Results A total of 31 patients, men (32.26%) and women (67.74%) with a mean age = 44.35 years (SD = 11.57 years, range = 20–66 years) were recruited. The BMI of the participants was 44.5 ± 6.57 kg/m2, MO started during adolescence (32.26%) or in adulthood (32.6%) Table 2. Interview protocol. Stage Subject Content/Example Questions Introduction Motives, reasons Learn about your experiences of social support since your inclusion in a bariatric surgery programme. Ethical issues Inform about voluntary participation, recording, consent, possibility of withdrawing and confidentiality. Beginning Introductory question Tell me about your experience with MO. Development Conversation guide How has it affected your relationship with your friends and other social relations? How has MO affected your perception of your body and your hobbies/free time? How has MO affected your life with your partner? How has MO affected your relationship with healthcare providers? 2.5. Rigor Credibility: participants and researchers were familiarised with the context of the study to ensure trust and obtain rich data. Various researchers participated in the codifica- tion, analysis and interpretation of the data. Transferability: the researchers described the experiences and context of the study in detail. Reliability and confirmability: the nurses did the transcriptions and then other members of the research group revised them, verified them and accepted the participants. Reflexivity: the researchers examined their own values and preconceptions about the issue at hand before describing the results. 2.6. Ethical Issues The study was carried out in accordance with the ethical standards established by the Helsinki Declaration. It was approved by the Ethical Committee of the University of Almería’s Nursing, Physiotherapy and Medicine Department (protocol number: 45/2018). Prior to the study, informed written consent and permission to record the interview were obtained. The participants were informed that their participation in the study was inde- pendent of the bariatric surgery programme. 2.4. Data Analysis Thematic analysis was carried out using Braun and Clarke’s [43] method with the following steps: (1) familiarisation with the data—data transcription, reading and re- reading and annotation of initial ideas; (2) generate initial codes—systematic codification of data groups; (3) find themes—convert codes into themes; (4) revise themes—verify codes with themes; (5) define themes—analyse the details of each theme; (6) elaborate the report—select examples of themes and subthemes, associate the analysis with the research question and generate the final report. 2.3. Data Collection What kind of formal and informal social support was missing for you after being included in a bariatric surgery programme? What role did technology play in terms of social support in a bariatric surgical programme? Closing Final question Is there anything else you would like to tell me? Appreciation Thank them for their participation, remind them that their testimony will be useful and place ourselves at their disposition. MO: morbid obesity. Table 2. Interview protocol. Content/Example Questions 2.3. Data Collection Nurses called participants who fulfilled the inclusion criteria to explain the objectives, respond to queries and request voluntary participation in the study. Nurses and partici- pants had established contact prior to the clinical care context. The nurses had a Master’s degree in qualitative research and they did not need further training. The participants agreed to be interviewed in a hospital meeting room outside of the surgical ward. Two nurses with five years of experience working in the bariatric surgery programme carried out in-depth interviews following the interview protocol (Table 2). Each participant did one individual, private, in-depth interview that lasted 56 min on average. The nurses collected field notes to describe the data. Before the interview, participants were informed about the study, signed consent was obtained and sociodemographic data of the participants were collected. The participants’ answers were recorded, transcribed and analysed using ATLAS.ti.9 (ATLAS.ti Scientific Software Development GmbH, Berlin, Germany). Data collection ceased when the researchers considered that they had reached data saturation. 4 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 nt. J. Environ. Res. Public Health 2021, 18, 6530 4 of 14 Table 2. Interview protocol. Stage Subject Content/Example Questions Introduction Motives, reasons Learn about your experiences of social support since your inclusion in a bariatric surgery programme. Ethical issues Inform about voluntary participation, recording, consent, possibility of withdrawing and confidentiality. Beginning Introductory question Tell me about your experience with MO. Development Conversation guide How has it affected your relationship with your friends and other social relations? How has MO affected your perception of your body and your hobbies/free time? How has MO affected your life with your partner? How has MO affected your relationship with healthcare providers? What kind of formal and informal social support was missing for you after being included in a bariatric surgery programme? What role did technology play in terms of social support in a bariatric surgical programme? Closing Final question Is there anything else you would like to tell me? Appreciation Thank them for their participation, remind them that their testimony will be useful and place ourselves at their disposition. MO: morbid obesity. 2.4. 3. Results A total of 31 patients, men (32.26%) and women (67.74%) with a mean age = 44.35 years (SD = 11.57 years, range = 20–66 years) were recruited. The BMI of the participants was 44.5 ± 6.57 kg/m2, MO started during adolescence (32.26%) or in adulthood (32.6%) Int. J. Environ. Res. Public Health 2021, 18, 6530 5 of 14 5 of 14 (Table 1). Three themes allowed for the description and understanding of the social support experienced by patients with MO in a bariatric surgery programme (Table 3). Table 3. Themes, subthemes and units of meaning. Themes Subthemes Units of Meaning Accepting the problem in order to ask for help Social stigma, the other weight of obesity Bullying during childhood, limitations imposed by others, everything is in my head, a look is worth more than a thousand words, understanding is better amongst equals. Acknowledging MO I do not identify with my body, my health comes first, physical limitations, acknowledging, self-punishment, food as comfort and social withdrawal. Bariatric surgery, a shared journey Need to speak, fear, uncertainty, my only hope, sharing experiences, sharing decisions and high expectations. The need for close support The partner, the key support figure for MO Emotional support, fear of being left, lack of communication, suspicion, fear of bariatric surgery and fear of complications. Necessary company, unpleasant scrutiny Feeling observed, discomfort, sharing experiences, feeling scrutinised, feeling heard, a link to reality, selfless help and concern for others. Appearing and sharing on social networks Looking for information, inspecting results and Whatsapp groups. Professional support: opposing feelings Lack of professional support Professional indifference, lack of information, lack of empathy and bariatric surgery is not offered The bariatric nurse “is always there” Trust, assurance, active listening, bariatric surgery team, nurse as a point of reference, 24/7 telephone contact and someone in whom to seek comfort. MO: morbid obesity. (Table 1). Three themes allowed for the description and understanding of the social support experienced by patients with MO in a bariatric surgery programme (Table 3). Table 3. Themes, subthemes and units of meaning. 3.1. Accepting the Problem in Order to Ask for Help Those with MO faced physical, psychological and social problems. Alongside the associated risks and comorbidites, MO was problematic in relation to the patient’s physical appearance, image, emotional state and the demands of daily life. Acknowledging the situation was key in being able to face it, ask for help and find social support. My colleagues are always looking out for me “leave that, I’ll do it,” “careful, you might fall, be careful”. I feel like they are pitying me . . . I don’t want that! (IDI24) 3.1.1. Social Stigma, the Other Weight of Obesity Those with MO found themselves on the receiving end of insulting comments, which could lead to feelings of frustration, shame and blame. This situation could happen as early as childhood, resulting in the victims becoming more and more sensitive and insecure. As one participant stated, it was possible to interpret any gesture or look as contemptuous: I think they look at me, . . . I don’t go unnoticed and my mind doesn’t rest. If I eat an ice-cream I think that everyone is thinking “well, that’s why she’s so fat”. I can’t go on 3.1.2. Acknowledging MO Obesity is a chronic disease and the majority of those affected took their time to acknowledge the problem. They did not feel capable of overcoming it or asking for help from professionals or their close social circles. Feelings of rejection and denial were very common in the early phases of acknowledgement, at which point, social support played an important role: The doctor said that I had to look for solutions but I didn’t want to acknowledge it or look f g for help . . . first you have to become aware of the situation yourself. (IDI23) for help . . . first you have to become aware of the situation yourself. (IDI23) Participants suffered from psychological and emotional disorders and despite being able to share the problem or ask for informal help, they did not find it easy. They recognised that they did not like themselves or feel liked by others, which limited their social life and led to them cutting themselves off. As one participant stated, at those times, it was easier to turn to food than to social support: I sometimes argued with my husband and said “right, now I’m going to have some I sometimes argued with my husband and said “right, now I’m going to have some I sometimes argued with my husband and said “right, now I’m going to have some chocolate”. Then I regretted it and even made myself sick so that I wouldn’t put on weight. (IDI1) chocolate”. Then I regretted it and even made myself sick so that I wouldn’t put on weight. (IDI1) They slowly started to lose contact with those in their social circle, they stopped taking part in leisure activities and it was difficult to find a partner. They found excuses not to leave the house, they withdrew into themselves and looked for help within their family: I used to go out more but now I don’t feel like it; I avoid going out with my friends and I I used to go out more but now I don’t feel like it; I avoid going out with my friends and I go to the beach with my family because I feel more comfortable with them. (IDI8) g f g g y f go to the beach with my family because I feel more comfortable with them. 3.1.2. Acknowledging MO (IDI8) MO presented a constant struggle to stay healthy and in shape, which varied de- pending on gender, according to the participants. Women associated MO with aesthetic problems; they rejected a body with which they did not identify and avoided looking in the mirror. Whilst they may recognise their physical limitations and the risk posed to their health, they placed more importance on the discomfort and shame related to their body image: I see myself as fat, deformed, I don’t recognise myself. When I get out of the shower, I look at myself in the mirror and I don’t want to see myself, I’m ashamed of my body. (IDI10) The men did not identify with their bodies either, but MO was more of a health issue for them. As well as the physical limitations and comorbidities, the exacerbation of prior conditions and the inability to carry out daily tasks were what worried them the most. As one participant said, acknowledging the gravity of the situation was the first step in asking for help: If I were fat but healthy I wouldn’t mind, the problem is that I’m not healthy. I don’t have a complex, I don’t hide myself, but it’s difficult to deal with alone . . . I need help! (IDI26) like this. (IDI1) Social networks, the media and society’s notion of beauty as being thin led to prejudice against people with MO. The interviewees found it difficult to buy clothes, find jobs and establish social relations, both in terms of leisure and finding a partner. The social stigma around MO had more of an impact than physical problems, which was exemplified by the pity people with MO face in relation to their limitations: Int. J. Environ. Res. Public Health 2021, 18, 6530 6 of 14 Those with MO found that understanding and empathy came from people who were in the same situation. Putting yourself in someone else’s shoes is difficult if you have never experienced the sorts of comments and blaming looks from society when faced with MO. p g y Those with MO turned to patient associations to talk about their problems and experi- ences and to find social support. 3.1.3. Bariatric Surgery, a Shared Journey After failed attempts to lose weight using other methods, opting for surgery and joining a bariatric surgery programme was a time in which those with MO preferred not to be alone. Participants said that bariatric surgery was not part of their original plan due to fear of the intervention, being judged and the cost of the process. In this phase, rather than family or friends, their partner was the key pillar of support when making these decisions: My wife would talk to me about obesity and I would change the conversation. She tried to help me understand that we had a serious problem, that we had to go to the doctor and even consider bariatric surgery. (IDI27) 7 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 Those in the bariatric surgery programme were scared and unsure of the process. If patients had a partner, they shared those feelings with the partner; they needed their partner’s positivity to improve their self-acceptance, for the partner to not feel embarrassed by them and for the partner to encourage them to continue with their daily activities. Those who did not have a partner shared their experiences with family and friends. As one participant said, while they were waiting for the intervention, their expectations of the surgery being a success were high. In fact, they expected a radical change in all areas of their life, which could lead to disappointment: I want to be thin, positive, focus on the good things. We (partner) talk about it and I tell him that everything will change, we are going to start from scratch, a new body, different sexuality, another life. (IDI21) 3.2. The Need for Close Support Those with MO needed support from those with whom they live. Diets, commutes, work and leisure all required effort for which social support was needed. However, they were often faced with a lack of understanding; their perceived notion of social stigma surrounding MO caused an internal conflict that led them to ask for help. 3.2.1. The Partner, the Key Support Figure for MO 3.2.1. The Partner, the Key Support Figure for MO 3.2.1. The Partner, the Key Support Figure for MO Those with MO suffered from stress, insecurity and low self-esteem; it was vital for their partners to accept them. Although the participants did not express it, they thought that their partners were not satisfied with them or their physical appearance, which affected their self-esteem and social or sexual relations. As one participant stated, having their partner’s support was the driving force they needed to opt for and face the surgical process together: He tells me that he met me like this (obese), that he loves me like this, that he’s not with me for my physical appearance but I know it’s impossible . . . I’m embarrassed to have sexual relations. (IDI6) The participants’ partners worried a great deal about the associated comorbidities. They feared an exacerbation of cardiovascular or respiratory problems (e.g., a heart attack). This led to the partner being in a permanent state of alert and to adapting their lifestyles to the person with MO. Likewise, the participants had a profound sense of guilt; they knew that their partner was looking out for them and they did not want them to suffer: She (wife) is scared of something happening to me, of me dying and leaving her alone. I try to not let my obesity affect my loved ones . . . but it’s difficult. (IDI29) However, this type of support was not universal, and some patients’ partners had made derogatory comments. As one man said, this led to a fear of being left, which, in turn, hastened their decision to enter a bariatric surgery programme. In summer, I wear more revealing clothes so my body is on show. If your partner looks at you and says “look at their bodies, not like yours” you think that they are going to leave you and you want to have the operation immediately! (IDI 31) My family says “don’t eat this, go for a walk, you need to endure it . . . ” Since I was put in the bariatric surgery programme, they monitor me and control me all day, it’s stifling. (IDI14) The patient association brought us together, it was like a big family. We were social and able to be ourselves, without feeling observed or rejected. (IDI19) The patient association brought us together, it was like a big family. We were social and able to be ourselves, without feeling observed or rejected. (IDI19) Losing this meeting point (MO patient associations shut down at a local level) had a negative impact on the participants’ self-esteem. They tried to replace it by using social networks, such as Facebook, online forums and, above all, Whatsapp, to inform themselves about advances, pre-operative experiences, post-operative complications and expectations of the post-surgery period. It was especially mothers with children with MO who turned to these means of communication: Now we use Whatsapp, that’s how we talk, laugh and cry. For a mother, sharing fears with someone who is going through the same as you, who is going to understand you, is priceless. (IDI11) 3.2.2. Necessary Company, Unpleasant Scrutiny Family and friends were social support for those with MO. Nonetheless, some partici- pants felt self-conscious, scrutinised and uncomfortable in their own homes. The family was aware of the severe risks involved with MO for the person diagnosed. They monitored them and hid food, making themselves responsible for the situation not worsening, which became even more evident when the person with MO was awaiting bariatric surgery: My family says “don’t eat this, go for a walk, you need to endure it . . . ” Since I was put in the bariatric surgery programme, they monitor me and control me all day, it’s stifling. (IDI14) Int. J. Environ. Res. Public Health 2021, 18, 6530 8 of 14 This situation provoked more anxiety, despair and avoidance behaviours. On occasion, they resorted to lies, hiding themselves, putting on alarms and waking up in the middle of the night to eat so that they were not judged in a negative way. They spent years feeling worried and monitored by their families. As one participant stated, although they knew it was for their health, over time, they avoided receiving advice from family members: When everyone at home talks about my MO, I get sick of it. I get up from the table and leave. Then I think and say . . . they’re right. (IDI28) Participants indicated that the support they received from their inner circle was fundamental in making decisions. The people close to them made them want to fight and persevere whilst waiting for their bariatric surgery. Their friends could be a source of support that was even greater than that of their families because they listened to them without judging them: When I’m feeling down, I turn to a friend and tell her my problems and let off steam. My family just repeat the doctor’s advice over and over, they don’t listen to me or understand me. (IDI6) 3.3. Professional Support: Opposing Feelings Healthcare professionals were the key source of formal support for educating patients and finding a solution to their problem. However, bariatric surgery was not the first choice of treatment, nor was it explained in doctor or nurse consultations. 3.2.3. Appearing and Sharing on Social Networks Those with MO participated in associations, which allowed for mutual support, understanding and health education. They shared their experiences, concerns and hopes, enabling them to feel less lonely and increase social contact. As one of the participants said, these associations were an extension of the safe space provided by close family, in which they could share their problems in a social context. The patient association brought us together, it was like a big family. We were social and able to be ourselves, without feeling observed or rejected. (IDI19) 3.3.1. Lack of Professional Support When patients acknowledged that they could not face MO alone, they looked for healthcare professionals to guide them in their treatment. However, as some participants said, they did not find the support they had expected in visits to the doctor, nurse or dietician. Many healthcare professionals did not show interest or did not have the neces- sary training to address MO. The patients note that the professionals did not empathise, listen actively or take their problem seriously, leading them to feel disheartened and less motivated: During a medical visit, the nurse weighed me on a kitchen scale. I felt awful. Is that why During a medical visit, the nurse weighed me on a kitchen scale. I felt awful. Is that why I go to the hospital? To be embarrassed? I stopped going (IDI23) 3.3.2. The Bariatric Nurse Is “Always There” From the first contact with the bariatric surgery team, their vision of professional support changed radically. As one participant said, they slowly gained the confidence and assurance that they needed, they felt accompanied, listened to, informed and knew that they could count on their professional support: Speaking to an expert outside of my family is liberating. I can be honest, express my feelings without anyone judging me . . . this team is just amazing! (IDI1) Along with the social support from family members and close friends, people with MO needed specialised professional support during the surgical process. The participants appreciated the specialist nurse who guided and accompanied them from entering the bariatric surgery programme up until the post-operative phase. They had access to a 24/7 phone service with the bariatric surgery nurse. It allowed them to ask questions, make appointments, book the surgery, inform themselves and share their fears. Family members also contacted the service to ask for information, calm their nerves or talk about their expectations: The specialist nurse manages the situation, always picks up the phone at any hour of the day. It makes you feel assured and confident, you know that you always have someone there to inform, help and advise you . . . someone who listens to your fears and worries. (IDI7) of patients sought consultation after advice from other patients or finding information on the internet: of patients sought consultation after advice from other patients or finding information on the internet: A neighbour told me that she knew people who had been operated, that it was a good team. I only want them to explain it to me properly . . . to lift that burden. (IDI19) I go to the hospital? To be embarrassed? I stopped going. (IDI23) Dieticians insisted that diet alone allows one to achieve the desired weight loss objective, and they did not support the idea of bariatric surgery. General practitioners or nurses did not inform about bariatric surgery in primary care services either. The majority Int. J. Environ. Res. Public Health 2021, 18, 6530 9 of 14 9 of 14 4. Discussion According to Leahy-Warren [40], informal support is key in making the decision to undergo surgery. In this phase, the partner is a fundamental support figure with whom to share worries, experiences and conflicts, thus offering assurance [48]. Support from one’s partner can improve the phase of awaiting surgery [38], especially if both partners are in the bariatric surgery programme [39]. In accordance with our results, some participants were on the receiving end of negative gestures or comments, leading to reduced communication and affection, as well as increased fear of being left [22]. Support from family is key for people with MO [49] and the more support they have, the more likely they are to consider bariatric surgery [27]. Our results reflect the overbearing attitudes of family members, which the people with MO reject, thus leading them to seek support amongst friends [50]. The quality of social support amongst close members of an inner circle can influence weight gain. In fact, strong social support can be a factor in avoiding weight gain, especially in men [51]. In terms of professional support, the participants criticised the information they received on visits to the doctor or nurse, citing a lack of communication, empathy and possibility to be referred to specialists in bariatric surgery. The patients turned to others who had undergone surgery, associations or the internet (web pages, forums, etc.) to inform themselves about the bariatric surgical programme [46]. Their negative opinion changed radically upon contacting the bariatric surgery team [37], as there was an improvement in information, communication, support and quality of life while waiting for bariatric surgery [38]. g y [ ] Social stigma was an added problem for people with MO [21], as social networks and the media promoted an idealised body image. The participants reported a lack of access to quality support for weight control [52], even though they received support from family and professionals every time they used social networks [53]. Social support could be more effective in long-term weight loss but there was a lack of evidence on the subject [24]. It could be important to encourage people with MO to participate in associations to create a shared social identity [36]. Online support groups can be useful for people with MO in a bariatric surgery programme, as they can discuss their problems and share their fears, expectations, challenges and experiences. 4. Discussion The objective of our study was to describe and understand the experiences of social support amongst people with MO included in a bariatric surgery programme. Leahy- Warren’s [40] theoretical framework allowed us to study how formal, informal and struc- tural support influenced their experiences. MO caused physical, psychological and social problems, as well as emotional and body image issues. According to our results, men regarded MO as a health issue that also affected social and amorous relationships [22]. Women, on the other hand, were more worried about the aesthetic facet, feeling stuck in a body that they rejected and did not want to reveal, which, in turn, deteriorated their social relations [21]. MO limited the daily activities of the patients and although they tried to gradually adapt [12], their frustration led them to look for solutions. The participants experienced an initial phase of denial in which they avoided conversations about MO. After failed attempts at losing weight using other methods, their isolation and frustration led them to join a bariatric surgery programme [44]. However, bariatric surgery is not always an option, some patients prefer other treatments to avoid risk [37] or to hide their intentions due to social or economic obstacles [45]. Various studies confirm the positive effects of social support on health and wellbeing during MO [26], highlighting the importance of the partner, family, extended family and professionals. Concurring with Willmer and Salzmann-Erikson [46], people with MO fear bariatric surgery, but they have high expectations for improving their body image, associated conditions and quality of life. The participants believed that losing weight would strengthen their personal identity, social relations and participation in public and professional life [33]. However, their expectations for positive results could lead to disappointment [44]. The role of weight bias in concerns over body image in bariatric surgery patients could jeopardise the results of their treatment. Obese people tend to percieve their weight incorrectly, even after bariatric surgery, patients seem unable to identify changes in their body image after massive weight loss [47]. Social support could positively influence body Int. J. Environ. Res. Public Health 2021, 18, 6530 10 of 14 10 of 14 image perception and interpersonal body comparisons after bariatric surgery, although additional studies should be carried out. image perception and interpersonal body comparisons after bariatric surgery, although additional studies should be carried out. 4. Discussion Healthcare providers must familiarise themselves with the content of these groups and be cautious when recommending them [35]. Participating in online forums and support groups does not predict stress or weight loss [28], but they could increase awareness for people with MO regarding their responsibility in achieving positive outcomes [29]. This social support can be useful for people with MO in a bariatric surgery programme, although it depends on their level of activity. Professionals must improve their understanding and develop and recommend online self-help groups [25]. The use of social networks, private groups or public pages requires careful examination because their effectiveness has not been demonstrated in experimental studies [54]. Together with early support for patients who take part in a bariatric surgery programme, life-long, individualised support may be required to optimise the effects of bariatric surgery. Receiving follow-up visits from a multidisciplinary medical team or joining support groups may be beneficial in counteracting weight gain after bariatric surgery [55]. 5. Conclusions Society’s high beauty standards, the media and social networks all contribute to prejudice towards people with MO. Social stigma, together with physical and psychological problems, demonstrate the need for social support. The majority of participants with MO were slow to acknowledge the problem and joining a bariatric surgery programme was neither an easy decision nor an option they wanted to face alone. They needed information, company and shared experiences. Those with MO had high expectations for bariatric surgery, imagining a radical change in their lives, which could lead to disappointment. 11 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 11 of 14 The partners of people with MO were the key social support figures for them as they needed support to face the surgical process. However, those with MO were sometimes on the receiving end of disrespectful comments, leading to fear of being left by their partners. The partners were in a constant state of alert, worrying about the potential exacerbation of comorbidities, which provoked feelings of blame amongst the people with MO. Family and friends were also members of the participants’ social support groups. Families were aware of the risks of living with MO and therefore monitored and scrutinised the person’s lifestyle. Those with MO felt self-conscious and uncomfortable, thus their friends substituted family as social support. Healthcare professionals were the main source of formal support, but people with MO did not find the support they expected in general practitioners, nurses or dieticians. Some healthcare professionals showed little interest in the issue and lacked specific training to be able to guide the person with MO in making decisions. The majority of people with MO reached the bariatric surgery consultant either through advice from people who had already undergone the surgery or information found on the internet. This situation changed dramatically upon the first contact with the bariatric surgery team as the people with MO felt confident, assured, accompanied, listened to and informed. Those with MO needed a specialised healthcare professional who centralised the information and accompanied them throughout the whole surgical process. 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Organising support groups among patients themselves, or with family or friends, managed by bariatric surgery nurses, could help to improve shortcomings and support decision making. Social support can also help to generate more realistic perspectives regarding outcomes after bariatric surgery in patients. The role of healthcare professionals, friends, partners and/or social networks as a means of support for weight control after bariatric surgery may be fundamental and should be investigated further. Providing images of before and after surgery, as well as explaining that the results are not the same in all patients, could be helpful to patients’ perceptions of their improvements. Author Contributions: Conceptualisation, J.G.-M., M.J.T.-S. and M.F.-M.; methodology, B.E.-F. and M.d.M.J.-L.; formal analysis, A.R.-M. and I.D.-S.; investigation, M.I.V.-M.; data curation, A.R.-M. and B.E.-F.; writing—original draft preparation, M.J.T.-S. and M.F.-M.; writing—review and editing, J.G.-M. and M.F.-M.; supervision, M.J.T.-S. and J.G.-M. All authors have read and agreed to the published version of the manuscript. Funding: Thanks to the Research Group Health Sciences CTS-451 and the Health Research Center from the University of Almería for their support. Institutional Review Board Statement: The study was conducted according to the guidelines of the 2013 Helsinki Declaration. The protocol was approved by the Ethics and Research Committee of the Department of Nursing, Physiotherapy and Medicine (Nº. 45/2018). Informed Consent Statement: Informed consent was obtained from all participants involved in the study. Informed Consent Statement: Informed consent was obtained from all participants involved in the study. Data Availability Statement: Data are available from the authors (J.G.-M., M.J.T.-S. and M.F.-M.) upon reasonable request. Acknowledgments: Our sincere gratitude is due to all participants. Acknowledgments: Our sincere gratitude is due to all participants. Conflicts of Interest: The authors declare no conflict of interest. 12 of 14 12 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 1. Mazer, L.; Morton, J.M. The Obesity Epidemic. In The SAGES Manual of Bariatric Surgery; Reavis, K., Barrett, A., Kroh, M., Eds.; Springer: Cham, Switzerland, 2018; pp. 81–92. References 2019, 20, 1608–1618. [CrossRef] [PubMed] 34. Pereira, M.G.; Faria, S.; Lopes, H. Quality of life one year after bariatric surgery: The moderator role of spirituality. Obes. Surg. 2019, 29, 1207–1215. [CrossRef] 35. Koball, A.M.; Jester, D.J.; Domoff, S.E.; Kallies, K.J.; Grothe, K.B.; Kothari, S.N. 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https://openalex.org/W3195438052
https://link.springer.com/content/pdf/10.1007/s00184-021-00837-y.pdf
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$$\ell _2$$-penalized approximate likelihood inference in logit mixed models for regional prevalence estimation under covariate rank-deficiency
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Metrika (2022) 85:459–489 https://doi.org/10.1007/s00184-021-00837-y Metrika (2022) 85:459–489 https://doi.org/10.1007/s00184-021-00837-y B Joscha Krause krause@uni-trier.de ℓ2-penalized approximate likelihood inference in logit mixed models for regional prevalence estimation under covariate rank-deficiency Joscha Krause1 · Jan Pablo Burgard1 · Domingo Morales2 Received: 1 October 2020 / Accepted: 28 July 2021 / Published online: 17 August 2021 © The Author(s) 2021 1 Department of Economic and Social Statistics, Trier University, Universitätsring 15, 54296 Trier, Germany 2 Operations Research Center, University Miguel Hernández de Elche, Elche, Spain 1 Introduction Regional prevalence estimation is an essential element of modern epidemiologic research (Branscum et al. 2008; Stern 2014; Burgard et al. 2019). Policymakers and health care providers need reliable information on regional disease distributions to plan comprehensive health programs. Depending on the disease of interest, corresponding figures may not be recorded in registries and must be estimated from survey data instead. However, national health surveys often lack in sufficient local observations due to limited resources. As a result, regional prevalence estimates based on survey data can be subject to unacceptable uncertainty due to large sampling variances. Small area estimation (SAE) solves this problem by linking a response variable of interest to statistically related covariates by means of a suitable statistical model. The obser- vations from multiple regions are combined and jointly used for model parameter estimation. Regional prevalence estimates are obtained via model prediction, which allows for an increase in the effective sample size relative to classical direct estimation. See Rao and Molina (2015) for an overview on SAE. In practice, the efficiency advantage of SAE methods over direct estimators is mainly determined by two aspects: (i) finding a suitable model type to describe the response variable, and (ii) having covariate data with explanatory power. Regarding the first aspect, since regional prevalence is usually stated as proportion (number of sick persons divided by the total number of persons), binomial, Poisson or negative binomial mixed models are canonical choices. The binomial-logit approach has been used for regional proportion estimation in the past, for instance by Molina et al. (2007), Ghosh et al. (2009), Chen and Lahiri (2012), Erciulescu and Fuller (2013), López-Vizcaíno et al. (2013), López-Vizcaíno et al. (2015), Burgard (2015), Militino et al. (2015), Chambers et al. (2016), Hobza and Morales (2016), Liu and Lahiri (2017) and Hobza et al. (2018). The Poisson or negative binomial mixed models were applied to estimate small area counts or proportions by Berg (2010), Chambers et al. (2014), Dreassi et al. (2014), Tzavidis et al. (2015) and Boubeta et al. (2016, 2017), among others. Marino et al. (2019) propose a semiparametric approach allowing for a flexible random effects structure in unit-level models. Ranalli et al. (2018) introduced benchmarking for logistic unit-level. Concerning the second aspect, medical routine data provided by official statistics or health insurance companies have been found to be promising data bases for regional prevalence estimation. Abstract Regional prevalence estimation requires the use of suitable statistical methods on epidemiologic data with substantial local detail. Small area estimation with medical treatment records as covariates marks a promising combination for this purpose. How- ever, medical routine data often has strong internal correlation due to diagnosis-related groupingintherecords.Dependingonthestrengthofthecorrelation,thespacespanned by the covariates can become rank-deficient. In this case, prevalence estimates suffer from unacceptable uncertainty as the individual contributions of the covariates to the model cannot be identified properly. We propose an area-level logit mixed model for regional prevalence estimation with a new fitting algorithm to solve this problem. We extend the Laplace approximation to the log-likelihood by an ℓ2-penalty in order to stabilize the estimation process in the presence of covariate rank-deficiency. Empiri- cal best predictors under the model and a parametric bootstrap for mean squared error estimation are presented. A Monte Carlo simulation study is conducted to evaluate the properties of our methodology in a controlled environment. We further provide an empirical application where the district-level prevalence of multiple sclerosis in Germany is estimated using health insurance records. Keywords Generalized linear mixed models · Laplace approximation · Multiple sclerosis · Prevalence mapping · Small area estimation 2 Operations Research Center, University Miguel Hernández de Elche, Elche, Spain 123 123 460 J. Krause et al. 1 Introduction Exemplary applications were provided by Tamayo et al. (2016), Burgard et al. (2019), and Breitkreutz et al. (2019). However, using medical routine data as covariates can be problematic, especially within logit mixed models. Medical treatment frequencies are typically recorded and coded into diagnosis groups, for instance on ICD-3 level (World Health Organization 2018). This context-related segmentation can induce strong correlation between med- ical treatment frequencies for diseases that are closely related in terms of comorbidity, such as diabetes and hypertension (Long and Dagogo-Jack 2011). If two or more diagnoses from the auxiliary data set are strongly correlated, the space spanned by the covariates can become rank-deficient. In that case, it is not possible to accurately sep- arate the individual contributions of the covariates to the description of the response variable. Model parameter estimates suffer from high variance and model predic- 123 ℓ2-penalized approximate... 461 tions for regional prevalence are not reliable. This is particularly problematic for logit mixed models, as model parameter estimation already relies on approximate inference in the absence of rank-deficiency (Breslow and Clayton 1993). The respective like- lihood integral does not have a closed-form solution, which requires techniques like the Laplace approximation to find a proper substitute as objective function. Therefore, when approximate inference is to be performed on a rank-deficient covariate space, methodological adjustments are required to allow for reliable results. In this paper, we propose a modification to the maximum likelihood Laplace (ML- Laplace) algorithm for model parameter estimation (e.g. Demidenko 2013; Hobza et al. 2018) in a logit mixed model under covariate rank-deficiency. We draw from theoretical insights on ridge regression (Hoerl and Kennard 1970) and extend the Laplace approximation to the log-likelihood function by the squared ℓ2-norm of the regression parameters (ℓ2-penalty). This adjustment reduces the variance of model parameter estimates considerably and improves approximate inference in the presence of strong covariate correlation. To the best of our knowledge, ℓ2-penalization has only been studied for standard ML estimation in fixed effect logit models, for instance by Schaefer et al. (1984), Cessie and Houwelingen (1992), and Pereira et al. (2016). We are not aware of corresponding studies for logit mixed models based on ML-Laplace estimation, especially not in the context of SAE. An area-level binomial logit mixed model for regional prevalence estimation is presented. 1 Introduction Following Jiang and Lahiri (2001) and Jiang (2003), we derive empirical best predictors (EBPs) under the model and present a parametric bootstrap estimator for their mean squared error (MSE). Thereafter, we state the Laplace approximation to the log-likelihood function and demonstrate ℓ2-penalized approximate likelihood (ℓ2-PAML) estimation of the model parameters. We further show how the tuning parameter for the ℓ2-penalty can be chosen in practice. A Monte Carlo simulation study is conducted to study the behavior of ℓ2-PAML estimation under different degrees of covariate correlation. And finally, the proposed methodology is applied to regional prevalence estimation in Germany. We use health insurance records of the German Public Health Insurance Company (AOK) and inpatient diagnosis frequencies of the Diagnosis-Related Group Statistics (DRG-Statistics) to estimate district-level multiple sclerosis prevalence. The remainder of the paper is organized as follows. In Sect. 2, we present the model and its EBP. We further address MSE estimation. In Sect. 3, we present the Laplace approximation and the technical details for ℓ2-PAML. Section 4 contains a Monte Carlo simulation study. Section 5 covers the application to regional prevalence estimation. Section 6 closes with some conclusive remarks. 2.1 Formulation For the subsequent derivation, we rely on model-based inference in a finite population setting. Let U be a finite population of size |U| = N. Suppose that U is partitioned into D domains Ud of size |Ud| = Nd. That is to say, U = ∪D d=1Ud, Ud1 ∩Ud2 = ∅, For the subsequent derivation, we rely on model-based inference in a finite population setting. Let U be a finite population of size |U| = N. Suppose that U is partitioned into D domains Ud of size |Ud| = Nd. That is to say, U = ∪D d=1Ud, Ud1 ∩Ud2 = ∅, 123 462 J. Krause et al. d1 ̸= d2, and D d=1 Nd = N. Let S be a sample of size |S| = n that is drawn from U. For simplicity, assume the sampling scheme is such that there are domain-specific subsamples Sd of size |Sd| = nd with fixed nd > 0 for all d = 1, ..., D. Thus, we have S = ∪D d=1Sd and D d=1 nd = n. Let y be a dichotomous response variable with potential outcomes {0, 1}. Denote the realization of y for some individual i ∈Ud by yid. Note that we use the same symbol for a random variable and its realizations in order to avoid overloading the notation. Define yd =  i∈sd yid as the sample total (count) of y in domain Ud. Let x = {x1, ..., x p} be a set of covariates statistically related to y. Denote xd as a 1 × p vector of aggregated (domain-level) realizations of x. Suppose that corresponding information is retrieved from administrative records and not calculated from the sample S. In what follows, we present an area-level logit mixed model for estimating the domain totals Yd =  i∈Ud yid or proportions pd = Yd/Nd of the response variable. Let us consider a set of random effects such that {vd : d = 1, . . . , D} are independent and identically distributed according to vd ∼N(0, 1). In matrix notation, we have normally distributed random effects v = col 1≤d≤D(vd) ∼ND(0, I D) (1) (1) and, hence, their probability density function (PDF) is stated as and, hence, their probability density function (PDF) is stated as fv(v) = (2π)−D/2 exp  −1 2 v′v  . 2.1 Formulation (2) (2) The model assumes that the distribution of the response variable yd, conditioned to the random effect vd, is yd|vd ∼Bin(nd, pd), d = 1, . . . , D, (3) (3) and that the natural parameter fulfills ηd = log pd 1 −pd = xdβ + φvd, d = 1, . . . , D, (4) (4) where φ > 0 is an standard deviation parameter, β = col1≤r≤p(βr) is the vector of regression parameters and xd = col′ 1≤r≤p(xdr). We complete the model definition by assuming that the domain-specific sample counts yd are independent when conditioned on the random effects v. Therefore, the conditional PDF of y = col1≤d≤D(yd) given v is stated as P(y|v) = D  d=1 P(yd|vd), P(yd|v) = P(yd|vd) = nd yd  pyd d (1 −pd)nd−yd, (5) (5) where where pd = exp {xdβ + φvd} 1 + exp {xdβ + φvd} = exp{ηd} 1 + exp{ηd}, 1 −pd = 1 1 + exp{ηd}. (6) (6) 123 123 ℓ2-penalized approximate... 463 Further, the unconditional PDF of y is Further, the unconditional PDF of y is P(y) =  RD P(y|v) fv(v) dv =  RD ψ(y, v) dv, (7) P(y) =  RD P(y|v) fv(v) dv =  RD ψ(y, v) dv, (7) P(y) =  RD P(y|v) fv(v) dv =  RD ψ(y, v) dv, (7) with (7) with with with with ψ(y, v) = (2π)−D 2 exp −v′v 2 D  d=1 nd yd exp {yd(xdβ + φvd)} 1 + exp {xdβ + φvd}nd = (2π)−D 2 D  d=1 nd yd  exp −v′v 2 exp  p  k=1 D  d=1 ydxdk βk + φ D  d=1 ydvd − D  d=1 nd log (1 + exp {xdβ + φvd})  . (8) ψ(y, v) = (2π)−D 2 exp −v′v 2 D  d=1 nd yd exp {yd(xdβ + φvd)} 1 + exp {xdβ + φvd}nd = (2π)−D 2 D  d=1 nd yd  exp −v′v 2 exp  p  k=1 D  d=1 ydxdk βk + φ D  d=1 ydv − D  d=1 nd log (1 + exp {xdβ + φvd})  . (8) 2.2 Prediction Hereafter, we obtain EBPs under the area-level logit mixed model introduced in Sect. 2.1. For this, wefirst derivebest predictors (BPs) inapreliminarysettingwhereall model parameters θ := (β′, φ) are assumed to be known. Then, the EBPs are obtained by replacing the full parameter vector θ by consistent estimators ˆθ := ( ˆβ ′, ˆφ). Note that calculating the EBP requires Monte Carlo integration over the random effect PDF, which can be computationally infeasible for some applications. Therefore, we also state two alternative predictors that do not rely on Monte Carlo integration and are easier to apply in practice. We start with the EBPs. Recall the definition of the conditional PDF P(y|v) from the last section. For the domain-specific component P(yd|vd), we can write P(yd|vd) = nd yd  pyd d (1 −pd)nd−yd = nd yd exp {yd(xdβ + φvd)} 1 + exp {xdβ + φvd}nd = exp log nd yd  + yd(xdβ + φvd) −nd log 1 + exp{xdβ + φvd}  .(9) The probability density function of v is The probability density function of v is The probability density function of v is f (v) = D  d=1 f (vd), f (vd) = (2π)−1/2 exp  −1 2 v2 d  . (10) (10) The BP of pd = pd(θ, vd) is given by the conditional expectation ˆpd(θ) = Eθ[pd|y]. Due to the conditional independence of the response realizations given the random effects, we have Eθ[pd|y] = Eθ[pd|yd] and The BP of pd = pd(θ, vd) is given by the conditional expectation ˆpd(θ) = Eθ[pd|y]. Due to the conditional independence of the response realizations given the random effects, we have Eθ[pd|y] = Eθ[pd|yd] and 123 123 464 J. Krause et al. Eθ[pd|yd] =  R exp{xdβ+φvd} 1+exp{xdβ+φvd} P(yd|vd) f (vd) dvd  R P(yd|vd) f (vd) dvd = Nd(yd, θ) Dd(yd, θ) = Nd(yd, θ) Dd(yd, θ), (11) (11) where Nd = Nd(yd, θ), Dd = Dd(yd, θ), Nd = Nd(yd, θ) and Dd = Dd(yd, θ) are functions of the model parameters and the domain-specific sample counts. 2.2 Prediction They are stated as follows: Nd =  R exp{xdβ + φvd} 1 + exp{xdβ + φvd} exp log nd yd  + yd xdβ + φydvd −nd log 1 + exp{xdβ + φvd}  f (vd) dvd, Dd =  R exp log nd yd  + yd xdβ + φydvd −nd log 1 + exp{xdβ + φvd}  f (vd) dvd, Nd =  R exp{xdβ + φvd} 1 + exp{xdβ + φvd} exp  φydvd −nd log 1 + exp{xdβ + φvd}  f (vd) dvd, Dd =  R exp  φydvd −nd log 1 + exp{xdβ + φvd}  f (vd) dvd. We can conclude that the EBP of pd is ˆpd(ˆθ). However, its quantification requires integration over the random effect PDF f (vd). As the logit mixed model belongs to the family of generalized linear mixed models, this cannot be performed analytically. Instead, we apply Monte Carlo integration and approximate the EBP as follows: 1. Estimate ˆθ = ( ˆβ, ˆφ). (β, φ) 2. For k = 1, . . . , K, generate v(k) d i.i.d. N(0, 1) and v(K+k) d = −v(k) d . 3 Calculate ˆpd(ˆθ) = ˆNd/ ˆDd where (β, φ) 2. For k = 1, . . . , K, generate v(k) d i.i.d. N(0, 1) and v(K+k) d = −v(k) d . ˆ ˆ ˆ d 3. Calculate ˆpd(ˆθ) = ˆNd/ ˆDd, where 3. Calculate ˆpd(ˆθ) = ˆNd/ ˆDd, where ˆNd = 1 2K 2K  k=1  exp{xd ˆβ + ˆφv(k) d } 1 + exp{xd ˆβ + ˆφv(k) d } exp  ˆφydv(k) d −nd log 1 + exp{xd ˆβ + ˆφv(k) d }   , ˆDd = 1 2K 2K  k=1 exp  ˆφydv(k) d −nd log  1 + exp{xd ˆβ + ˆφv(k) d }  . ˆDd = 1 2K 2K  k=1 exp  ˆφydv(k) d −nd log  1 + exp{xd ˆβ + ˆφv(k) d }  . The EBP of pd can be used to obtain the predictor ˆYd = Nd ˆp(ˆθ) of the domain total Yd. The EBP of pd can be used to obtain the predictor ˆYd = Nd ˆp(ˆθ) of the domain total Yd. We now state two alternative predictors that do not rely on Monte Carlo integration. The first is a synthetic predictor. 2.3 Mean squared error estimation In order to assess the reliability of the obtained predictions for pd, we use the mean squared error. It is generally characterized by MSE( ˆpd) = E[( ˆpd −pd)2]. However, MSE( ˆpd) cannot be quantified directly and must be estimated instead. For this, we apply a parametric bootstrap as demonstrated by González-Manteiga et al. (2007) and Boubeta et al. (2016). It is performed as follows. 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ ′, ˆφ). 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ ′, ˆφ). 2. Repeat B times with b = 1, ..., B: 2. Repeat B times with b = 1, ..., B: (a) Generate v(b) d ∼N(0, 1), y(b) d ∼Bin(nd, p(b) d ), d = 1, . . . , D, where p(b) d = exp  xd ˆβ+ ˆφv(b) d  1+exp  xd ˆβ+ ˆφv(b) d . (b)   (b) For each bootstrap sample, calculate the estimator ˆθ (b) and the EBP ˆp(b) d = ˆp(b) d (ˆθ (b)) as stated above. (b) For each bootstrap sample, calculate the estimator ˆθ (b) and the EBP ˆp(b) d = ˆp(b) d (ˆθ (b)) as stated above. 3. Output: mse( ˆpd) = 1 B B b=1 ˆp(b) d −p(b) d 2. 2.2 Prediction It is characterized by a regression-synthetic prediction from the area-level logit mixed model without considering the random effect. On that note, the synthetic predictor of pd is obtained according to ˜psyn d = exp{xd ˆβ} 1 + exp{xd ˆβ} , (12) (12) which constitutes the synthetic predictor ˜Y syn d = Nd ˜psyn d for Yd. The plug-in predictor is obtained along the same lines, but includes the random effects vd as well as the which constitutes the synthetic predictor ˜Y syn d = Nd ˜psyn d for Yd. The plug-in predictor is obtained along the same lines, but includes the random effects vd as well as the ℓ2-penalized approximate... 465 variance parameter φ. For the prediction of pd, we have variance parameter φ. For the prediction of pd, we have ˜p plug d = exp{xd ˆβ + ˆφ ˆvd} 1 + exp{xd ˆβ + ˆφ ˆvd} , (13) (13) where ˆvd is a predictor for the random effect vd. We describe how to calculate the corresponding predictors in Sect. 3. Finally, the plug-in predictor of Yd is ˜Y plug d = Nd ˜p plug d . where ˆvd is a predictor for the random effect vd. We describe how to calculate the corresponding predictors in Sect. 3. Finally, the plug-in predictor of Yd is ˜Y plug d = Nd ˜p plug d . 3 Penalized model parameter estimation In this section, it is demonstrated how model parameter estimation in the area-level logit mixed model under covariate rank-deficiency is performed. The foundation of our estimation strategy is the ML-Laplace algorithm (e.g. Demidenko 2013; Hobza et al. 2018). That is to say, the integrals in the likelihood function are approximated via the Laplace method and the result is maximized with a Newton-Raphson algorithm. However, in light of the comments in Sect. 1 and prior to maximization, we extend the approximated likelihood function by the squared ℓ2-norm of β to account for the negative effects of covariate rank-deficiency. With this, we obtain a penalized version approximated likelihood, which is then maximized to obtain reliable model parameter estimates.Werefertothisprocedureasℓ2-penalizedapproximatemaximumlikelihood (ℓ2-PAML) estimation. 123 J. Krause et al. 466 3.1 Laplace approximation We first perform the Laplace approximation of the likelihood function. Let h : R →R be a continuously twice differentiable function with a global maximum at x0. This is to say, assume that h′(x0) = 0 and h′′(x0) < 0. A Taylor series expansion of h(x) around x0 yields h(x) = h(x0) + h′(x0)(x −x0) + 1 2h′′(x0)(x −x0)2 + o |x −x0|2 ≈h(x0) + 1 2h′′(x0)(x −x0)2. (14) The univariate Laplace approximation is The univariate Laplace approximation is The univariate Laplace approximation is  ∞ −∞ eh(x) dx ≈  ∞ −∞ eh(x0) exp  −1 2 −h′′(x0) (x −x0)2 dx = (2π)1/2 −h′′(x0) −1/2eh(x0)  ∞ −∞ exp  −1 2  x−x0 (−h′′(x0))−1/2 2 (2π)1/2 −h′′(x0) −1/2 d = (2π)1/2 −h′′(x0) −1/2eh(x0). (15  ∞ −∞ eh(x) dx ≈  ∞ −∞ eh(x0) exp  −1 2 −h′′(x0) (x −x0)2 dx = (2π)1/2 −h′′(x0) −1/2eh(x0)  ∞ −∞ exp  −1 2  x−x0 (−h′′(x0))−1/2 2 (2π)1/2 −h′′(x0) −1/2 dx (2 )1/2 h′′( ) −1/2 h(x0) (15) = (2π)1/2 −h′′(x0) −1/2eh(x0)  ∞ −∞ exp  2  (−h′′(x0))−1/2   (2π)1/2 −h′′(x0) −1/2 dx = (2π)1/2 −h′′(x0) −1/2eh(x0). (15) = (2π)1/2 −h′′(x0) −1/2eh(x0). (15) (15) Let us now approximate the log-likelihood of the area-level logit mixed model. Recall that v1, . . . , vD are independent and identically distributed according to vd ∼N(0, 1), and that Let us now approximate the log-likelihood of the area-level logit mixed model. Recall that v1, . . . , vD are independent and identically distributed according to vd ∼N(0, 1), and that yd|vd ind ∼Bin(nd, pd), pd = pd(vd) = exp {xdβ + φvd} 1 + exp {xdβ + φvd}, d = 1, . . . , D. Thus, y1, . . . , yD are unconditionally independent with marginal probability density Thus, y1, . . . 3.1 Laplace approximation (18) From there, we can state the log-likelihood function under the model, which is given by From there, we can state the log-likelihood function under the model, which is given by l = D  d=1 ld = log P(yd). l = D  d=1 ld = log P(yd). Using the results of the Laplace approximation, we obtain Using the results of the Laplace approximation, we obtain ld ≈l0d(θ) = log nd yd  −1 2 log ξ0d −v2 0d 2 +yd(xdβ + φv0d)nd log 1 + exp{xdβ + φv0d} , (19) ld ≈l0d(θ) = log nd yd  −1 2 log ξ0d −v2 0d 2 +yd(xdβ + φv0d)nd log 1 + exp{xdβ + φv0d} , (19) (19) where p0d = pd(v0d) and ξ0d = 1 + φ2nd p0d(1 −p0d). where p0d = pd(v0d) and ξ0d = 1 + φ2nd p0d(1 −p0d). where p0d = pd(v0d) and ξ0d = 1 + φ2nd p0d(1 −p0d). 3.1 Laplace approximation , yD are unconditionally independent with marginal probability density ∞ P(yd) =  ∞ −∞ P(yd|vd) f (vd) dvd =  ∞ −∞ nd yd  exp  yd(xdβ + φvd} −nd log 1 + exp{xdβ + φvd}  ·(2π)−1/2 exp{−1 2v2 d} dvd = (2π)−1/2 nd yd  ·  ∞ −∞ exp  −v2 d 2 + yd(xdβ + φvd) −nd log 1 + exp{xdβ + φvd}  dvd (2 ) 1/2 nd   ∞  h( )  d (16 P(yd) =  ∞ −∞ P(yd|vd) f (vd) dvd =  ∞ nd  exp  yd(xdβ + φvd} −nd log 1 + exp{xdβ + φvd}  P(yd) =  ∞ −∞ P(yd|vd) f (vd) dvd =  ∞ −∞ nd yd  exp  yd(xdβ + φvd} −nd log 1 + exp{xdβ + φvd}  ·(2π)−1/2 exp{−1 2v2 d} dvd = (2π)−1/2 nd yd  ·  ∞ −∞ exp  −v2 d 2 + yd(xdβ + φvd) −nd log 1 + exp{xdβ + φvd}  dvd = (2π)−1/2 nd yd   ∞ −∞ exp  h(vd)  dvd, (16) where h(vd) = −v2 d + yd(xdβ + φvd) −nd log 1 + exp{xdβ + φvd} (17) P(yd) =  ∞ −∞ P(yd|vd) f (vd) dvd =  ∞ ∞ P(yd|vd) f (vd) dvd =  ∞ −∞ nd yd  exp  yd(xdβ + φvd} −nd log 1 + exp{xdβ + φvd}  ·(2π)−1/2 exp{−1 2v2 d} dvd = (2π)−1/2 nd yd  ∞ 2 ·  ∞ −∞ exp  −v2 d 2 + yd(xdβ + φvd) −nd log 1 + exp{xdβ + φvd}  dvd = (2π)−1/2 nd yd   ∞ −∞ exp  h(vd)  dvd, (16) = (2π)−1/2 nd yd   ∞ −∞ exp  h(vd)  dvd, (16) (16) (16) here where h(vd) = −v2 d 2 + yd(xdβ + φvd) −nd log 1 + exp{xdβ + φvd} . (17) (17) 123 123 467 ℓ2-penalized approximate... Note that for the maximizer of h(·), denoted by v0d, the first derivative is h′(v0d) = 0, and the second derivative is characterized by h′′(v0d) < 0. By applying (15) in vd = v0d, we get P(yd) ≈ nd yd  ·  1 + φ2nd pd(v0d)(1 −pd(v0d)) −1/2 · exp  −v2 0d 2 + yd(xdβ + φv0d) −nd log 1 + exp{xdβ + φv0d}  . Step 1: Log-likelihood approximation In order to maximize h(vd), we need to quantify its first and second derivatives. These are h′(vd) = −vd + φ  yd −nd pd(vd)  (21) h′′(vd) = −  1 + φ2nd pd(vd)(1 −pd(vd))  (22) (21) (22) for all d = 1, ..., D. The Newton-Raphson algorithm maximizes h(vd) = h(vd, θ), defined in (17), for fixed θ = (β′, φ) = θ0. The updating equation is v(k+1) d = v(k) d −h′(v(k) d , θ0) h′′(v(k) d , θ0) , (23) (23) where k denotes an iteration of the procedure. where k denotes an iteration of the procedure. where k denotes an iteration of the procedure. 3.2 ℓ2-penalized approximate maximum likelihood The approximated log-likelihood function is expanded by the squared ℓ2-norm of the regression coefficients β to account for strong correlation between covariates in x1, . . . , xD. We obtain the penalized maximum likelihood problem ˆθ = argmax θ∈Rp+1 l pen(θ), l pen(θ) = D  d=1 l0d(θ) −λ∥β∥2 2, (20) (20) where l0d(θ) is defined in (19) and λ > 0 is a predefined tuning parameter. Maximiza- tion is performed via a Newton-Raphson algorithm. However, note that the Laplace approximations of l1, ...,lD depends on the maximizers of h(v1), ..., h(vD), which in turn depend on l1, ...,lD. Therefore, the maximization of (20) must contain two steps that are performed iteratively and conditional on each other. The first step is the approximation of the log-likelihood by maximizing h(v1), ..., h(vD). The second step is the maximization of l pen(θ) given the results of the first step. This is demonstrated hereafter. 123 J. Krause et al. 468 ep 1: Log-likelihood approximation Step 2: Penalized maximization ∂η0dr ∂φ = 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2 0d)∂p0d ∂φ = φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2 0d)], ∂η0d ∂βr = φ2v0dndxdr p0d(1 −p0d)[1 −6p0d + 6p2 0d], ∂η0d ∂φ = 2nd p0d(1 −p0d) + 2φnd1 −2p0d)∂p0d ∂φ ∂ ∂η0dr ∂φ = 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2 0d)∂p0d ∂φ = φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2 0d)], ∂ ∂η0dr ∂φ = 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2 0d)∂p0d ∂φ = φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2 0d)], ∂η0d = φ2v0dndxdr p0d(1 −p0d)[1 −6p0d + 6p2 0d], +2φnd(1 −2p0d)p0d(1 −p0d)v0d + φ2ndv0d(1 −6p0d + 6p2 0d)∂p0d ∂φ = nd p0d(1 −p0d)[2 + 2φ(1 −2p0d)v0d + 2φ(1 −2p0d)v0d +φ2v2 0d(1 −6p0d + 6p2 0d)]. For the domain-specific likelihood component l0d, this yields to ∂2l0d ∂β2r = −1 2 ∂η0dr ∂βr ξ0d −η2 0dr ξ2 d −ndx2 dr p0d(1 −p0d), ∂2l0d ∂β2r = −1 2 ∂η0dr ∂βr ξ0d −η2 0dr ξ2 d −ndx2 dr p0d(1 −p0d), ∂2l0d ∂βs∂βr = −1 2 ∂η0dr ∂βs ξ0d −η0drη0ds ξ2 d −ndxdr xdjs p0d(1 −p0d), ∂2l0d ∂φ∂βr = −1 2 ∂η0dr ∂φ ξ0d −η0drη0d ξ2 d −v0dndxdr p0d(1 −p0d), ∂2l0d ∂φ2 = −1 2 ∂η0d ∂φ ξ0d −η2 0d ξ2 d −v2 0dnd p0d(1 −p0d). ∂2l0d ∂βs∂βr = −1 2 ∂η0dr ∂βs ξ0d −η0drη0ds ξ2 d −ndxdr xdjs p0d(1 −p0d), ∂2l0d ∂φ∂βr = −1 2 ∂η0dr ∂φ ξ0d −η0drη0d ξ2 d −v0dndxdr p0d(1 −p0d), ∂2l0d ∂φ2 = −1 2 ∂η0d ∂φ ξ0d −η2 0d ξ2 d −v2 0dnd p0d(1 −p0d). As for the first partial derivatives applying these equations to all domain-specific likelihood components l01, ...,l0D and considering the ℓ2-penalty, we end up with As for the first partial derivatives applying these equations to all domain-specific likelihood components l01, ...,l0D and considering the ℓ2-penalty, we end up with ∂2l pen ∂β2r = D  d=1 ∂2l0d ∂β2r −2λ, ∂2l pen ∂βs∂βr = D  d=1 ∂2l0d ∂βs∂βr , ∂2l pen ∂φ∂βr = D  d=1 ∂2l0d ∂φ∂βr , ∂2l pen ∂φ2 = D  d=1 ∂2l0d ∂φ2 . (25) (25) For r, s = 1, . . . , p + 1, define the components of the score vector For r, s = 1, . . . Step 2: Penalized maximization We continue with maximizing the penalized approximate log-likelihood function. Regarding the first partial derivatives of l pen with respect to β1, ..., βp and φ, it holds that ∂p0d ∂βr = xdr p0d(1 −p0d) = xdr(p0d −p2 0d), ∂p0d ∂φ = v0d p0d(1 −p0d) = v0d(p0d −p2 0d), η0dr = ∂ξ0d ∂βr = φ2ndxdr[p0d −3p2 0d + 2p3 0d], η0d = ∂ξ0d ∂φ = 2φnd p0d(1 −p0d) + φ2nd(1 −2p0d)∂p0d δφ = φnd p0d(1 −p0d)[2 + φ(1 −2p0d)v0d]. For the domain-specific likelihood component l0d, this yields to ∂l0d ∂βr = −1 2 η0dr ξ0d + (yd −nd p0d)xdr, ∂l0d ∂φ = −1 2 η0d ξ0d + (yd −nd p0d)v0d. With the application of these equations to all domain-specific likelihood components l01, ...,l0D and the consideration of the ℓ2-penalty, we finally obtain With the application of these equations to all domain-specific likelihood components l01, ...,l0D and the consideration of the ℓ2-penalty, we finally obtain ∂l pen ∂βr = D  d=1 ∂l0d ∂βr −2λβr, ∂l pen ∂φ = D  d=1 ∂l0d ∂φ . (24) (24) 123 ℓ2-penalized approximate... 469 For the second partial derivatives, it holds that For the second partial derivatives, it holds that For the second partial derivatives, it holds that ∂η0dr ∂βs = φ2ndxdr xdjs[p0d(1 −p0d) −6p2 0d(1 −p0d) + 6p3 0d(1 −p0d)] = φ2ndxdr xdjs p0d(1 −p0d)[1 −6p0d + 6p2 0d], βs = φ2ndxdr xdjs p0d(1 −p0d)[1 −6p0d + 6p2 0d], ∂η0dr ∂φ = 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2 0d)∂p0d ∂φ = φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2 0d)], ∂η0d ∂βr = φ2v0dndxdr p0d(1 −p0d)[1 −6p0d + 6p2 0d], ∂η0d ∂φ = 2nd p0d(1 −p0d) + 2φnd1 −2p0d)∂p0d ∂φ +2φnd(1 −2p0d)p0d(1 −p0d)v0d + φ2ndv0d(1 −6p0d + 6p2 0d)∂p0d ∂φ = nd p0d(1 −p0d)[2 + 2φ(1 −2p0d)v0d + 2φ(1 −2p0d)v0d +φ2v2 0d(1 −6p0d + 6p2 0d)]. Step 2: Penalized maximization , p + 1, define the components of the score vector U0r = ∂l pen ∂βr , U0p+1 = ∂l pen ∂φ , (26) (26) 123 123 123 J. Krause et al. 470 as well as the Hessian matrix as well as the Hessian matrix as well as the Hessian matrix H0rs = H0sr = ∂2l pen ∂βs∂βr , Hrp+1 = Hp+1r = ∂2l pen ∂φ∂βr , H0p+1p+1 = ∂2l pen ∂φ2 . (27) (27) In matrix form, we have U0 = U0(θ) = col 1≤r≤p+1(U0r) and H0 = H0(θ) = In matrix form, we have U0 = U0(θ) = col 1≤r≤p+1(U0r) and H0 = H0(θ) = (H0rs)r,s=1,...,p+1. The Newton-Raphson algorithm maximizes l pen(θ), with fixed vd = v0d, d = 1, . . . , D. Let k denote the index of iterations. The corresponding updating equation is θ(k+1) = θ(k) −H−1 0 (θ(k))U0(θ(k)). (28) (28) Complete ℓ2-PAML algorithm (c) k ←k + 1. 3. Output: ˆθ = θ(k), ˆvd = v(k) d , d = 1, . . . , D. We remark that the output of the ℓ2-PAML algorithm gives estimates ˆθ of the model parameters θ and mode predictions ˆv of the random effects vd, d = 1, ..., D. We remark that the output of the ℓ2-PAML algorithm gives estimates ˆθ of the model parameters θ and mode predictions ˆv of the random effects vd, d = 1, ..., D. Complete ℓ2-PAML algorithm The final algorithm containing both steps is performed as follows. 1. Set the initial values k = 0, θ(0), θ(−1) = θ(0) + 1, v(0) d = 0, v(−1) d = 1, d = 1, . . . , D. 2. Until ∥θ(k) −θ(k−1)∥2 < ε1, |v(k) d −v(k−1) d | < ε2, d = 1, . . . , D, do 1. Set the initial values k = 0, θ(0), θ(−1) = θ(0) + 1, v(0) d = 0, v(−1) d = 1, d = 1, . . . , D. (k) (k 1) (k) (k 1) 1. Set the initial values k = 0, θ(0), θ(−1) = θ(0) + 1, v(0) d = 0, v(−1) d = 1, d = 1, . . . , D. (k) (k 1) , , + , d , d 1, . . . , D. 2. Until ∥θ(k) −θ(k−1)∥2 < ε1, |v(k) d −v(k−1) d | < ε2, d = 1, . . . , D, do , , 2. Until ∥θ(k) −θ(k−1)∥2 < ε1, |v(k) d −v(k−1) d | < ε2, d = 1, . . . , D, do (a) Apply algorithm (23) with seeds v(k) d , d = 1, . . . , D, convergence tolerance (a) Apply algorithm (23) with seeds v(k) d , d = 1, . . . , D, convergence tolerance ε2 and θ = θ(k) fixed. Output: v(k+1) d , d = 1, . . . , D. (a) Apply algorithm (23) with seeds v(k) d , d = 1, . . . , D, convergence tolerance ε2 and θ = θ(k) fixed. Output: v(k+1) d , d = 1, . . . , D. (a) Apply algorithm (23) with seeds v(k) d , d = 1, . . . , D, convergence tolerance ε2 and θ = θ(k) fixed. Output: v(k+1) d , d = 1, . . . , D. d ε2 and θ = θ(k) fixed. Output: v(k+1) d , d = 1, . . . , D. k (b) Applyalgorithm(28)withseedθ(k),convergencetoleranceε1 andv0d = v(k+1) d fixed, d = 1, . . . , D. Output: θ(k+1). (b) Applyalgorithm(28)withseedθ(k),convergencetoleranceε1 andv0d = v(k+1) d fixed, d = 1, . . . , D. Output: θ(k+1). (c) k ←k + 1. (b) Applyalgorithm(28)withseedθ(k),convergencetoleranceε1 andv0d = v(k+1) d fixed, d = 1, . . . , D. Output: θ(k+1). 3.3 Tuning parameter choice and information criterion In the technical descriptions of Sect. 3.2, we assumed that the tuning parameter λ had been defined prior to model parameter estimation. In practice, it has to be found empirically from the sample data. Note that this aspect is crucial for the effectiveness of the proposed method. On the one hand, if λ is chosen too small, the ℓ2-PAML approach cannot sufficiently stabilize model parameter estimates in the presence of covariate rank-deficiency. On the other hand, if λ is chosen too large, the shrinkage induced by penalization dominates the optimization problem and resulting model parameter estimates are heavily biased. Finding an appropriate value for the tuning parameter is oftendoneviagridsearch,ascanbeseenforinstanceinBergstraandBengio(2012)and Chicco (2017). We define a sequence of candidate values {λq}Q q=1, where λq > λq+1. For each candidate value λq model parameter estimation as demonstrated in Sect. 3.2 is performed. The results of model parameter estimation have to be evaluated by a 471 ℓ2-penalized approximate... suitable goodness-of-fit measure. For our application, we choose the non-corrected Bayesian information criterion (BIC; Schwarz 1978). Alternative measures would be the generalized cross-validation criterion (Craven and Wahba 1979) or the Akaike information criterion (Akaike 1974). For given candidate value λq, let ˆβ(λq) and ˆφ(λq) be the estimators of β and φ, respectively. Further, let ˆvd(λq) be the mode predictor of vd. The Laplace non-corrected BIC is given by BIC(λq) = p log(D) −2lapp(λq), (29) (29) where the second term is the Laplace approximation (19) to the log-likelihood, that is lapp(λq) = lapp  ˆβ(λq), ˆφ(λq), ˆv1(λq), . . . , ˆvD(λq)  = D  d=1 log nd yd  + D  d=1 −1 2 log ˆξd(λq) −ˆvd(λq)2 2 +  yd(xd ˆβ(λq) + ˆφ(λq)ˆvd(λq)) −nd log 1 + exp{xd ˆβ(λq) + ˆφ(λq)ˆvd(λq)}  , where ˆξd(λq) = 1 + ˆφ(λq)2nd ˆpd(λq)(1 −ˆpd(λq)), ˆpd(λq) = exp  xd ˆβ(λq) + ˆφ(λq)ˆvd(λq)  1 + exp  xd ˆβ(λq) + ˆφ(λq)ˆvd(λq) . For all λ1, ..., λQ, the following algorithm is performed: For all λ1, ..., λQ, the following algorithm is performed: 1. Apply the ℓ2-PAML algorithm to obtain ˆθ(λq) and ˆv1(λq), ..., ˆvD(λq). 2. Calculate ˆpd(λq) and ˆξd(λq), d = 1, ..., D. 1. Apply the ℓ2-PAML algorithm to obtain ˆθ(λq) and ˆv1(λq), ..., ˆvD(λq). 1. Apply the ℓ2-PAML algorithm to obtain ˆθ(λq) and ˆv1(λq), ..., ˆvD(λq). 2. Calculate ˆpd(λq) and ˆξd(λq), d = 1, ..., D. 3. Calculate BIC(λq) according to (29). 3.3 Tuning parameter choice and information criterion q q 3. Calculate BIC(λq) according to (29). After the algorithm is finalized, the optimal tuning parameter λopt can be defined as the candidate value that minimizes the BIC. After the algorithm is finalized, the optimal tuning parameter λopt can be defined as the candidate value that minimizes the BIC. However, due to the non-convexity of the underlying optimization problem for ℓ2- PAML estimation, the behavior of the BIC along the tuning parameter sequence can be volatile to the extent that it may be characterized by multiple local minima. Therefore, we further apply cubic spline smoothing by defining BIC(λ) = f (λ) + ϵq, where f (λ) is a twice differentiable function and ϵq ∼N(0, ψ). The cubic spline estimate ˆf of the function f is obtained from solving the optimization problem min f ∈F Q  q=1 BIC(λq) −f (λq) 2 + δ  f ′′(λ)2 dλ, (30) (30) 123 472 J. Krause et al. J. Krause et al. where F = { f : f is twice differentiable} denotes the class of twice differentiable functions and δ > 0 is a smoothing parameter. After ˆf has been obtained, the optimal tuning parameter value λopt is defined as the minimizer of the smoothed function, that is is λopt = argmin λ∈{λq}Q q=1 ˆf (λ). (31) (31) 4.1 Setup Hereafter, the performance of the ℓ2-PAML approach is evaluated under controlled conditions. For this, we conduct a Monte Carlo simulation study with K = 500 iterations that are indexed by k = 1, ..., K. We generate synthetic data according to yd ∼Bin(nd, pd), pd = exp  β0 + xdβ1 + φvd  1 + exp  β0 + xdβ1 + φvd , β0 = −0.2, β1 = 0.315, d = 1, ..., D, where nd = 100, 15 as column vector of five ones, and φ = 0.4. The random effect vd is drawn from a standard normal, as defined in Sect. 2.1. For the covariate vector xd, we consider four different settings {A, B, C, D} with respect to the dependency between the auxiliary variables. This is done in order to test the methodology under different covariate correlation situations. In the A-setting, we have orthogonal covariates that are generated according to xrd ∼U(0.7, 1.2), r = 1, ..., 5. For the remaining three settings, we choose x1d ∼U(0.7, 1.2), xrd = α(zd + ρx1d), zd ∼U(0, 0.2), r = 2, ..., 5, where ρ is a parameter controlling the dependency between x1d and xrd, and α is a parameter harmonizing the variance of the random variables over settings. In the B-setting, there is medium correlation with 20-50% on a percentage scale for the product-moment correlation coefficient. For the C-setting, we have correlation with about 50-75%. And in the D-setting, we have a strong correlation with 80-90%. Note that the latter mimics situations of quasi rank-deficiency, which are of special interest. In addition to covariate correlation, we let the total number of areas D vary over scenarios in order to evaluate the method under different degrees of freedom. Overall, we consider 8 simulation scenarios: A.1 : D = 50, A.2 : D = 100, B.1 : D = 50, ρ = 0.3, α = 2.0, B.2 : D = 100, ρ = 0.3, α = 2.0, C.1 : D = 50, ρ = 0.9, α = 1.5, C.2 : D = 100, ρ = 0.9, α = 1.5, D.1 : D = 50, ρ = 1.5, α = 0.7, D.2 : D = 100, ρ = 1.5, α = 0.7. 4.2 Model parameter estimation results The target of this subsection is to study the fitting behavior of the ℓ2-PAML algorithm. Define θ := (β0, β′ 1, φ). For a given estimator ˆθr ∈ˆθ of the model parameter θr, r = 1, ..., p + 1, we consider the following performance measures: Bias( ˆθr) = 1 K K  k=1  ˆθ(k) r −θr  , MSE( ˆθr) = 1 K K  k=1  ˆθ(k) r −θr 2 , (32) (32) where θ(k) r is the value that ˆθr takes in the k-th iteration of the simulation and θr denotes the true value. As θr = 0.3 for all components of β1, we average the perfor- mance measures for the regression parameters. Table 1 contains the results for model parameter estimation. We start with the regression parameters β = (β0, β′ 1)′. It can be seen that the ℓ2-PAML algorithm obtains more efficient estimates than the ML-Laplace approach. Its MSE is significantly smaller in all considered scenarios. The largest efficiency gains are obtained in the D-scenarios, which include strong covariate correlation. This could be expected from theory, as the ℓ2-penalty was introduced by Hoerl and Kennard (1970) in order to improve the fitting behavior in these settings. However, we also see that under orthogonal covariates (A-scenarios), the ℓ2-PAML algorithm still outperforms the ML-Laplace approach. This is because approximate likelihood inference introduces additional uncertainty to model parameter estimation. Here, the ℓ2-penalty stabilizes the shape of the objective function, which allows for efficiency gains even without covariate correlation. Yet, the increased efficiency comes at the cost of an increased bias. The slope parameters β1, which are penalized while applying the ℓ2-PAML algorithm, are estimated with larger bias relative to the ML-Laplace method. Please note that this is in line with theory. Hoerl and Kennard (1970) showed that the ℓ2-penalty affects the bias-variance trade-off the researcher typically encounters in ML estimation. It increases the bias in order to reduce the variance, which ultimately allows for a smaller MSE when the regularization parameter λ is chosen appropriately. This is also becomes evident when looking at the distribution of regression param- eter estimates. Figure 1 shows boxplots of the absolute deviation | ˆβr −βr|, βr ∈β1, over all Monte Carlo iterations and for different simulation scenarios. 4.1 Setup A.1 : D = 50, A.2 : D = 100, B.1 : D = 50, ρ = 0.3, α = 2.0, B.2 : D = 100, ρ = 0.3, α = 2.0, C.1 : D = 50, ρ = 0.9, α = 1.5, C.2 : D = 100, ρ = 0.9, α = 1.5, D.1 : D = 50, ρ = 1.5, α = 0.7, D.2 : D = 100, ρ = 1.5, α = 0.7. 12 3 473 ℓ2-penalized approximate... The objective is to estimate the domain proportion pd, d = 1, ..., D. We compare two model parameter estimation approaches for the logit mixed model described in Sect. 2.1: a non-penalized approach that is obtained from maximizing lapp (Laplace-ML), and the ℓ2-penalized approach through maximizing l pen (ℓ2-PAML), as described in Sect. 3. We evaluate the simulation outcomes with respect to three aspects: (i) model parameter estimation, (ii) domain proportion prediction, and (iii) MSE estimation based on the parametric bootstrap in Sect. 2.3. The results are sum- marized in the following subsections. 4.2 Model parameter estimation results In each quarter, the distribution yielded by the ℓ2-PAML algorithm is displayed on the left, while the the one obtained by the ML-Laplace algorithm is located on the right. We see that the 123 123 474 J. Krause et al. Table 1 Model Parameter Estimation Results Scen Method Bias(β0) MSE(β0) Bias(β1) MSE(β1) Bias(φ) MSE(φ) A.1 ML-Laplace −0.2760 2.3958 0.0059 0.4972 0.1691 0.0344 A.1 ℓ2-PAML 0.6752 1.4878 −0.1889 0.1908 0.1832 0.0416 A.2 ML-Laplace −0.3861 1.1191 0.0317 0.2364 0.1870 0.0381 A.2 ℓ2-PAML 0.5021 0.9434 −0.1522 0.1215 0.1985 0.0430 B.1 ML-Laplace −0.3128 0.6994 0.0132 0.9795 0.1699 0.0345 B.1 ℓ2-PAML 0.2271 0.5077 −0.1197 0.3004 0.1819 0.0406 B.2 ML-Laplace −0.3086 0.3347 0.0136 0.4284 0.1898 0.0390 B.2 ℓ2-PAML 0.1501 0.2963 −0.1007 0.1534 0.2014 0.0441 C.1 ML-Laplace −0.3380 0.8412 0.0060 3.1709 0.1621 0.0325 C.1 ℓ2-PAML 0.4065 0.8252 −0.0979 0.7336 0.1806 0.0409 C.2 ML-Laplace −0.3096 0.3974 0.0087 1.7727 0.1813 0.0359 C.2 ℓ2-PAML 0.2329 0.3875 −0.0674 0.3062 0.1931 0.0413 D.1 ML-Laplace −0.3345 0.7745 0.0186 11.7489 0.1641 0.0328 D.1 ℓ2-PAML 0.2697 0.6943 −0.0939 3.1089 0.1852 0.0416 D.2 ML-Laplace −0.2907 0.3461 0.0128 5.6289 0.1868 0.0380 D.2 ℓ2-PAML 0.1016 0.3210 −0.0615 1.7118 0.2021 0.0448 12 ℓ2-penalized approximate... 475 l2-PAML ML-Laplace 0.0 1.0 2.0 Scenario A.1 Absolute deviation 0 1 2 3 Scenario B.1 Absolute deviation 0 2 4 6 8 Scenario C.1 Absolute deviation 0 5 10 15 Scenario D.1 Absolute deviation l2-PAML ML-Laplace l2-PAML ML-Laplace l2-PAML ML-Laplace Fig. 1 Absolute deviation of regression parameter estimates l2-PAML ML-Laplace 0.0 1.0 2.0 Scenario A.1 Absolute deviation 0 1 2 3 Scenario B.1 Absolute deviation l2-PAML ML-Laplace Absolute deviation 0 5 10 15 Scenario D.1 Absolute deviation l2-PAML ML-Laplace stimates 0 2 4 6 8 Scenario C.1 Absolute deviation l2-PAML ML-Laplace Scenario D.1 Fig. 1 Absolute deviation of regression parameter estimates boxes and whiskers of the ℓ2-PAML algorithm are much shorter than those of the ML- Laplace method. This implies that the deviations from the true value are much smaller under penalization for the vast majority of cases. Accordingly, the fitting behavior is overall stabilized. Concerning φ, the results are different. The standard deviation parameter estima- tion is not influenced by the covariate correlation. An intuitive explanation for this phenomenon is that p0d is not affected by the collinearity of xd, and that the diagonal element H0p+1p+1 of the Hessian matrix depends on xd only through p0d. 4.3 Domain proportion prediction results The target of this subsection is to investigate the behavior of the EBP of pd, d = 1, ..., D. We consider absolute bias, MSE, relative absolute bias, and relative root mean squared error as performance measures. For a domain proportion prediction in the k-th iteration of the simulation study, define ¯pd = 1 K K  k=1 p(k) d , RBd = K k=1 | ˆp(k) d −p(k) d | K| ¯pd| , ¯pd = 1 K K  k=1 p(k) d , RBd = K k=1 | ˆp(k) d −p(k) d | K| ¯pd| , REd =  1 K K k=1( ˆp(k) d −p(k) d )2 | ¯pd| , d = 1, . . . , D. REd =  1 K K k=1( ˆp(k) d −p(k) d )2 | ¯pd| , d = 1, . . . , D. Further, let Further, let Bd = 1 K K  k=1 | ˆp(k) d −p(k) d |, Ed = 1 K K  k=1 ( ˆp(k) d −p(k) d )2, d = 1, . . . , D. The performance measures are then given by The performance measures are then given by ABias = 1 D D  d=1 Bd, RABias = 1 D D  d=1 RBd, MSE = 1 D D  d=1 Ed, RRMSE = 1 D D  d=1 REd. The results obtained from the simulation study are summarized in Table 2. We observe that ℓ2-PAML improves domain total prediction performance in terms of all consid- ered performance measures and for all implemented simulation scenarios, including those without covariate correlation. This is in line with the simulation results for model parameter estimation from the last subsection. The ℓ2-penalty stabilizes the estimation performance even for orthogonal covariates due to the necessary Laplace approximation. However, the strongest efficiency gains in terms of the MSE relative to the ML-Laplace algorithm are obtained in the C- and D-scenarios, where we have strong covariate correlation. Against the backhground of Hoerl and Kennard (1970), this could be expected from theory, as ℓ2-penalization is known to be particularly useful in the presence of (quasi-)multicollinearity. Overall, we can conclude that the ℓ2-PAML algorithm not only improves model parameter estimation, but also domain total prediction in any setting. 4.2 Model parameter estimation results This is why, we expect that the asymptotic behavior of the ML-Laplace and ℓ2-PAML estimators of φ will be not (or almost not) affected by the covariate correlation. Concerning the comparison of the two fitting algorithms, the ℓ2-PAML approach increases the efficiency of regression parameter estimation. On the other hand, the efficiency of standard deviation parameter estimation is impaired relative to the ML- Laplace approach. In general, both methods overestimate the true value. This is likely due the involved Laplace approximation in both algorithms. It is known to induce bias to model parameter estimation, as for instance addressed by Jiang (2007), p. 131. However, the bias for the ℓ2-PAML algorithm is larger, as it implements additional shrinkage of the regression parameters through the ℓ2-penalty. The regression parame- ter estimates are drawn to zero (to some extent), which causes a larger proportion of the target variable’s variance to be attributed to the random effect. This leads to a stronger overestimation of the random effect standard deviation. Nevertheless, we will see in the next subsection that the efficiency advantage in regression parameter estimation overcompensates the loss in standard devation parameter estimation accuracy. 123 476 J. Krause et al. 4.4 Mean squared error estimation results Krause et al. Table 3 Mean Squared Error Estimation Results Scen MSE mse RBias RRMSE A.1 0.001735 0.001687 −0.027801 0.097148 A.2 0.001729 0.001676 −0.030432 0.089698 B.1 0.001879 0.001851 −0.014858 0.090956 B.2 0.001851 0.001842 −0.004771 0.085181 C.1 0.001300 0.001275 −0.019039 0.171667 C.2 0.001333 0.001276 −0.042769 0.168694 D.1 0.001605 0.001613 0.004951 0.124678 D.2 0.001640 0.001610 −0.018371 0.119636 with respect to practice, a RRMSE ranging from 8.5% to 17.2% is a solid result for uncertainty estimation. with respect to practice, a RRMSE ranging from 8.5% to 17.2% is a solid result for uncertainty estimation. with respect to practice, a RRMSE ranging from 8.5% to 17.2% is a solid result for uncertainty estimation. 4.4 Mean squared error estimation results The target of this subsection is to study the performance of the parametric bootstrap for MSE estimation. We employ B = 500 bootstrap replicates in order to approximate 123 477 ℓ2-penalized approximate... Table 2 Domain Proportion Prediction Results Scen Method ABias MSE RABias RRMSE A.1 ML-Laplace 0.033342 0.001781 0.047429 0.059960 A.1 ℓ2-PAML 0.032920 0.001735 0.046830 0.059188 A.2 ML-Laplace 0.033094 0.001749 0.047128 0.059465 A.2 ℓ2-PAML 0.032911 0.001729 0.046870 0.059132 B.1 ML-Laplace 0.034811 0.001922 0.053363 0.066946 B.1 ℓ2-PAML 0.034416 0.001879 0.052758 0.066201 B.2 ML-Laplace 0.034372 0.001872 0.052849 0.066259 B.2 ℓ2-PAML 0.034180 0.001851 0.052557 0.065877 C.1 ML-Laplace 0.028710 0.001344 0.036400 0.046199 C.1 ℓ2-PAML 0.028228 0.001300 0.035794 0.045434 C.2 ML-Laplace 0.028748 0.001355 0.036663 0.046597 C.2 ℓ2-PAML 0.028539 0.001333 0.036399 0.046227 D.1 ML-Laplace 0.032102 0.001652 0.044746 0.056371 D.1 ℓ2-PAML 0.031651 0.001605 0.044116 0.055569 D.2 ML-Laplace 0.032197 0.001663 0.045118 0.056794 D.2 ℓ2-PAML 0.031972 0.001640 0.044802 0.056401 the prediction uncertainty under the model. For a MSE estimate in the k-th iteration of the simulation study, define MSEd = 1 K K  k=1 ( ˆY (k) d −Y (k) d )2, msed = 1 K K  k=1 mse( ˆY (k) d ), mse = 1 D D  d=1 msed, where ˆY (k) d and mse( ˆY (k) d ) are the EBP of Yd and its bootstrap MSE estimator (see Sect. 2.3), respectively. We consider the following performance measures where ˆY (k) d and mse( ˆY (k) d ) are the EBP of Yd and its bootstrap MSE estimator (see Sect. 2.3), respectively. We consider the following performance measures RBias = 1 D D  d=1 msed −MSEd MSEd , RRMSE = 1 D  d=1  1 D D d=1(msed −MSEd)2 MSEd Table 3 summarizes the simulation results. We see that the parametric bootstrap estima- tor shows a decent performance overall. There is a slight tendency for underestimation. However, with a relative bias of less then 4.3% for approximate likelihood inference with a generalized linear mixed model, this is negligible. With regards to the RRMSE, we see that the parametric bootstrap is more efficient under orthogonal covariates (A- scenarios) and medium correlation (B-scenarios). In the C- and D-scenarios, which employ stronger covariate correlation, the RRMSE becomes larger. This is in line with the results of Sect. 4.2. In these scenarios, the model parameter estimates are subject to larger variation, which affects the bootstrap due to its parametric construction. Yet, 3 478 J. 5.1 Data description and model specification In what follows, we apply the ℓ2-PAML approach to estimate the regional prevalence of multiple sclerosis in Germany. For this, we consider the German population of the year 2017. It is segmented into 401 administrative districts and contains about 82 million individuals. The districts correspond to the domains in accordance with Sect. 2.1. The required demographic information is retrieved from the German Federal Statistical Office and based on the methodological standards described in Statistisches Bundesamt (2016). As model response y, we define a binary variable with realizations yid =  1 person has multiple sclerosis 0 else yid =  1 person has multiple sclerosis 0 else for some i ∈Ud. The objective is to estimate pd = Yd/Nd with Yd =  i∈Ud yid for all German districts. In order to define whether a person has multiple sclerosis, we rely on an intersectoral disease profile provided by the Scientific Institute Institute of the AOK (WIdO). It is based on multiple aspects, including medical descriptions, inpatient diagnoses, and ambulatory diagnoses. The necessary sample counts for y are based on health insurance records provided by the AOK. In particular, we use district- level prevalence figures of the AOK insurance population in 2017 that are based on the intersectoral disease profiles. The AOK insurance population is the biggest statutory health insurance population of the country with roughly 26 million individuals in 2017 (AOK Bundesverband 2018). Note that the German health insurance system has a rather unique separation between statutory and private health insurance. Usually, this has to be accounted for in order to produce reliable prevalence estimates. However, Burgard et al. (2019) showed that model-based inference using covariate data with sufficient explanatory power can overcome this issue. 479 ℓ2-penalized approximate... As auxiliary data source, we use district-level inpatient diagnosis frequencies of the German DRG-Statistics that are provided by the German Federal Statistical Office (Statistisches Bundesamt 2017). The data set contains figures on how often a given disease has been recorded in hospitals within a year. Both main and secondary diag- noses are considered. With respect to diagnosis grouping, the records are provided on the ICD-3 level (World Health Organization 2018). Note that the DRG-Statistics are a full census of all German hospitals. Thus, the corresponding records cover the entire population, as required for the model derivation in Sect. 2.1. 5.1 Data description and model specification However, a drawback of the data set’s richness is that we have to choose a suitable set of predictors x out of approximately 3000 potential covariates. Naturally, it is not feasible to apply an exhaustive stepwise strategy that is often used in the context of variable selection, as for instance demonstrated by Yamashita et al. (2007). Instead, we apply a heuristic strategy based on the premise that the objective is to find a covariate subset with sufficient explanatory power for our purpose. First, we isolate the 20 variables of the DRG-Statistics that have the strongest correlation with the AOK records on G35, which is multiple sclerosis on the ICD-3 level. The variables are arranged in decreasing order with respect to their correlation. Next, we use the ℓ2-PAML algorithm from Sect. 3.2 to perform model parameter estimation for p covariates, where p ∈{2, 3, ..., 20}. That is to say, we start with the 2 covariates that have the strongest correlation to G35, and then sequentially increase the number of predictors up to 20. For every result of model parameter estimation, we calculate the Laplace non-corrected BIC in (29). Then, we select the covariate subset that corre- sponds to the model fit which minimizes the BIC. The BIC curve over all considered covariate set cardinalities is displayed in Fig. 2. We see that the curve has an odd evolution over the covariate sets. This can be attributed to three reasons. Firstly, due to the non-linearity of the link function, the covariate sorting is guaranteed to orga- nize the covariates in descending order with regards to their relevance for the target variable. Secondly, due to the strong correlation between them, the covariate contri- butions interfer with each other. That is to say, when including an additional covariate into the active set, the contributions of the previously contained covariates can change considerably. And finally, as already addressed in Sect. 3.3, the non-convexity of the optimization problem further leads to irregularities in the BIC curve. Despite these issues, the BIC curve has a clear minimum that is located at p = 9. Therefore, we isolate the 9 DRG-Statistics variables that have the strongest correlation with the AOK records on G35. Thereafter, we perform a parametric bootstrap to esti- mate the standard deviation of each model parameter estimate ˆθ j ∈ˆθ, j = 1, ..., p+1, to evaluate its significance in terms of the p-value. 5.1 Data description and model specification The parametric bootstrap is described as follows: 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ ′, ˆφ). 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ ′, ˆφ). 2. Repeat B times with b = 1, ..., B: 2. Repeat B times with b = 1, ..., B: 2. Repeat B times with b = 1, ..., B: (a) Generate v(b) d ∼N(0, 1), y(b) d ∼Bin(nd, p(b) d ), d = 1, . . . , D, where p(b) d = exp  xd ˆβ+ ˆφv(b) d  1+exp  xd ˆβ+ ˆφv(b) d . (b) For each bootstrap sample, calculate the estimator ˆθ (b). 123 123 J. Krause et al. 480 5 10 15 20 979780 979790 979800 979810 Number of Predictors Bayesian Information Criterion Fig. 2 BIC over covariate set cardinalities 3. Output: sd( ˆθ j) =  1 B B b=1 ˆθ(b) j −1 B B k=1 ˆθ(k) j 2, j = 1, ..., p + 1. Based on the estimated standard deviations, we calculate test statistics for a sequence of t-tests under the null hypothesis H0 : θ j = 0, j = 1, ..., p + 1. For a given θ j ∈θ, the test statistic is given by t j = ˆθ j/sd( ˆθ j) and follows a standard normal distribution. The test statistic values are located in the pdf of the standard normal to obtain their respective p-values. We delete every predictor that corresponds to a model parameter that is not relevant on at least a 10% significance level. The entire procedure is summarized hereafter: 5 10 15 20 979780 979790 979800 979810 N b f P di t Bayesian Information Criterion Number of Predictors Fig. 2 BIC over covariate set cardinalities 3. Output: sd( ˆθ j) =  1 B B b=1 ˆθ(b) j −1 B B k=1 ˆθ(k) j 2, j = 1, ..., p + 1. Based on the estimated standard deviations, we calculate test statistics for a sequence of t-tests under the null hypothesis H0 : θ j = 0, j = 1, ..., p + 1. For a given θ j ∈θ, the test statistic is given by t j = ˆθ j/sd( ˆθ j) and follows a standard normal distribution. 123 5.1 Data description and model specification The test statistic values are located in the pdf of the standard normal to obtain their respective p-values. We delete every predictor that corresponds to a model parameter that is not relevant on at least a 10% significance level. The entire procedure is summarized hereafter: Based on the estimated standard deviations, we calculate test statistics for a sequence of t-tests under the null hypothesis H0 : θ j = 0, j = 1, ..., p + 1. For a given θ j ∈θ, the test statistic is given by t j = ˆθ j/sd( ˆθ j) and follows a standard normal distribution. The test statistic values are located in the pdf of the standard normal to obtain their respective p-values. We delete every predictor that corresponds to a model parameter that is not relevant on at least a 10% significance level. The entire procedure is summarized hereafter: 1. Find the 20 covariates with the strongest correlation to y g 2. Perform model parameter estimation for p ∈{2, 3, ..., 20} predictors 3. Find the number of predictors that minimizes the BIC 4. For the BIC-minimal predictor set, perform a parametric bootstrap to estimat standard deviations for the model parameter estimates 5. Perform t-tests to evaluate their significance and delete insignificant predictors The proposed strategy yields us the final covariate set x which consists of p = 5 predictors. The selected covariates are briefly characterized as follows: • X1: G43 (Migraine, secondary diagnosis) • X2: M20 (Acquired deformities of fingers and toes, main diagnosis) • X3: E66 (Overweight and obesity, main diagnosis) • X4: E04 (Other nontoxic goiter, main diagnosis) • X5: G35 (Multiple sclerosis, secondary diagnosis) Please note that the association of these variables with multiple sclerosis is the result of district-level correlation. It does not directly imply person-level comorbidities in 123 481 ℓ2-penalized approximate... Table 4 Estimation results for final model specification Parameter Estimate Std.Dev. p-value 95%-Conf.Int. β0 −5.79912 0.00640 0.00000 [−5.81167; −5.78657] β1 −0.00092 0.00009 0.00000 [−0.00110; −0.00073] β2 0.00026 0.00009 0.00380 [0.00009; 0.00043] β3 −0.00030 0.00007 0.00001 [−0.00043; −0.00017] β4 −0.00081 0.00006 0.00000 [−0.00093; −0.00068] β5 0.00154 0.00009 0.00000 [0.00136; 0.00173] φ 0.10939 0.00551 0.00000 [0.09860; 0.12019] a medical sense. Applying the ℓ2-PAML algorithm on the final covariate set yields us the final model specification that we use for regional prevalence estimation. It is summarized in Table 4. 5.2 Results Let us now investigate the results of prevalence estimation. The national prevalence D d=1 Yd/ D d=1 Nd · 100% is estimated at 0.296%. Based on the parametric boot- strap, we calculate a 95% confidence interval of [0.293%; 0.300%]. This implies that the estimated total number of persons with multiple sclerosis ranges approximately from 239000 to 246000, which is in line with reference figures on this topic. The Cen- tral Research Institute of Ambulatory Health Care in Germany estimated that in 2017 about 240000 individuals had multiple sclerosis (Müller 2018). The regional distribu- tion of prevalence estimates on the district-level is displayed in Fig. 3. We observe a prevalence discrepancy between the western and eastern parts of Germany. The esti- mated prevalence in western Germany are overall higher than in eastern Germany. Further, we observe regional clustering with higher prevalence in the central-northern and central-southern parts of Germany. This is also consistent with reference studies. Similar patterns have been found by Central Research Institute of Ambulatory Health Care in Germany (Müller 2018) and Petersen et al. (2014). Overall, the estimates are plausible in both level and geographic distribution. Figure 3 shows the distributions of district-level prevalence estimates for the EBPs under both ℓ2-PAML and the classical ML-Laplace method. The ML-Laplace results are displayed in black, the ℓ2-PAML results are plotted in red. We see that the means of the distributions are almost identical. However, the ℓ2-PAML distribution shows considerably less variance than the ML-Laplace distribution. This is in line with both theory and the simulation study, which both suggest stabilizing effects through ℓ2- penalization. This is further evident when looking at the summarizing quantiles of both predictive distributions. They are displayed in Table 5 . We see that the ℓ2-PAML estimates are more more focussed around the mean and do not show as strong of outliers at the tails of the distribution compared to ML-Laplace. Figure 5 displays the root mean squared error estimates rmse( ˆpd) = mse( ˆpd) for the prevalence estimates in Fig. 3, where mse( ˆpd) is obtained from the paramet- ric bootstrap procedure described in Sect. 2.3. It becomes evident that there are no obvious spatial patterns in the RMSE estimates. We neither observe a particular depen- dency on the domain sizes nor on the prevalence estimates themselves. 5.1 Data description and model specification The confidence intervals for the parameters are calculated according to ˆθ j ± t(D,1−α/2)sd( ˆθ j), j = 1, ..., p + 1, where t(·) is the corresponding quantile of t-distribution with D degrees of freedom and significance level α. The BIC value of the upper model specification is 979754 and therefore even better than the optimal fit with p = 9 in Fig. 2. This suggests that the used model specification was a reasonable choice given the considered data basis. Further, observe that the estimated value for the standard deviation parameter φ is considerably larger than all slope parameters β1, ..., β5. This implies that the random effects v1, ..., vD are clearly evident in the empirical distribution of p1, ..., pD. Therefore, we can conclude that using a mixed effect model in this context was a necessary choice. We further look at the internal correlation structure of the considered predictors in order to assess the demand for ℓ2-penalization in the application. For this, we look at the empirical correlation matrix for the five selected DRG-Statistics variables. It is given as follows: ϱxx = ⎛ ⎜⎜⎜⎜⎝ 1.00 0.95 0.93 0.85 0.94 0.95 1.00 0.94 0.87 0.95 0.93 0.94 1.00 0.88 0.95 0.85 0.87 0.88 1.00 0.88 0.94 0.95 0.95 0.88 1.00 ⎞ ⎟⎟⎟⎟⎠ ϱxx = ⎛ ⎜⎜⎜⎜⎝ 1.00 0.95 0.93 0.85 0.94 0.95 1.00 0.94 0.87 0.95 0.93 0.94 1.00 0.88 0.95 0.85 0.87 0.88 1.00 0.88 0.94 0.95 0.95 0.88 1.00 ⎞ ⎟⎟⎟⎟⎠ We observe that (beside the main diagonal elements), the correlation values range from 0.85 to 0.95, or 85% to 95% on a percentage scale. This suggests that the internal correlation structure is very strong and comparable to the D-scenarios of our simulation study. Therefore, we conclude that using ℓ2-penalization is reasonable in this context. However, note that some of this correlation is due to the size as a result of district- level aggregation. Again, this does not directly resemble medical comorbidity on an individual level. 123 482 J. Krause et al. Table 5 Quantiles of the EBP distributions Method Min 0.25 0.50 Mean 0.75 Max ML-Laplace 0.171% 0.259% 0.297% 0.301% 0.336% 0.513% ℓ2-PAML 0.209% 0.269% 0.295% 0.300% 0.324% 0.471% 5.2 Results However, with respect to the overall level of RMSE estimates, we can conclude that our estimates are more efficient than direct estimates ˆpdir d = yd/nd, d = 1, ..., D, that are exclu- sively obtained from the health insurance records. Their standard deviation is given by sd( ˆpdir d ) =  ˆpdir d (1 −ˆpdir d ). 123 ℓ2-penalized approximate... 483 0.200% to 0.245% 0.245% to 0.290% 0.290% to 0.335% 0.335% to 0.370% 0.370% and higher 0.200% to 0.245% 0.245% to 0.290% 0.290% to 0.335% 0.335% to 0.370% 0.370% and higher Fig. 3 Results of prevalence estimation A one-to-one comparison of rmse( ˆpd) and sd( ˆpdir d ) per domain is visualized in Fig. 6. The ordinate measures sd( ˆpdir d ) and the abscissa measures rmse( ˆpd). The red line marks the bisector, which indicates equality between the two. We observe that rmse( ˆpd) is always smaller than sd( ˆpdir d ) by quite a margin. Thus, given the reason- able performance of the parametric bootstrap for MSE estimation in the simulation, we can conclude that our estimates mark an improvement over the direct estimates. There is a slight positive relation between the two measures. That is to say, a relatively large sd( ˆpdir d ) is accompanied by a relatively large rmse( ˆpd) on expectation. However, the trend is only vaguely visible. Finally, let us look at the distribution of random effect predictions over domains. They are visualized in Fig.7. The bars of the histogram correspond to the probability density of the mode predictors in the respective interval of the support. The red line is 123 484 J. Krause et al. 0.001 0.002 0.003 0.004 0.005 0 200 400 600 800 1000 Distribution of predictions N = 401 Bandwidth = 0.0001798 Density ML−Laplace l2−PAML Fig. 4 Comparision of the EBPs Distribution of predictions 0.001 0.002 0.003 0.004 0.005 0 200 400 600 800 1000 Distribution of predictions N = 401 Bandwidth = 0.0001798 Density ML−Laplace l2−PAML Fig. 4 Comparision of the EBPs Fig. 4 Comparision of the EBPs the result of a kernel density estimation over their realized values. We observe that the distribution is very close to normal. This is in line with the theoretical developments from Sect. 2.1. Overall, it can be concluded that the ℓ2-PAML approach in the area- level logit mixed model was a sensible choice for the considered application. the result of a kernel density estimation over their realized values. We observe that the distribution is very close to normal. This is in line with the theoretical developments from Sect. 2.1. Overall, it can be concluded that the ℓ2-PAML approach in the area- level logit mixed model was a sensible choice for the considered application. 6 Conclusion Regional prevalence estimation is an important issue to monitor the health of the population and for planning capacities of a health care system. A good covariate on the prevalence of a disease can be typically obtained from medical treatment records such as the DRG-Statistics in Germany. We proposed a new small area estimator for regional prevalence that copes with two major issues in this context. First, typically health surveys do not have a large sample and the sample size is mainly dedicated to allow for the estimation of national figures. Within regional entities, therefore, the sample size is very small. Applying classical design based or model assisted estimators on these small sample sizes leads to very high standard errors for many regions. Our small area estimator is capable of overcoming this issue by using a model based approach. The second problem we tackle is, that the best covariates at hand, typically have high correlations between each other. This leads to numerical problems inhibiting the exploitation of these covariates. To overcome this problem we propose to use a ℓ2-penalization approach. This leads to the need for revising the parameter estimation procedure and to adapt it to the new requirements. We provide therefore ℓ2-penalized approximate... 485 0.005 to 0.010 0.010 to 0.015 0.015 to 0.020 0.020 to 0.025 0.025 and higher Fig. 5 Results of RRMSE estimation Fig. 5 Results of RRMSE estimation a novel Laplace approximation to a logit mixed model with ℓ2 regularization. This estimation procedure is applicable for other purposes such as classical logit mixed model estimation with ℓ2-penalization. The prevalence estimation maps of Sect. 5 show some clusters of small areas with high or low prevalence. This fact indicates that modeling spatial correlation by intro- ducing, for example, simultaneous autoregressive random effects, might benefit the final predictions. Combining this additional generalization with the robust penalized approach is thus desirable. However, it is not an easy theoretical task and deserves future independent research. In a Monte Carlo simulation study we show that the proposed estimation approach ℓ2-PAML yields stable parameter estimates even under strong correlations of the covariates. This simulation results underpin the theoreti- cal arguments. Finally, we applied this newly derived estimator to the prediction of 123 486 J. Krause et al. 486 J. Krause et al. Declarations Conflict of interests The authors declare they have no competing interests. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 6 Conclusion 0.01 0.02 0.06 0.07 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.03 0.04 0.05 Standard Deviation, Direct Estimator Root Mean Squared Error, l2-PAML Fig. 6 Comparision of estimation uncertainty Domain-Level Random Effect Realizations Value Density -3 -2 -1 0 1 2 3 0.0 0.1 0.2 0.3 0.4 Fig. 7 Distribution of random effect mode predictions 0.01 0.02 0.06 0.07 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.03 0.04 0.05 Standard Deviation, Direct Estimator Root Mean Squared Error, l2-PAML Fig. 6 Comparision of estimation uncertainty Domain-Level Random Effect Realizations Value Density -3 -2 -1 0 1 2 3 0.0 0.1 0.2 0.3 0.4 Fig 7 Distribution of random effect mode predictions Domain-Level Random Effect Realizations Value Density -3 -2 -1 0 1 2 3 0.0 0.1 0.2 0.3 0.4 Fig. 7 Distribution of random effect mode predictions Domain-Level Random Effect Realizations Domain-Level Random Effect Realizations Value -3 -2 -1 0 1 2 3 0.0 Fig. 7 Distribution of random effect mode predictions 123 Fig. 7 Distribution of random effect mode predictions 123 123 123 ℓ2-penalized approximate... 487 district-level multiple sclerosis prevalence and obtained estimates with a considerably low root mean squared error. Hence, we recommend using our new approach for the regional prevalence estimation. district-level multiple sclerosis prevalence and obtained estimates with a considerably low root mean squared error. Hence, we recommend using our new approach for the regional prevalence estimation. Funding Open Access funding enabled and organized by Projekt DEAL. This research is supported by the Spanish grant PGC2018-096840-B-I00, by the grant “Algorithmic Optimization (ALOP) - Graduate School 2126”fundedbytheGermanResearchFoundation,aswellastheresearchproject“Gesundheitsatlas”funded by the Scientific Institute of the German Public Health Insurance Company. Data availability The demographic data as well as the DRG-Statistic data used in this study are available on request from the German Federal Statistical Office. The health insurance records are property of the German Public Health Insurance Company and subject to special privacy restrictions under the national law. 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Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance
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Queen's University Belfast - Research Portal: Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Queen s University Belfast Research Portal: Link to publication record in Queen's University Belfast Research Portal y Link to publication record in Queen's University g Copyright 2020 the authors. Copy g 0 0 e au o s This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Scraton, P. (2020). Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance. International Journal for Crime, Justice and Social Democracy, 9(4), 1-15. https://doi.org/10.5204/ijcjsd.1686 Scraton, P. (2020). Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance. International Journal for Crime, Justice and Social Democracy, 9(4), 1-15. https://doi.org/10.5204/ijcjsd.1686 Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access This research has been made openly available by Queen's academics and its Open Research team. 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Document Version: Document Version: Publisher's PDF, also known as Version of record Published in: I i l J International Journal for Crime, Justice and Social Democracy Document Version: Publisher's PDF, also known as Version of record Abstract Emerging from critical conferences in the early 1970s involving academic researchers, community-based workers and activists, critical social research challenged the role and legitimacy of mainstream social sciences in their support of social orders fractured by class, ‘race’, sectarianism, gender, sexuality and age. This article opens with a brief reflection on the emergence and consolidation of critical social theory as the foundation and context for research that challenges state-institutionalised power and authority. It draws on long-term, in-depth primary research into the operational policies and practices of policing and incarceration, exploring the profound challenges involved in bearing witness to the ‘pain of others’. Recounting personal testimonies ‘from below’, revealing institutionalised deceit and pursuing ‘truth recovery’, it argues that dissenting voices are the foundation of hope, resistance and transformation. Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton1 Queen’s University Belfast, United Kingdom Phil Scraton1 Phil Scraton1 Queen’s University Belfast, United Kingdom Queen’s University Belfast, United Kingdom Open Access Thi h Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love t this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 IJCJ&SD 9(4) 2020 ISSN 2202-8005 Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Introduction In his classic text Ways of Seeing, the artist John Berger encouraged wide public debate about how selectivity afforded to images ‘establish our place in the surrounding world’. The photographer’s ‘choice of subject’, the painter’s ‘marks … on the canvas or paper’ and the consequent ‘perception or appreciation of an image’ are filtered by ‘our way of seeing’ (Berger 1972: 10). Apparently impulsive, the immediacy and spontaneity of observation are informed by prior knowledge and understanding. Reflecting on her experiences as a war journalist witnessing brutality and brutalisation, Susan Sontag (2003: 125–126) observes that outsiders can neither ‘understand’ nor ‘imagine how dreadful, how terrifying and how normal’ are the ‘ordeals of others’. She was driven by a determined resolve to reveal the reality of ‘faraway suffering’, informing and encouraging interventions that might ‘do something to alleviate it’. Situating herself in the presence of those ordeals, photographing and reporting the deprivations endured by civilians caught in the crossfire, Sontag introduced an international audience to a ‘way of seeing’ that otherwise would have remained invisible. While distinct in focus and content, Berger and Sontag illustrate the significance of proximity, interpretation and representation in informing knowledge and understanding. Seemingly unique, the ‘caught in the moment’ gaze of the individual, however, cannot be understood in isolation from its broader social, political and economic contexts. An instant in time and place is framed by the prevailing ideologies and their contestation. As Foucault (1980) demonstrates, through mechanisms, techniques and procedures central to the maintenance and legitimacy of the state’s established order, ‘knowledge’ embedded in official discourse reflects and reproduces ‘regimes of truth’. In this context, ‘truth’ is manufactured, transmitted and reproduced. Acts and events are ascribed meaning, understanding is inhibited, and agency is diminished. A key component of critical social research is its interrogation of the processes through which legitimacy is ascribed to specific events thus elevating interpretation to fact. It enquires ‘beneath the surface of appearances’ to reveal ‘how social systems really work’ and ‘how ideology or history conceal the processes which oppress and control people’ (Harvey 1990: 6). Revealing the mechanisms through which knowledge is ascribed legitimacy, critical social research questions the premises on which public information is considered reliable, challenging the claims made for ‘value-free’ ‘scientific’ analysis within social sciences. Further, it explores the processes through which dissenting accounts emerge and their significance in the advancement of alternative discourses. Introduction Thus, it ‘rejects the premise that knowledge … is value-free or value-neutral’ but ‘derived and reproduced, historically and contemporaneously, in the structural relations of inequality and oppression that underpin established social orders’ (Scraton and Chadwick 2013: 107). This article focuses on distinct but related research conducted over four decades in the United Kingdom, reflecting the significance of personal and collective testimonies in recovering truth otherwise lost in the interminable processes, obstructive investigations and deceitful obfuscations of official procedures and discourse. Gathering testimonies, derived in ‘being there’ and ‘bearing witness’, reflects Sontag’s mission as an investigative journalist. It also establishes critical social research as fundamental to knowledge acquisition and truth recovery. Taking Berger literally, what follows identifies and engages the ‘view from below’ or, in the case of incarceration, the ‘view from inside’. Researching within and alongside complex communities enduring social, political and economic marginalisation challenges the power dynamic and academic conceit inherent in mainstream social sciences. Keywords State power; legitimacy; regimes of truth; structural inequality; critical social research; incarceration; Ireland; Hillsborough. Please cite this article as: Scraton P (2020) Fractured lives, dissenting voices, recovering ‘truth’: Frontiers of research and resistance. International Journal for Crime, Justice and Social Democracy 9(4): 1-15. https://doi.org/10.5204/ijcjsd.1686 Except where otherwise noted, content in this journal is licensed under a Creative Commons Attribution 4.0 International Licence. As an open access journal, articles are free to use with proper attribution. ISSN: 2202- 8005 © The Author(s) 2020 Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance www.crimejusticejournal.com Power, Legitimacy and State Authority For, the ‘sociological imagination’ was ‘not a fashion or an aesthetic choice’ but a means through which ‘the struggles of oppressed people for equality, self-determination and social justice’ could be advanced (Krisberg 1975: 19). Having previously focused on ‘how social actors define each other and their environments’, Becker (1974: 60) recognised the significance of structural ‘differentials in the power to define’ invested in ‘elites, ruling classes, bosses, adults, men, Caucasians, superordinate groups generally’. They were discernible in ‘primitive forms of control’ and, more significantly, ‘by controlling how people define the world’. Davis (1975: 205) identified inequality as essential to the success of advanced capitalism, evident and reproduced in the complex intersections of ‘class, ethnicity, sex and political and economic’ relations. Those holding political and economic power set an agenda ‘detrimental to the interests and needs’ of the powerless. This emerging body of work was neither homogenous nor uncontested, identifying as ‘new’, ‘radical’ or ‘critical’ criminology (Taylor, Walton and Young 1973, 1975). It affirmed the ‘diverse and unique world of everyday life, the claimed location of interactionists, yet it adapted and contextualized new deviancy theory’ prioritising ‘the dynamics of power and the processes which underpinned its legitimacy’ (Scraton and Chadwick 1991: 165). Built on these foundations, critical analysis of the State’s definition and operational dynamics of ‘reasonable’ force (policing) and ‘humane’ containment (prisons) prioritises the processes through which public order, social discipline and civil obedience are sustained. State authority is the concrete manifestation of the relationship between power and legitimacy. All states, whatever their form, claim authority for institutional powers to govern and regulate their subjects. In exercising institutional power, legitimacy is secured and maintained through regulatory processes embodied in the administration of the ‘rule of law’—policing, courts, incarceration. State authority is absolute and determining, albeit mediated by the extent to which distinct regimes accommodate participatory democracy. It holds the ‘monopoly of the legitimate use of physical force within a given territory’ (Weber 2009: 77–78, emphasis in original), institutionalised in the relationships between geographical boundaries, political autonomy and legal jurisdiction. Poulantzas (1978: 81) argued that the State’s monopoly on the legitimate use of violence ‘underlies the techniques of power and mechanisms of consent: it is inscribed in the web of disciplinary and ideological devices; and even when not directly exercised, it shapes the materiality of the social body upon which domination is brought to bear’. Power, Legitimacy and State Authority Identifying key moments in the emergence and consolidation of critical social theory and its application to the ‘ordeals of others’ formed the underlying premise of the text Power, Conflict and Criminalisation (Scraton 2007). It acknowledged the radical shift in social sciences that had challenged academic orthodoxies, not least the contributions of critical interventionists in the USA compelled to face down the fierce threat of McCarthyism. In this hostile climate within sociology, Wright Mills (1959: 20) was determined to establish an alternative discourse to the ‘inhibitions, obscurities and trivialities’ underpinning the ‘bureaucratic techniques’ and ‘methodological pretensions’ useful to ‘the corporation, IJCJ&SD 2 2020 9(4) IJCJ&SD 2 2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance army and the state’. Functionalist orthodoxy, bereft of ‘sociological imagination’ and leaving political- economic interests undeclared, served and serviced post-war regeneration reinforcing mainstream, traditional hierarchical values as norms. These were values that sustained social stratification, gendered families, racial superiority, homogenous sexuality, compliant youth and law-abiding citizens. army and the state’. Functionalist orthodoxy, bereft of ‘sociological imagination’ and leaving political- economic interests undeclared, served and serviced post-war regeneration reinforcing mainstream, traditional hierarchical values as norms. These were values that sustained social stratification, gendered families, racial superiority, homogenous sexuality, compliant youth and law-abiding citizens. Within a decade, in a highly charged public address, Howard Becker (1967: 240) considered critical social analysis was ‘caught in a crossfire’ of political values. State institutions and mega-corporations had financed and incorporated sociological research to help deliver their exploitative agendas. Calling for observational research into polarised social divisions, sufficiently immersed ‘to have a perspective’, Becker (1967: 240) challenged the mantra of ‘value-freedom’, demanding that social scientists identify ‘whose side we are on’. Alvin Gouldner, also rejected the proposition that social science research could claim value-neutrality, reiterating the significance of structural relations evident in manifestations of power, legitimacy and authority. As ‘new’ capitalism advanced, functionalist academic research had spawned a generation of economic expansionists, social engineers and welfare technicians. Railing against social research that patronised the ‘concrete and smaller worlds’ of everyday life with researchers cast as ‘zoo- keepers of deviance’, Gouldner (1973: x) argued for ‘reflexive’ analysis locating social and cultural relations in material history and the political-economic present. Power, Legitimacy and State Authority The threat of physical punishment, from incarceration through to solitary confinement and state execution, is internalised through ‘mechanisms of fear’. For Hall (1985), the conjuncture identified by Poulantzas as authoritarian statism more appropriately should be understood as authoritarian populism, generated by the purposeful construction and manipulation of popular consent. In their highly influential work, Hall et al. (1978: 319) proposed that the underlying crisis in the UK www.crimejusticejournal.com IJCJ&SD 3 2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance economy had generated crises ‘in political legitimacy, in social authority, in hegemony and in class struggle and resistance’. economy had generated crises ‘in political legitimacy, in social authority, in hegemony and in class struggle and resistance’. What followed was a relentless, dogmatic commitment—in government policy supported by compliant mainstream media—to regulate popular dissent, community-based movements and increasingly strong trade union resistance. It represented a ‘qualitative shift in the balance and relations of force: the law, the police, administrative regulation, public censure’ (Hall et al. 1978: 278). What had been progressive, in retrospect modest, advances in welfare, education, social housing and workers’ rights were redefined within a right-wing media as responsible for social upheaval, economic decline and the subversion of democracy. Within a year of its 1979 election, the British Conservative Government introduced extensive surveillance and regulatory mechanisms across social policy, welfare support and criminal justice alongside trade union regulation. In 1969, the British Army had been deployed on the streets of Northern Ireland, ostensibly to quell civil unrest and to work alongside the secret services and the Royal Ulster Constabulary. Direct rule by the British Government was imposed in 1972 with the virtual suspension of civil rights. This included internment without trial, implementation of emergency powers, progressing non-jury trials and withdrawing ‘special category status’ from politically affiliated prisoners. Within the UK, ‘laws became increasingly prohibitive and repressive, the police operated outside the checks and balances of political accountability, sentencing hardened and prison conditions worsened’ (Scraton 2007: 228). This revealed the authoritarian state’s reaction to criminalise ‘an unlimited reservoir of acts’ while characterising its interventionism as ‘warfare’ (Christie 1994: 24)—a war on drugs, terrorism, unions and welfare claimants. This relentless shift to authoritarianism consolidated the New Right’s political agenda. Power, Legitimacy and State Authority Having presided over the deaths of ten prisoners on hunger strike in the North of Ireland and inner-city uprisings in England’s Black communities, Prime Minister Margaret Thatcher projected her UK Government’s ideological purpose: At one end of the spectrum are the terrorist gangs within our borders, and the terrorist states which finance and arm them. At the other are the hard Left, operating inside our system, conspiring to use union power and the apparatus of local government to break, defy and subvert the law … now the mantle has fallen on us to conserve the very principle of Parliamentary democracy and the rule of law itself. (Thatcher 1984) Legitimate trade union action and local authority opposition were placed on a spectrum alongside terrorism. The die was cast. Structural constraints on individuals, families and communities marginalised by class, gender, sexuality, ‘race’ and age hardened under Thatcherism and, in the USA—its closest political-ideological ally— Reaganism. These overarching determining contexts shackled potential, diminished hope and hobbled agency. Having introduced draconian legislation, the UK Government used criminal justice agencies to stifle dissent from within Black communities, trades unions, young people and politically affiliated prisoners in the North of Ireland. It declared war on Argentina claiming ‘ownership’ of the Malvinas and legitimacy for its intervention. As state authoritarianism consolidated its political and ideological mission, critical social research confirmed its guiding principles: ‘bearing witness, gathering testimonies, sharing experiences, garnering the view from below and exposing the politics and discourses of authoritarianism’ (Scraton 2007: 240). IJCJ&SD 4 2020 9(4) IJCJ&SD 4 2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance The ‘Healthy Prison’ Oxymoron In the early 1970s, I taught a Criminology class in the Victorian-built Walton Prison, now HMP Liverpool. Guards were consciously intimidating, some openly wearing National Front pins. The jail was rat-infested and stank of urine. Without night access to toilets, prisoners, three to a cell, wrapped excreta in newspaper and threw it from cell windows. Each morning, it was collected by prisoners on the ‘shit detail’. These were the conditions endured by prisoners often locked down 23 in every 24 hours. Strangeways, now HMP Manchester, was Walton’s Victorian sibling. MOBS KILL SEX INMATES AND BURN PRISON 11 DIE IN JAIL RIOT (Daily Mirror) Unattributed allegations had been encouraged by inducements, remaining uncontested by official negotiators inside the prison throughout. The failure to deny became the substance of verification. Lord Justice Woolf was appointed to lead a comprehensive inquiry and make recommendations for systemic change in prison regimes, management and staffing prioritising prisoner rehabilitation. Intended to underpin root-and-branch reform throughout the prison estate, his report made 12 recommendations and 204 proposals. They were not realised. Twenty-five years later, reflecting on the failure to liberalise prison regimes, he condemned ‘competition between politicians’ to be ‘tough on crime … for that read prisoners’ (The Guardian 1 April 2015). Throughout the post-Strangeways decades, reflecting the profound authoritarian shift discussed earlier, successive Conservative and Labour Governments cast the net of criminalisation ever-widely, imposing increasingly longer jail sentences. Most prisons functioned not as places of reform and progression but as human warehouses—sites of regression. The inhumane conditions that prevailed in Strangeways persisted: overcrowded and unhygienic cells, weak management, aggressive regulation and minimal opportunity for work/education. What were the institutionalised consequences of this failure? In 2017, the Strangeways Independent Monitoring Board supplied the answer: ‘decent, humane, safe accommodation in which prisoners might find a degree of self-respect’ were unfeasible, given ‘the squalid, vermin-infested, damp environment more reminiscent of Dickensian England’ (IMB 2017: 7).2 Prisoners were subjected to ‘vermin in the residential accommodation’, a ‘plague of rats’ in the shower block, with bedding, clothing and belongings ‘soiled by vermin’. Residential areas were ‘unhygienic, unhealthy, environmentally dangerous’ (IMB 2017: 18). The inhumanity and neglect, against which the 1990 protesters had railed, prevailed. An inspection at another Victorian prison, HMP Liverpool, condemned its ‘abject failure … to offer a safe, decent and purposeful environment’.3 Its regime was ‘unacceptably poor’, the prison ‘so dirty, infested and hazardous to health’ it ‘could not be cleaned’. Inspectors ‘could not recall having seen worse living conditions’ (HMCIP 2018: 5–6). Three decades beyond the Strangeways uprising and Woolf’s mild reformism, conditions in Manchester and Liverpool prisons, among others, remain uninhabitable. Meaningful work and recreation are unavailable to most prisoners languishing for excessive periods in locked cells, their physical and mental health compromised. The severe criticisms levelled at these regimes by monitors and inspectors are consistent with devastating inspection assessments at Northern Ireland’s main male prison, Maghaberry. In 2015, persistent long-term overcrowding in ‘unfit for purpose’ accommodation was condemned by the prison’s independent monitors. MOBS KILL SEX INMATES AND BURN PRISON 11 DIE IN JAIL RIOT (Daily Mirror) 20 DEAD SEX OFFENDERS HANGED AND CASTRATED (Manchester Evening News) On the morning of 2 April 1990, gruesome headlines dominated the front pages of UK tabloids. A hastily constructed perimeter fence encircled the Strangeways prison as live radio and television confidently reported the extremity of prisoners’ violence. They claimed a rampaging mob had destroyed a wing of the insanitary and overcrowded jail, set a fire, raided prison hospital drugs cabinets and slaughtered fellow prisoners. A small group occupied the prison’s roof, hurling tiles and masonry at officers below. As days passed, the headlines continued: ‘Carnage in the Cages’; ‘Quake Britain’; ‘Tortured’; ‘Lynched in Jail Horror’; ‘Prison Mob Hang Cop’; ‘Bodies cut up and dumped in sewer’. The reported execution of 11 became 20, then 30. As non-participant prisoners were dispersed to other prisons, further gruesome details emerged. Sex offenders had been targeted, put before kangaroo courts, beaten, castrated and executed, their bodies butchered. By day, negotiations faltered; by night, hovering helicopters blasted music to exhaust the protesters. As the unprecedented occupation continued for 25 days, calls for military intervention mounted. Prisoners shouted grievances from the rooftop, holding aloft their unequivocal message scrawled on a white sheet: NO DEAD. Whatever the Home Office negotiators knew, they remained silent regarding casualties. It transpired that while some prisoners jailed for sex offences had been assaulted, allegations of torture and death were false. Gruesome events, graphically transmitted in media quotes from unidentified witnesses, had not happened. There had been no executions, no prisoners hung from balconies, none butchered nor castrated. In an instant, a myth had been created, and it remained uncorrected for several weeks. As the small dishevelled group retreated from their occupation, well aware they faced severe punishment, the media fell silent. The ‘Strangeways Massacre’ had been the most remarkable story in contemporary English penal history. Condemned by Prison Governor Brendan Friel as an ‘explosion of evil’, its promotion had been engineered, a direct consequence of unofficial reports and off-the-record briefings by prison IJCJ&SD 5 2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance officials, guards and their association. Unattributed allegations had been encouraged by inducements, remaining uncontested by official negotiators inside the prison throughout. The failure to deny became the substance of verification. officials, guards and their association. MOBS KILL SEX INMATES AND BURN PRISON 11 DIE IN JAIL RIOT (Daily Mirror) Most prisoners, particularly the vulnerable, were offered no purposeful activity. A landing culture dominated by boredom, drug-taking, bullying and self-harm was the norm. Within a debilitating atmosphere of depression, mental ill-health pervaded, treatment programs were non-existent, and ‘at-risk’ prisoners were poorly monitored. Maghaberry’s deficiencies were endemic. A full independent inspection followed (HMCIP/CJINI 2015). In three of four assessment categories— safety, respect and dignity, purposeful activity (World Health Organisation 1999)—the prison was ranked ‘poor’. Its management, conditions and regime were trapped in a downward spiral endangering prisoners’ lives. The Chief Inspector condemned the prison as ‘one of the worst prisons [we] have ever seen and the most dangerous … Dickens could write about Maghaberry without batting an eyelid’ (BBC News 5 November 2015). Unsafe and unstable, Northern Ireland’s main prison had descended into crisis. The severe condemnation of Manchester, Liverpool and Maghaberry prisons reflects institutional failures in the operational policies and practices prevalent throughout most prisons: overcrowding, long periods of lockdown, minimal work opportunities, drug use, bullying and hopelessness. These are the ingredients of revolt. In the aftermath of Strangeways, rather than positive regimes emerging to stimulate hope and recovery, punitive regimes of fear and degradation prevailed. It is a direction of travel consistent with the USA. In analysing California’s ‘distinctive new form and function of the prison’, Simon (2007: 142–143) www.crimejusticejournal.com IJCJ&SD 6 2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance considers it ‘a space of pure custody, a human warehouse or even a kind of waste management facility’. Populist politicians relentlessly promote all prisoners as inherently ‘dangerous’, recasting harsh rhetoric as a commitment to protecting ‘the wider community’. In this regressive climate, the ‘transformation of the prisoner through penitence, discipline, intimidation, or therapy’ has been abandoned in favour of incarcerating ‘people whose propensity for crime makes them appear an intolerable risk for society’. As Simon (2007: 142–143) concludes, maintaining ‘order’ within the ‘toxic-waste-dump prison increasingly relies on total segregation of the prisoners considered to be the most threatening’. It offers no correctional pretence and no commitment to reform—only an inhumane reality of unconditional containment, social isolation and persistent lockdown. What are the consequences of such institutionalised inhumanity? MOBS KILL SEX INMATES AND BURN PRISON 11 DIE IN JAIL RIOT (Daily Mirror) As Sim (2009: 130–131) states, prisons have an ‘extraordinary capacity for inflicting pain and inculcating fear, and if need be terror, into the lives of the confined’. Through its inherent culture, incarceration generates ‘social harm’ derived in ‘systemic discourses of punitive degradation’ further damaging the ‘lives of those whose sense of psychological and material well-being’ has been ‘fractured by their pre- and post-prison experiences’ (Sim 2009: 130–131). Regarding penal expansionism’s populist manifesto, Law’s research with women prisoners in the USA demonstrates that mass incarceration plays to a banal ‘tough on crime’ agenda. It has resulted in ‘the criminalization of more activities, leading to higher rates of arrest, prosecution and incarceration while shifting money and resources away from other public entities—education, housing, health care, drug treatment and other societal supports’ (Law 2009: 169). Gilmore (2007: 13) questions how incarceration is ‘supposed to produce stability through controlling what counts as crime’. She reflects on centuries ‘of experience into conflicting and generally overlapping explanations for why societies decide they should lock people out by locking them in’ (2007: 13). Put directly, ‘those locked up cannot make trouble outside’. Penal reality is a ‘mix of care, indifference, compulsory training, and cruelty to people in cages’ (Gilmore 2007: 13). For most prisoners subjected to this reality, ‘the acquisition of sobriety and skills’ and the adoption of ‘alternative lifestyles’ are unrealisable. Liberal reformist claims for prisoner rehabilitation have little traction in retributive regimes prioritising incapacitation. Rehabilitation is reduced to aspirational rhetoric, overwhelmed by a persistent and growing wave of ‘penal populism … dehumanising criminals as dangerous and irredeemable outsiders’ giving ‘legitimacy to a whole array of symbolic and draconian practices’ (Di Giorgi 2013: 34). Additional to dissuading the offender from committing further crimes, the severity of the prison sentence and the inhumane conditions in which it is served are proposed as deterrents to others. Neither objective is achieved. Thus, penal reformism is exposed as deceit; that via some magical process, conducted within a punitive and destructive environment of institutionalised deprivation, prisoners emerge not only unscathed by captivity but equipped to face a hostile world. Such groundless assumptions neglect the harm and consequences inflicted on prisoners ‘in our name’. Prisons create and reproduce ‘populations of incomplete and wounded lives … inside the prison or outside the prison’ (Quinney 2006: 270). MOBS KILL SEX INMATES AND BURN PRISON 11 DIE IN JAIL RIOT (Daily Mirror) ‘Prisoner’ or ‘non-prisoner’, within the walls or in the community, ‘no-one escapes the damage caused by the fact that prison exists’ (Quinney 2006: 270). Its deprivations and consequences are ‘pervasive’. Whether ‘economic, social, psychological’ or ‘spiritual’, the ‘injuries caused by the prison are shared by all’ (Quinney 2006: 270). Yet the pains of confinement are generated and sustained in ‘our’ name, financed through ‘our’ financial contributions. Given the emotional, social and political dislocation inflicted selectively on individuals and communities, the full societal cost cannot be reduced to economics. It is ironic that in times of severe political-economic crisis, an increasingly popular liberal reformist defence for incarceration is that it provides a safe harbour from destitution—poverty, homelessness, alcohol and drugs. While prisons might provide marginal respite from harsh realities in communities, they are places that reinforce impoverishment while encouraging prescribed and illicit drug dependency. Half a century has passed since Goffman (1968: 24–25) observed that those entering prison from relatively stable circumstances are ‘immediately stripped of support’ while subjected to ‘a series of abasements, IJCJ&SD 7 2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance degradations, humiliations and profanations of self’ inflicted ‘around the clock’. Relentless incarceration generates a dislocated state of ‘permanent civil dispossession’, abrogating prisoners’ political ‘rights’. Through objectifying procedures imposed on arrival—‘photographing, weighing, fingerprinting, assigning numbers, searching, listing personal possessions for storage, undressing, bathing, disinfecting, haircutting, issuing institutional clothing, rules’—the prisoner is ‘shaped and coded into an object that can be fed into the administrative machinery of the establishment’ (Goffman 1968: 25–26). Institutionalised objectification underpinned the Strangeways uprising, generating persistent volatility against penal authoritarianism. Our research into the uprising and hostage-taking in Scotland’s Peterhead Prison recorded prisoners’ testimonies revealing the parlous conditions they endured (Scraton, Sim and Skidmore 1991). Multiple deprivations were exposed in a deficient regime within a remote jail built in the 1880s. The ‘totality of the institution’ was ‘underwritten by a degree of totality or absolutism in power relations which virtually strip the prisoner of civil rights, personal consultation and democratic process’ while imposing an ‘unrelenting imposition of authority’ (Scraton, Sim and Skidmore 1991: 61–62). As Mandela (1994: 340–341) concluded, prisons are ‘designed to break one’s spirit and destroy one’s resolve’. MOBS KILL SEX INMATES AND BURN PRISON 11 DIE IN JAIL RIOT (Daily Mirror) Their regimes purposefully ‘exploit every weakness, demolish every initiative, negate all signs of individuality—all with the idea of stamping out that spark that makes each of us human and each of us who we are’. The ‘challenge for every prisoner’ is ‘how to survive prison intact, how to emerge from a prisoner undiminished’. Distinctive in time and specific in circumstances, what unifies these accounts is the ‘view from inside’. In applying the principles of critical analysis, bearing witness to the events and gathering testimonies from those suffering the institutional deprivations and arbitrary punishments administered behind closed doors, ‘truth’ is recovered. These testimonies, collected independently and over decades, provide a consistent and informed counter to official versions propagandised relentlessly at stage-managed press conferences and in evidence to official inquiries. Once headlined in public discourse, however, the propaganda becomes virtually impossible to unravel and reverse. From War to Peace: Women’s Incarceration in Northern Ireland The 1998 Northern Ireland (Sentences) Act was central to the settlement and established the legislative framework to release on licence prisoners affiliated to a paramilitary organisation. In 2003, the Human Rights Commission initiated in-depth research into the conditions experienced by non-politically affiliated women prisoners held in a high-security unit within Northern Ireland’s largest male jail (Scraton and Moore 2005). As the initial report was published, women prisoners were transferred to a medium-security unit within a male young offenders’ institution, and the research was extended (Scraton and Moore 2007). The research revealed a prison estate unfit for purpose. Incarcerating women in a male jail imposed severe restrictions on their movement, association, work and educational opportunities. With no prison staff recruited since the early 1990s and the early release of politically affiliated prisoners, there remained a surplus of mid-career, predominantly male, Protestant prison managers and officers. Many had limited experience of prisoner contact, few with women prisoners. A culture of non-engagement persisted; contact was reactive rather than proactive in conditions where often distressed and vulnerable women—and young men—were locked down for long periods without opportunities to participate in constructive activities. Further, serious failures in oversight and monitoring were institutionalised. In-depth interviews revealed that the women’s lives in their communities often amounted to ‘virtual’ imprisonment. Many were locked into poverty, child-rearing, primary parenting and caring responsibilities in neighbourhoods ravaged by illicit and prescription drugs. Over one-third of Northern Ireland’s children live on or below the poverty line, affecting schooling, health and welfare. Held in a prison regime designed, administered and prioritised to accommodate men reflected women’s experiences of marginalisation within their communities. Negotiating the regime exacerbated the profound impact of maintaining family contact, particularly with children, and the often-traumatising experiences of ‘family’ visits. On their release, women—particularly mothers—labelled ‘former prisoner’ were subjected to stigma, humiliation and shame associated with their incarceration. Ciara was 16, a child in an adult jail. She self-harmed, and no part of her visible body was free from tears or cuts. Her only clothing was a canvas pinafore dress—its Velcro fastening removed to prevent her scouring her skin. Held in the prison hospital for nine days, she had been returned to a punishment and segregation cell. It was bare except for a solid concrete plinth, a canvas blanket and a cardboard potty as a toilet. From War to Peace: Women’s Incarceration in Northern Ireland In 2003, under its unique powers of entry, the Northern Ireland Human Rights Commission visited HMP Maghaberry, widely considered one of the ‘most complex’ prisons in Europe due to its diverse population: men; women; remand; short-term/medium-term/life sentence prisoners; asylum seekers; sex offenders; ‘political’ prisoners. Commissioners, profoundly concerned by the conditions and regime they experienced, initiated research specifically focused on women prisoners. From 1969, for almost three decades, the six counties of Northern Ireland were at war, occupied by the British military and secret intelligence services. During the Conflict, in a population of approximately 1.6 million, 3636 people died, including over 2000 civilians (McKittrick et al. 2004: 1477). Hillyard et al. (2003) note that 88,000 households lost a close relative; a further 50,000 households had a resident injured. As a direct consequence of sectarian intimidation and threats, approximately 28,000 people lost employment, and 54,000 families were relocated. Throughout the Conflict, the North was policed and regulated by special powers and non-jury trials. In August 1971, the internment of ‘political’ activists without trial was introduced and institutionalised; 2357 men and women were arrested and subjected to harsh interrogation (McEvoy 2001). Boyle, Hadden and Hillyard (1975: 32) note that in imposing Direct Rule powers, the UK Government established a ‘new system of arrest and detention’. Initially, those prisoners claiming ‘political’ affiliation were granted ‘special category status’. In 1976, however, the British State reclassified politically affiliated prisoners as ‘ordinary’ criminals. In response, Republican prisoners refused to wear prison clothes or conform to prison rules. Draped only in coarse blankets, they were confined to strip cells. Following a series of beatings by guards, they embarked on a ‘dirty protest’, smearing excrement on cell walls. Hunger strikes followed, and 10 men died (McKeown 2001; Scraton 2020). IJCJ&SD 8 2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance The politics of incarceration throughout the Conflict were driven by the State’s steadfast commitment to criminalising and containing politically affiliated prisoners. In April 1998, following four years of uneasy peace negotiations, the UK and Irish Governments signed the Good Friday Agreement, setting the constitutional foundation for devolution powers to a democratically elected Northern Ireland Assembly. From War to Peace: Women’s Incarceration in Northern Ireland Unsurprisingly, she had attempted to hang herself: I hear voices, [they] tell me to cut myself. And I release the pain as well. It’s terrible, so it is. You sleep and you keep changing positions ... Just look what they make me go to the toilet in. That’s for night time … It’s a disgrace … They don’t give me underwear or nothing … just a wee sanitary towel and that’s it … stuck in the cell with a camera ... no privacy or nothing. Your dignity’s taken away. They just said, ‘It’s your own fault you’re behind the door’. (Moore and Scraton 2014: 134–136) Such were the conditions that led to prisoners self-harming and taking their own lives. The research interviews and observation further exposed the fiction of penal reformism and the lip-service paid to the ‘healthy prison’ agenda. Women prisoners were judged and treated according to their perceived femininity and the tropes associated with female conformity. They were consistently disrespected, not least when escorted within the prison, walking a gauntlet of shouted sexist abuse from young men in the male blocks. Invariably, abuse focused on their bodies and appearance. With workshops and education prioritised for young male prisoners, purposeful or creative activity for women prisoners was minimal. Most suffered mental ill-health, often as a consequence of imprisonment within a deficient regime. Ciara’s comments demonstrate how women’s vulnerability was heightened through punitive IJCJ&SD 9 2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance isolation. Further, the claims made about rehabilitation, resettlement and reintegration on release amounted to cruel deceit. isolation. Further, the claims made about rehabilitation, resettlement and reintegration on release amounted to cruel deceit. As McCulloch and Scraton (2009: 11) propose, ‘the violence of incarceration is historically, socially and culturally imprinted on the foundations of the prison’. For Ciara and the women interviewed, the pain of incarceration was a combination of mounting depression, mental ill-health and physical debilitation. The research developed to include the incarceration of men and young people within the context and legacy of the Conflict (Scraton 2015). Focusing on institutional and interpersonal power relations, it questioned the political legitimacy afforded to carceral regimes, their management and operation. From War to Peace: Women’s Incarceration in Northern Ireland Limited educational provisions, inadequate or insufficient opportunities for skills development, long periods of lockdown and mental ill-health extended throughout the penal estate. The research findings were supported by reports from the Independent Monitoring Boards and the Inspectorates. The cumulative evidence presented above challenges the social, political and ideological justifications advanced for prisons as places of opportunity where individuals—many suffering from mental ill-health, deep-seated addictions and inescapable poverty—can exit prison gates with hope and purpose. It exposes the operational reality of life in jail as oppressive, contextualised by policies of containment, security and institutional control rather than opportunity, safety and personal development. The abject failure of penal reform contradicts claims for the legitimacy of incarceration, thus returning the focus to decarceration and abolition. In seeking the eventual abolition of mass imprisonment, however, Davis (2003: 103–104) argues, the challenge of campaigning for ‘more humane, habitable environments’ while not ‘bolstering the permanence of the prison system’. Its resolution requires sociological imagination and political will to create a ‘constellation of alternative strategies and institutions … revitalization of education at all levels’ alongside a ‘health system providing free physical and mental health care to all’ (Davis 2003: 103–104). On the road to abolition, reducing the prison population—decarceration—requires a societal commitment to developing and funding a ‘justice system based on reparation and reconciliation rather than retribution and vengeance’. Hillsborough: Justice Delayed is Justice Denied In the Liverpool Daily Post (18 April 1989), John Williams was unequivocal: ‘gatecrashers’ had ‘wreaked their fatal havoc’; ‘uncontrolled fanaticism and mass hysteria’ had ‘literally squeezed the life out of men, women and children’. It was ‘yobbism at its most base’ as ‘Scouse killed Scouse’. The allegations that followed became increasingly sinister. Unnamed police officers claimed that while rescuing the dying, they were assaulted by Liverpool fans who stole from the dead. The Star (18 April 1989) claimed that ‘Ticketless thugs staged crush to gain entry’. They had ‘attacked an ambulance man, threatened firemen and punched and urinated on policemen as they gave the kiss of life to stricken victims’. The following day, the allegations were echoed by five national newspapers: ‘Dead Fans Robbed by Drunk Fans’ (Daily Star); ‘They were drunk and violent and their actions were vile’ (Mail); ‘Police Accuse Drunken Fans: Police saw “sick spectacle of pilfering from the dying”’ (Express); ‘Fury as police claim fans robbed victims’ (Mirror); ‘Police tell MP of attacks on them as they helped injured’ (Telegraph). The Sun cleared its front page: ‘THE TRUTH’: ‘Some fans picked pockets of victims; Some fans urinated on the brave cops; Some fans beat up PC giving life kiss’. A ‘high-ranking police officer’ claimed that a dying young woman had been sexually abused: ‘fans were just acting like animals’. In the public’s mind, the die was cast. Four months later, the Home Office Inquiry published an interim report concluding that overcrowding was the ‘main cause’ of the disaster, the ‘main reason’ being a serious failure in police control (Taylor 1989). Contrary to earlier press coverage, the report berated the Match Commander and senior police officers for their collective failure on the day, exacerbated by their evasiveness before the inquiry. Preliminary inquest proceedings heard uncontested pathology findings on each of the deceased. The Coroner ordered that blood alcohol levels of all who died, including children, should be recorded. His unprecedented decision further fuelled the allegation that alcohol consumption had contributed to the deaths. In November 1991, following the Director of Public Prosecutions’ decision not to proceed with criminal prosecutions, the full inquests were resumed. Remarkably, the Coroner imposed a 3.15 pm cut-off on evidence. Without taking medical opinion or providing clinical evidence, he claimed that by that time all who died would have suffered irreversible fatal injuries. It was a claim without substance. Hillsborough: Justice Delayed is Justice Denied On 15 April 1989, as an FA Cup semi-final kicked off at Sheffield’s Hillsborough Stadium, compression in a tightly-packed crowd on standing terraces led to the death of 96, mostly young Liverpool soccer fans. Four hundred were seriously injured, thousands traumatised. To relieve congestion outside, police officers had opened an exit gate allowing over 2000 fans to enter an unfamiliar stadium. Descending a steep tunnel, they emerged at the rear of already overcrowded pens—fences to the sides, to the front and no means of retreat. As people were dying, the Police Commander told officials that fans had forced entry. Broadcast internationally, his lie blamed fans for the disaster. Further elaborated, the unsubstantiated police version dominated the headlines: fans had arrived ‘late’, without tickets, many drunk, violent and abusive. These unproven allegations deflected responsibility, then infected the investigations and inquests, the latter concluding that the deaths were ‘accidental’. In this hostile climate, the Hillsborough Project was founded. Its research provides a critical analysis of the causes, circumstances and aftermath. The day after the disaster, Prime Minister Margaret Thatcher, her Press Secretary Bernard Ingham and Home Secretary Douglas Hurd arrived in Sheffield. Subsequently, Ingham wrote that the Chief Constable told Thatcher, ‘there would have been no Hillsborough if a mob, who were clearly tanked up, had not tried to force their way into the ground’ (Personal Correspondence, dated 13 July 1994). Within days, allegations from senior police officers and local Conservative politicians dominated media coverage, and a public inquiry was appointed, led by Lord Justice Taylor (Coleman et al. 1990; Scraton, Jemphrey and Coleman 1995). Accepting the veracity of police briefings, the press minimised accounts from survivors and witnesses that painted a markedly different picture. IJCJ&SD 10 2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance The relentless condemnation of fans’ behaviour, vilified for causing death and physical injury, devastated already traumatised survivors. On 17 April, the Star claimed the tragedy was the consequence of a ‘crazed surge’. The Yorkshire Post reported ‘thousands of latecomers’ without tickets had caused a ‘fatal charge’. Writing in the Evening Standard (17 April 1989) Peter McKay condemned ‘the tribal passions of Liverpool supporters’ who had ‘literally killed themselves and others to be at the game’. Jacques Georges, the UEFA President, condemned ‘beasts waiting to charge into the arena’ (Liverpool Echo, 17 April 1989). Hillsborough: Justice Delayed is Justice Denied Further, in consultation with police advisers, the Coroner selected witnesses, including local residents, publicans and police officers, whose collective testimony affirmed that ticketless, drunk and aggressive fans had arrived at the stadium determined to force entry. Five months later, following his selective summary of evidence from 230 witnesses, he directed the jury to a majority ‘accidental death’ verdict. It devastated the families and survivors. A bereaved mother stated, ‘The inquests were a farce. The Coroner clearly directed the jury … he got what he wanted’. Another mother stated: ‘… the jury could only arrive at one verdict after the Coroner’s performance’ (Scraton 2016: 219). The reversal of the public inquiry findings was welcomed by South Yorkshire senior police officers and the Chairman of the Police Federation who addressed bereaved families, live on television, ‘What more do they want?’ Using an unfortunate metaphor, another senior police officer involved proclaimed that it was ‘time to bury the past’ (Scraton, Jemphrey and Coleman 1995: 261). Two years later, a High Court judicial review confirmed the Coroner’s direction as ‘impeccable’. In 1997, an unprecedented judicial ‘scrutiny’ of ‘new’ evidence was initiated by the Labour Government. Despite compelling evidence that police statements had been altered by a team appointed within the South Yorkshire Police, it concluded that there was no justification for a further public inquiry (Stuart-Smith 1998). By this time, two detailed alternative reports had been published by the Hillsborough Project (Coleman et al. 1990; Scraton, Jemphrey and Coleman 1995). Their findings were consolidated by Jimmy IJCJ&SD 11 2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Res Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance McGovern’s award-winning 1996 drama–documentary Hillsborough and a comprehensive analysis, Hillsborough: The Truth (Scraton 1999, 2016). The analysis revealed the full extent and impact of altered police statements, which had been ordered by the South Yorkshire Police and its solicitor. Despite widespread publicity, this extensive body of research and analysis was ignored for a further decade. In 2009, the families’ persistent campaign led to the appointment of a unique independent panel of inquiry. Scraton drafted the application, headed its research and was the primary author of its comprehensive report. The panel’s researchers analysed two million documents from 80 organisations and individuals. Hillsborough: Justice Delayed is Justice Denied Two years later, it delivered a comprehensive report: its 12 chapters and 153 findings established a catalogue of official failures (Hillsborough Independent Panel 2012). The research exonerated the fans. New criminal investigations followed, alongside a review of policing by the Independent Police Complaints Commission (now the Independent Office for Police Conduct). The Attorney-General quashed the inquest verdicts, ordering new inquests. Following five preliminary hearings, supported by legal aid, full inquests ran for two years, presided over by a High Court Judge. In 2016, the jury returned a verdict of unlawful killing. In addition, it recorded egregious failings: 10 by the stadium owners, architects, safety engineers and ambulance service; 15 by the South Yorkshire Police. The verdict exonerated the fans, overturning the accidental death verdict so heavily influenced by the Coroner’s direction a quarter of a century earlier.4 Throughout two decades, senior South Yorkshire police officers, the Chief Constable and their lawyers, the Police Federation, Members of Parliament and civilian witnesses closely associated with the police purposefully and collectively attributed full responsibility for the disaster to fans, including survivors and those who died. Their statements generated and sustained a persistent myth regarding fans’ culpability, influencing the media, the Coroner, appeal court judges, the judicial scrutiny of ‘new’ evidence, government ministers and senior politicians. The detailed analysis and substantial recommendations of the Hillsborough Project’s 1990 and 1995 reports, precursors to the Independent Panel’s comprehensive findings two decades later, were ignored. Finally, the 2016 verdict silenced the highly vocal and much- publicised vilification directed against those who survived Hillsborough. However, those involved in the construction and promulgation of the myth failed to accept responsibility or apologise for the additional pain and suffering inflicted on bereaved families and survivors. Critical Research: The ‘Echo’ and the ‘Answer’ This brief excursion into my critical work on prisons, the legacy of civil war in the North of Ireland and three decades of investigative research into the Hillsborough aftermath demonstrates a driving imperative to recover truth and pursue justice through ‘bearing witness’, accessing alternative accounts, analysing ‘hidden’ data and recording abuses of state power. In challenging the obfuscations of ‘official discourse’ and ‘scientific’ knowledge, investigative research positions the immediacy of ‘personal troubles’ within their broader context as ‘public issues’ (Wright Mills 1959). Despite its claims for value-neutrality, official discourse is no guarantor of ‘truth’. Its legitimacy is derived in the status quo and the maintenance of dominant vested interests. Rather, the truth must be sought in alternative oppositional accounts: the ‘view from below’. In its recovery, truth holds the powerful—whether private or state agencies, corporations or individuals—to account. It does so on ‘their’ terms, using the rule of law and, where appropriate, engaging international standards and building oppositional alliances. Its fulfilment, however, necessitates a ‘fundamental shift in the structural relations and determining contexts of power which marginalize and exclude [individuals] from effective participation in their destinies’ (Scraton and Haydon 2002: 326). While diverse in focus, the research discussed here exposes the myth of value-freedom, demonstrating that regulatory institutions do not achieve the value-neutrality they claim. Rather, these institutions are woven into the fabric of inequality, marginalisation and criminalisation, which is evident in the power imbalance derived in and sustained by the determining contexts of class, ‘race’, gender and sexuality. Multiple deprivations are rooted in endemic poverty and structural inequality. There is consistent evidence in cross-jurisdictional prison research that deprivation and punishment are bedfellows. Working-class communities disproportionately endure cuts in employment, essential services, health care, IJCJ&SD 12 2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance welfare and education while living in poor, high-density housing, reliant on food banks. In June 2017, 72 people were killed and many more bereaved and displaced as a consequence of a foreseeable fire in a neglected social housing scheme administered by one of London’s most affluent boroughs. The Grenfell Tower fire is testimony to institutional failure in state–community relations and public consultation (Bulley, Edkins and El-Enany 2019). It is a failure derived, legitimated and reproduced in the structural relations of inequality and oppression that constitute, maintain and reproduce the established socio- economic order and its state institutions. Critical Research: The ‘Echo’ and the ‘Answer’ While this article was under review, it became dramatically apparent that a highly contagious and deadly disease, COVID-19, was escalating rapidly into a pandemic. With no known vaccine, it claimed many lives, closed down entire cities and towns, factories and businesses, shops and social venues. As people isolated in their homes, instructed by the State to end all direct personal contact, it appeared no one was immune from contracting the virus. Inevitably and unsurprisingly, however, it continues to impact disproportionately on the most marginalised communities where physical, spatial and social distancing are not possible, and medical capacity is minimal (Davis 2020; Giroux 2020). Unrelated, but at the height of the pandemic, in Minneapolis a White police officer was filmed kneeling on the neck of a prostrate Black man while other officers watched on passively and approvingly. Under the knee, George Floyd died. Under the banner ‘Black Lives Matter’, his death triggered unprecedented international protests directed against racist policing and colonial heritage—the visible manifestation of racist oppression. Clearly distinct in context and circumstances, the impact of the Grenfell fire, the marginalisation laid bare by a pandemic and the use of state violence against the Black community have shared roots in societies whose administration of state power and political and economic reproduction are dependent on maintaining and sustaining structural inequalities. Critical social research does not ‘hover in the air’ above seemingly random events unfolding ‘on the ground’ and managed by state institutions. As this article demonstrates, working with families and communities campaigning for ‘justice’ for loved ones in prison, in the aftermath of disasters or conflict reflects Sontag’s commitment to deploying her photographic skills as a chronicler of oppression, exposing the pain of ‘others’. Within communities riven by social, political and economic exclusion and ravaged by prescription and street drugs, the persistent challenge is to establish programs that share knowledge, consult inclusively and support active participation across the lifespan. These programs should celebrate diversity while eliminating fear and defeating discrimination in all its complex, interrelated forms. They should respond with alacrity, compassion and resources to mental ill-health crises. As Giroux (2002) states, defeating exclusion requires generating and sustaining a politics of hope derived in the advancement of learning and the creation of opportunity. Correspondence: Phil Scraton, Professor Emeritus, School of Law, Queen’s University, Main Site Building, Belfast, BT7 1NN, Northern Ireland. Email: p.scraton@qub.ac.uk Critical Research: The ‘Echo’ and the ‘Answer’ g llsborough Independent Panel web-site includes its report and all supporting documents: 4 The Hillsborough Independent Panel web-site includes its report and all supporting documents: https://discovery.nationalarchives.gov.uk/details/r/C14176659 See also: http://hillsboroughinquests.independent.gov.uk/ (temporarily unavailable at the time of publishing due to ongoing legal proceedings). g p p pp g https://discovery.nationalarchives.gov.uk/details/r/C14176659 See also: http://hillsboroughinquests.independent.gov.uk/ (temporarily unavailable at the time of publishing due to ongoing legal proceedings). References Brighton: Harvester Wheatsheaf Foucault M (1980) Power/Knowledge: Selected Interviews and Other Writings 1972–1977. Brighton: Wheatsheaf. Gilmore RW (2007) Golden Gulag: Prisons, Surplus, Crisis, and Opposition in Globalizing California. Berkeley: University of California Press. y Giroux HA (2002) Global capitalism and the return of the garrison state: Rethinking hope in the age of uncertainty. ARENA Journal 19. Giroux HA (2002) Global capitalism and the return of the garrison state: Rethinking hope in the age of uncertainty. ARENA Journal 19. Giroux HA (2020) The COVID-19 pandemic is exposing the plague of neo-liberalism. TruthOut. https://truthout org/articles/the-covid-19-pandemic-is-exposing-the-plague-of-neoliberalism/ Giroux HA (2020) The COVID-19 pandemic is exposing the plague of neo-liberalism. TruthOut. https //truthout org/articles/the covid 19 pandemic is exposing the plague of neoliberalism/ Giroux HA (2020) The COVID-19 pandemic is exposing the plague of neo-liberalism. TruthOut. h // h / i l / h id 19 d i i i h l f lib li ( ) p p g p g https://truthout.org/articles/the-covid-19-pandemic-is-exposing-the-plague-of-neoliberalis Goffman E (1968) Asylums: Essays on the Social Situation of Mental Patients and Other Inmates. Harmondsworth: Penguin. Goffman E (1968) Asylums: Essays on the Social Situation of Mental Patients and Other Inmates. Harmondsworth: Penguin. g Gouldner A (1973) Foreword. In Taylor I, Walton P and Young J (eds) The New Criminology. London: Routledge and Kegan Paul. Gouldner A (1973) Foreword. In Taylor I, Walton P and Young J (eds) The New Criminology. London: Routledge and Kegan Paul. Hall S (1985) Authoritarian populism: A reply to Jessop et al. New Left Review 151: 115–124. Hall S (1985) Authoritarian populism: A reply to Jessop et al. New Left Review 151: 115–124. Hall S, Critcher C, Jefferson T, Clarke J and Roberts B (1978) Policing the Crisis. London: Macmillan. Harvey L (1990) Critical Social Research. London: Sage. Hill b h I d d t P l (2012) Hill b h L d HMSO Hall S (1985) Authoritarian populism: A reply to Jessop et al. New Left Review 151: 115–124. Hall S, Critcher C, Jefferson T, Clarke J and Roberts B (1978) Policing the Crisis. London: Macmillan. Harvey L (1990) Critical Social Research. London: Sage. ( ) y f Hall S, Critcher C, Jefferson T, Clarke J and Roberts B (1978) Policing the Crisis. London: Macmilla Harvey L (1990) Critical Social Research London Sage Hall S, Critcher C, Jefferson T, Clarke J and Roberts B (1978) Policing the Crisis. London: Macmillan. References BBC News (2015) Maghaberry prison ‘most dangerous’ ever visited says chief inspector. BBC News (2015) Maghaberry prison ‘most dangerous’ ever visited says chief inspector. https://www.bbc.co.uk/news/uk-northern-ireland-34733832 BBC News (2015) Maghaberry prison ‘most dangerous’ ever visited says https://www.bbc.co.uk/news/uk-northern-ireland-34733832 Becker H (1967) Whose side are we on? Social Problems 14(3): 239–247. https://doi.org/10.2307/799147 Becker H (1974) Labelling theory reconsidered. In Rock P and McIntosh M (eds) Deviance and Social Control. London: Tavistock. Berger J (1972) Ways of Seeing. Harmondsworth: Penguin. Berger J (1972) Ways of Seeing. Harmondsworth: Penguin. Boyle K, Hadden T and Hillyard P (1975) Law and the State: The Case of Northern Ireland. London: Martin Robertson. Boyle K, Hadden T and Hillyard P (1975) Law and the State: The Case of Northern Ireland. London: Martin Robertson. Bulley D, Edkins J and El-Enany N (eds) (2019) After Grenfell: Violence, Resistance and Response. London: Pluto Press. Bulley D, Edkins J and El-Enany N (eds) (2019) After Grenfell: Violence, Resistance and Response. London: Pluto Press. Christie N (1994) Crime Control as Industry. 2nd ed. London: Routledge. Coleman S, Jemphrey A, Scraton P and Skidmore P (1990) Hillsborough and After: The Liverpool Experience. Liverpool: Liverpool City Council. Coleman S, Jemphrey A, Scraton P and Skidmore P (1990) Hillsborough and After: The Liverpool Experience. Liverpool: Liverpool City Council. Davis A (2003) Are Prisons Obsolete? New York: Seven Stories Press. Davis A (2003) Are Prisons Obsolete? New York: Seven Stories Press. Davis M (2020) The Monster Enters: COVID-19, Avian Flu and the Plagues of Capitalism. New York: OR ( ) g f p Davis NJ (1975) Social Constructions of Deviance: Perspectives and Issues in the Field. Dubuque: WC Brown Co. Di Giorgi A (2013) Prisons and social structure in late-capitalist societies. In Scott D (ed) Why Prison? Cambridge: Cambridge University Press. J ( ) f p q Di Giorgi A (2013) Prisons and social structure in late-capitalist societies. In Scott D (ed) Why Prison? Cambridge: Cambridge University Press. g y Dorfman A (2004) Manifesto for Another World: Voices from Beyond the Dark. New York: Seven Stories Press. Foucault M (1980) Power/Knowledge: Selected Interviews and Other Writings 1972–1977. Brighton: Harvester g y Dorfman A (2004) Manifesto for Another World: Voices from Beyond the Dark. New York: Seven Stori Dorfman A (2004) Manifesto for Another World: Voices from Beyond the Dark. New York: Seven Stories Press. Foucault M (1980) Power/Knowledge: Selected Interviews and Other Writings 1972–1977. Critical Research: The ‘Echo’ and the ‘Answer’ Alongside a commitment to social and economic justice, as the research in prisons and with young people in the North of Ireland demonstrates, is the creation of inclusive dialogue prioritising critical thought and informing political action. In a well-quoted passage, Dorfman (2004: 10) states that those who bear witness to torture, degradation or the suffering of others could not ‘live with themselves if they did nothing … could not stain their lives by remaining silent’. Witnessing suffering yet ‘doing nothing’ redefines observers as ‘accomplices’—so it is with critical social research. In challenging the ‘darkness of apathy’ and ensuring that the ‘voices’ of the marginalised are eminent, the excluded ‘receive the echo and answer’ that is their right. Correspondence: Phil Scraton, Professor Emeritus, School of Law, Queen’s University, Main Site Building, Belfast, BT7 1NN, Northern Ireland. Email: p.scraton@qub.ac.uk IJCJ&SD 13 2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance 1 School of Law, Queens University Belfast, p.scraton@qub.ac.uk. This article was first presented as a plenary presentation to the Interdisciplinary Workshop on State Violence, Macquarie University, Australia in December 2018. It is derived from three substantial research projects spanning three decades: The Violence of Incarceration; The Context, Circumstances and Consequences of the Hillsborough Disaster; Childhood, Transition and Marginalisation. While distinct in focus, the projects share an imperative of critical social research—foregrounding and contextualising the ‘view from below’ or, in the case of the prison research, the ‘view from inside’. Many thanks to the organisers of and participants in the Workshop, co-researchers on the projects, to the reviewers whose comments were very helpful and, as ever, to Deena Haydon. 2 Independent Monitoring Boards (IMBs) are established at all prisons, independent of prison management, with powers of entry to monitor operational policies and practices and hear prisoners’ complaints or concerns. 3 Prison inspections are conducted usually at five-year intervals and over two weeks at all prisons by Her Majesty’s Inspectorate of Prisons (HMIP), an independent body headed by a Chief Inspector (HMCIP). Their reports provide an assessment based on the World Health Organisation’s ‘Healthy Prison’ criteria. 3 Prison inspections are conducted usually at five-year intervals and over two weeks at all prisons by Her Majesty’s Inspectorate of Prisons (HMIP), an independent body headed by a Chief Inspector (HMCIP). Their reports provide an assessment based on the World Health Organisation’s ‘Healthy Prison’ criteria. HMCIP (2018) Report on an Unannounced Inspection of HMP Liverpool 4–15 September 2017. www.justiceinspectorates.gov.uk/hmiprisons/wp-content/uploads/sites/4/2018/01/HMP-Liverpool-Web- 2017.pdf Moore L and Scraton P (2014) The Incarceration of Women: Punishing Bodies, Breaking Spirits. London: Palgrave Macmillan. McKittrick D, Kelters S, Feeney B, Thornton C and McVea D (2004) Lost Lives: The Stories of the Men, Women and Children Who Died as a Result of the Northern Ireland Troubles. Edinburgh and London: Mainstream Publishing. Moore L and Scraton P (2014) The Incarceration of Women: Punishing Bodies, Breaking Spirits. London: Palgrave Macmillan. McKittrick D, Kelters S, Feeney B, Thornton C and McVea D (2004) Lost Lives: The Stories of the Men, Women and Children Who Died as a Result of the Northern Ireland Troubles. Edinburgh and London: Mainstream Publishing. Moore L and Scraton P (2014) The Incarceration of Women: Punishing Bodies, Breaking Spirits. London: Palgrave Macmillan. f g g Moore L and Scraton P (2014) The Incarceration of Women: Punishing Bodies, Breaking Spirits. London: Palgrave Macmillan. Poulantzas N (1978) State, Power, Socialism. London: Verso. Poulantzas N (1978) State, Power, Socialism. London: Verso. Quinney R (2006) The life inside: Abolishing the prison. Contemporary Justice Review 9(3): 269–2 Quinney R (2006) The life inside: Abolishing the prison. Contempo Scraton P (2007) Power, Conflict and Criminalisation. London: Routledge. Scraton P (2015) Prisons and imprisonment in Northern Ireland. In McAlinden A-M and Dwyer C (eds) Criminal Justice in Transition: The Northern Ireland Context. Oxford: Hart Publishing. Scraton P (2015) Prisons and imprisonment in Northern Ireland. In McAlinden A-M and Dwyer C (eds) Criminal Justice in Transition: The Northern Ireland Context. Oxford: Hart Publishing. J g Scraton P (1999/ 2016) Hillsborough: The Truth. London: Penguin Random House. Scraton P (1999/ 2016) Hillsborough: The Truth. London: Penguin Random House. 999/ 2016) Hillsborough: The Truth. 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Sontag S (2003) Regarding the Pain of Others. New York: Picador. g ( ) g g f Stuart-Smith, LJ (1998) Scrutiny of evidence relating to the Hillsborough Football Stadium Disaster. London: Home Office, Cmnd 3878. Stuart-Smith, LJ (1998) Scrutiny of evidence relating to the Hillsborough Football Stadium Disaster. London: Home Office, Cmnd 3878. y J ( ) g p , p Taylor I, Walton P and Young J (1973) The New Criminology, London: Routledge and Kegan Paul. y J ( ) g p p Taylor I, Walton P and Young J (1973) The New Criminology, London: Routledge and Kegan Paul. , g J ( ) gy, g g I, Walton P and Young J (eds) (1975) Critical Criminology. London: Routledge and Kegan Paul. Thatcher, M. (1984) The Carlton Lectures https://www.margaretthatcher.org/document/105799 The Guardian (2015) Lord Woolf: 25 years on from Strangeways, prisons are still in crisis. https://www.theguardian.com/society/2015/apr/01/lord-woolf-strangeways-prison e Guardian (2015) Lord Woolf: 25 years on from Strangeways, prisons are still in crisis. Guardian (2015) Lord Woolf: 25 years on from Strangeways, prisons are still in crisis. https://www.theguardian.com/society/2015/apr/01/lord-woolf-strangeways-prisons-still-crisis /www.theguardian.com/society/2015/apr/01/lord-woolf-strangeways-prisons-still-crisis Weber M (2009) [1958] From Max Weber: Essays in Sociology. (Gerth HJH and Wright Mills C eds). Abingdon/New York: Routledge. World Health Organisation (1999) Mental Health Provision in Prisons: A Consensus Statement. Copenhagen: WHO Regional Office. Regional Office. Wright Mills C (1959) The Sociological Imagination. 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https://openalex.org/W3091756168
https://www.granthaalayahpublication.org/journals/index.php/granthaalayah/article/download/IJRG20_B09_3731/1352
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RESEARCH AND EDUCATION IN THE MEDICAL AREA
International journal of research - granthaalayah
2,020
cc-by
4,169
ABSTRACT The training of health professionals poses a great challenge in developing countries around the world. Medicine of the XXI century, represented in health careers, has a direct relationship with scientific and technical development, involves, observation, the search for knowledge about the problems that affect the health and biological well-being of the human being. This work implies a great ethical commitment to society, hence the importance of strengthening research capacity in both undergraduate and graduate studies. Developing human talent is a task that must be carried out continuously from the beginning of the medical career. Preserving life and preventing serious complications in the vulnerable population due to known or unknown pathologies is an unavoidable function of all health personnel, this is only achieved by training future professionals in the best way to guarantee the survival of future generations against the pathogenic microorganisms that can cause death. Teachers must implement the necessary strategies to motivate new professionals. Curiosity and interest must be strengthened throughout all medical careers. Article Citation: MSC. Alida Bella Vallejo López, DRA. Josefina Ramirez Amaya, MSC. Juana Kou Guzman, ING. Cesar Noboa, MSC. Oswaldo Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila. (2020). RESEARCH AND EDUCATION IN THE MEDICAL AREA. International Journal of Research -GRANTHAALAYAH, 8(9), 43-48. , , Oswaldo Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila. (2020). RESEARCH AND EDUCATION IN THE MEDICAL AREA. International Journal of Research -GRANTHAALAYAH, 8(9), 43-48. Received Date: 31 August 2020 Accepted Date: 24 August 2020 Keywords: Research Education Health Medicine © 2020 The Author(s). 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. 43 RESEARCH AND EDUCATION IN THE MEDICAL AREA MSC. Alida Bella Vallejo López *1 , DRA. Josefina Ramirez Amaya 2 , MSC. Juana Kou Guzman 3 , ING. Cesar Noboa-Teran 4 , MSC. Oswaldo Francisco Zavala Palacios 5 , MSC. Narcisa Liliana Durán Ávila 6 *1, 2, 3, 4, 5 Guayaquil University, Faculty of Medical Sciences, Ecuador 6 Laboratory Jesus De Nazareth, Faculty of Medical Sciences, Ecuador DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 ISSN (Online): 2350-0530 International Journal of Research -GRANTHAALAYAH ISSN (Print): 2394-3629 September 2020, Vol 8(09), 43 – 48 DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 RESEARCH AND EDUCATION IN THE MEDICAL AREA MSC. Alida Bella Vallejo López *1 , DRA. Josefina Ramirez Amaya 2 , MSC. Juana Kou Guzman 3 , ING. Cesar Noboa-Teran 4 , MSC. Oswaldo Francisco Zavala Palacios 5 , MSC. Narcisa Liliana Durán Ávila 6 *1, 2, 3, 4, 5 Guayaquil University, Faculty of Medical Sciences, Ecuador 6 Laboratory Jesus De Nazareth, Faculty of Medical Sciences, Ecuador DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 ISSN (Online): 2350-0530 International Journal of Research -GRANTHAALAYAH ISSN (Print): 2394-3629 September 2020, Vol 8(09), 43 – 48 DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 RESEARCH AND EDUCATION IN THE MEDICAL AREA MSC. Alida Bella Vallejo López *1 , DRA. Josefina Ramirez Amaya 2 , MSC. Juana Kou Guzman 3 , ING. Cesar Noboa-Teran 4 , MSC. Oswaldo Francisco Zavala Palacios 5 , MSC. Narcisa Liliana Durán Ávila 6 *1, 2, 3, 4, 5 Guayaquil University, Faculty of Medical Sciences, Ecuador 6 Laboratory Jesus De Nazareth, Faculty of Medical Sciences, Ecuador DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 ISSN (Online): 2350-0530 ISSN (Print): 2394-3629 International Journal of Research -GRANTHAALAYAH September 2020, Vol 8(09), 43 – 48 DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 Article Type: Research Article 2. BACKGROUND Research is as old as humanity and has grown over time, like science to the present day. The emergence of the great philosophical, scientific and mathematical movements to understand the great phenomena of the world, from a deeper and more rational vision, had a great impulse after Christ. Among the most prominent figures of scientists in the history of mankind who made contributions to scientific research, we can mention Galileo Galilei, considered the father of the scientific method, Hippocrates known as the father of medicine, among others. The word investigate comes from the Latin investigare derived from vestigiun which means to look for a clue. (Universia México 2016) affirms that: Research is a quality of the human being, for the search for knowledge and truth. Investigating means searching and discovering, in order to develop new theories in different disciplines or modify existing ones. But this capacity, if it already exists in each person, must be developed, and the best way to do it is by promoting it from youth, through the development of academic papers. For Carlos Borsotti (1989) “… research is a process through which an attempt is made to respond to scientific problems, through systematic procedures, which include the production of valid and reliable information”. Therefore, research should be a fundamental educational practice for all students, especially in the medical education curriculum, both undergraduate and graduate. g g Since Wilhelm Von Humboldt (1810) added research units to the university process, the value of the institutional mission of the University has increased, by generating new knowledge, to transcend and not remain stuck in theories considered as absolute truths. (Deiters, H. 1960). ( ) This is another way to enrich future professionals educationally, to prepare them with the capacity for inquiry, to face the problems and challenges of science and society. (Naranjo R. 2013) "The research allows to expand knowledge, based on data on any topic of social or scientific interest for academic purposes" (Coelho, 2019). Research requires time and dedication, the researcher must be interested in a topic and have a curiosity to investigate, in order to solve a puzzle. So, although all human beings have the ability to investigate, not everyone is interested in doing it, so it is clear that to investigate there must be motivation. Research and Education in The Medical Area The benefits obtained range from economic growth to the discovery of new technologies that provide greater comfort to people and new treatments for diseases. Thousands of investigations could be mentioned that have changed the world, generating comforts. Going through the creation of the microscope, vaccines, X-rays, the decomposition of the atom, the study of DNA and the creation of new academic areas to learn about various topics, nothing would be the same, if it had not been investigated. (Vallejo A. 2020). In a globalized context, research plays an important role in the training process of future professionals in the health area, considering that the area of medicine is constantly updating knowledge, methodologies and procedures, to improve the quality of healthcare processes. In addition, they must be informed of new health problems that arise in the world, considering that viruses, bacteria and microbial agents evolve and appear with increasing aggressiveness. gg The current trend in the world is to conduct research of public utility, especially when it comes to biomedical research. Institutions must support not only theoretical academic training, but also training in the pillars of research such as the concepts of basic science and statistics, the mechanism by which the relevance of a medical or scientific study is presented or established, regardless of the area in which it has been developed. The impulse for the development of research, on the part of doctors, must begin with profound modifications in the study plans of medical careers. The way we teach medicine and its related careers must be changed. Teaching should be presented as a guide to understanding, developing critical thinking, achieving independence of judgment, and preparing for change. Every medical student must be prepared to prevent and attend to the priority health problems of the population in an ethical framework, but also to develop their eventual capacity to carry out research, regardless of their final destination: generalist, specialist, Ph DV (Norero, Colomba. 2000). International Journal of Research -GRANTHAALAYAH 1. INTRODUCTION Research plays a decisive role in the learning process, to increase student knowledge, allowing comprehensive academic development. Research is considered an activity aimed at obtaining significant findings that increase human knowledge and science, promotes the development of skills and the discovery of new facts, technological advances. Bijarro, Francisco (2007): It allows to develop improvements in all areas of knowledge; Thanks to research, it was possible to discover various inventions that are indispensable to society today. In this way, the researcher can understand the needs and problems that influence man's life. Research expands knowledge. Scientific development brings with it a cultural evolution for a better adaptation to the modern world. 43 Research and Education in The Medical Area Research and Education in The Medical Area RESEARCH IN THE MEDICAL AREA Medicine, like all science, needs research to ensure advances in knowledge. Research in medicine contributes to: understanding the biological processes of the human being, carrying out prevention, detection, diagnosis, treatment and looking for the causes of diseases. Medical practice also includes the control of the health problems of the population. To contribute to scientific advancement, there are various types of health research, such as biomedical, epidemiological, clinical, and health education research at the individual and population levels. It is important to recognize that there are health problems of various kinds, and to face them, learning in research during professional training must be valued. All research must be carried out in accordance with scientific and ethical principles, both in the theoretical and in the experimental part, always having as a priority to avoid risks of all kinds to the life and integrity of people. If there were no researchers in the medical area, we would not understand the complexity of the human body, nor would the function of each organ, the cure or treatment of diseases not have been found, there would be no vaccines, there would be no known DNA. They would not perform surgeries. Man is always exposed to diseases to investigate. Research takes time, dedication and sacrifice and, in most cases, it is not rewarded. This leads to the need to train educators and other related professionals to learn theories, methods and research techniques, so that they can analyze and interpret in their professional contexts. (Martinez, G., 2007) Society must understand and recognize that research is important in the life of man, it is the impulse that those who are inclined to investigate need. Medical personnel have the fundamental responsibility to protect, ensure, restore and guarantee health, this mission can only be effectively fulfilled by well-trained professionals both academically and ethically, with a vocation for service and with the desire to investigate to expand their services every day, services, knowledge. That the doctor is up to date will always be a professional, moral and social obligation. 2. BACKGROUND With the evolution of technologies, man adapts to new ways of communicating, studying and researching, everyone gets involved in one way or another in this new scheme of society, the digital age has come to stay, and while it has great advantages, it also has some disadvantages. This is evident in those people who have not yet mastered technology and computing, however it is important to join the new trend to constantly update. 44 MSC. Alida Bella Vallejo López, DRA. Josefina Ramirez Amaya, MSC. Juana Kou Guzman, ING. Cesar Noboa, MSC. Oswaldo Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila Currently the world trend is network connectivity. Internet allows to know more quickly all the studies, discoveries and changes that occur every minute, knowledge evolves and science advances, everyone watches this change carefully. Despite this, few are interested in investigating. One of the highlights of information technology is that it turns out to be a very useful medium for researchers. The new era brings the creation of the "virtual university", before which some projections indicate the disappearance of the structure of the traditional university in its current configuration. The reason for this is that the "virtual university" has several advantages over the traditional one, as it encourages collaboration between universities and professors who are in remote locations, allowing students to easily access top-notch libraries from different institutions. (Akyeampong, 1998) The number of medical researchers has decreased significantly in recent years, with a significant decrease in the interest of students to continue researching in their professional lives. "There is constant global concern about the decline of so-called 'medical researchers.' Students no longer perceive a career with solid research bases as viable for their professional future” (Herrera Miranda GL, 23 (2)). International Journal of Research -GRANTHAALAYAH 3. WHY DO UNIVERSITIES NEED TO INVESTIGATE? Research and education complement each other, especially in universities; since research is one of the fundamental pillars in the training of students, even more so in the area of health, because it allows increasing scientific production. The entities that regulate the quality of education in various countries of the world demand that education should be promoted beyond the classroom, the evaluation of a training center also depends to a large extent on how much it contributes to universal knowledge. These contributions help to improve the world by discovering and exploring new ways of solving everyday problems in the social field or of a biological nature in the area of medicine. By definition, the university is the institution dedicated to higher education, (one that provides specialized knowledge of each branch of science) that is made up of several faculties and that grants the corresponding academic 45 Research and Education in The Medical Area Research and Education in The Medical Area degrees. For his part (Albert, 2007) considers that “Research in general is an activity aimed at acquiring new knowledge; regardless of what that knowledge is, it can refer to the arts, letters or knowledge”. degrees. For his part (Albert, 2007) considers that “Research in general is an activity aimed at acquiring new knowledge; regardless of what that knowledge is, it can refer to the arts, letters or knowledge”. The university is the best place to promote scientific activity. In this way, it is possible to enrich production systems, obtain updated information and attract economic resources, as well as establish scientific projects with greater risk and potential return (Nuñez & Montalvo, 2014). Universities must impact, leave a mark in a region or country, beyond graduates, and one way to do this is through research. (Mazuera Arias, R. 2016). In the health area, the Medicine career and its related careers have the obligation to know health issues, so teaching, the most relevant content and research are important for better learning. The student should not be a data bank, but a restless being, capable of questioning and solving problems (Norero V., Colomba. 2000). The development of investigative competences allows expanding the field of knowledge. In the 21st century, transformations are required to improve the educational-institutional model. International Journal of Research -GRANTHAALAYAH 3. WHY DO UNIVERSITIES NEED TO INVESTIGATE? (Gómez, J.; Diéguez, P.; Gómez 2014) Universities are committed to promoting research, governments are favored by developing technological and scientific inventions for tomorrow, (Becker & Pera Espinoza, 2019) express that “the economic success or failure of a nation no longer depends on technology or machinery that he owns, but what he does with his people”. This suggests that the population generates economic and technological income for the benefit of a nation thanks to its contribution to the people through its research and that by working on this research topic, jobs are generated, increasing the value in the productive and economic system to improve, quality of life in all areas. 4. VISION OF THE WORLD The countries' commitment must be aimed at promoting all fields of science, generating new knowledge and deepening existing ones. Any complete investigation will always be the starting point of a new investigation, expanding more and more the imagination, ideas, theories, of the phenomena that surround us. The United Nations Educational, Scientific and Cultural Organization (UNESCO, 2013), in addition to China, the European Union, Japan, the Russian Federation and the United States still account for 72% of the world's research population. While in Latin America and South Asia they concentrate 8% and 23.3% of the world population of researchers, respectively, the more or less comparable mode is: 3.6% in the first and 3.1% in the second, which represents a low rate of researchers. The National Science Foundation (National Science Board, 2018) published that the United States leads the world in research and development (R&D) spending with 496,000 million dollars (26% of the world total), followed by China with 21% ($ 408 billion). In all areas of the university educational community, research work will always be productive for those involved, and it would be much more effective if it were implemented in the curricula, throughout all university careers. It is urgent, then, to promote research as a resource for the autonomous production of knowledge, which depends not only on the creation of individual and group expertise and learning, but also on the efficient management and organization of investigative processes In developed countries there is evidence of the support given to researchers, however in developing countries much remains to be done. A change of mentality to facilitate administrative procedures such as the provision of infrastructure, in addition to financial support, this process should be seen as an investment, which will benefit the country. A change in the focus of the subjects or courses taught in undergraduate becomes necessary to achieve a consolidated course of research fundamentals, as well as the dedication of a fixed time during the undergraduate career to achieve the necessary motivation in the student and that you undertake a research project on your own (or with a suitable mentor) and ultimately stand out. (Ávila, M.J. 2014) SOURCES OF FUNDING This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. 5. CONCLUSIONS The scientific advances achieved in the new century are the result of the work carried out by researchers, in all areas of knowledge. Research in the new educational context is an indispensable factor for Higher Education Institutions, teachers must involve students in scientific activities, to strengthen the learning of future professionals. 46 MSC. Alida Bella Vallejo López, DRA. Josefina Ramirez Amaya, MSC. Juana Kou Guzman, ING. Cesar Noboa, MSC. Oswaldo Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila MSC. Alida Bella Vallejo López, DRA. Josefina Ramirez Amaya, MSC. Juana Kou Guzman, ING. Cesar Noboa, MSC. Oswaldo Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila In the medical area, there is a great responsibility to protect the life and health of the population, hence the importance of promoting a culture of researchers with ethical values, who provide new knowledge. The countries that investigate generate knowledge that benefits society with technological and economic development and progress. All countries must adopt government and financial policies to support training programs and research skills development, providing infrastructure, legal resources and incentives to researchers, which will result in the well-being of the entire population. ACKNOWLEDGMENT None. CONFLICT OF INTEREST The author have declared that no competing interests exist. The author have declared that no competing interests exist. International Journal of Research -GRANTHAALAYAH REFERENCES [1] Akyeampong, D. (octubre de 1998). La educación superior y la investigación: Desafíos y oportunidades. Conferencia mundial sobre la educación superior. UNESCO, París [1] Akyeampong, D. (octubre de 1998). La educación superior y la investigación: Desafíos y oportunidades. Conferencia mundial sobre la educación superior. UNESCO, París [2] Ávila, J.M. & Rodríguez-Restrepo, A. (Noviembre 2014). La importancia de la investigación en el pregrado de medicina. MedWave 14 (10), pp. 1-12. Recuperado de: https://www.medwave.cl/link.cgi/Medwave/Revisiones/RevisionTemas/6032 [3] Albert, M. (2007). La Investigación Educativa: Claves Teóricas. Madrid: McGraw-Hill Interamericana de España. Obtenido de: http://webcache.googleusercontent.com/search?q=cache:http://dip.una.edu.ve/mpe/022metodologiaII/le cturas/Unidad%2520I/Albert_La_Investigacion.pdf [3] Albert, M. (2007). La Investigación Educativa: Claves Teóricas. Madrid: McGraw-Hill Interamericana de España. Obtenido de: http://webcache.googleusercontent.com/search?q=cache:http://dip.una.edu.ve/mpe/022metodologiaII/le cturas/Unidad%2520I/Albert_La_Investigacion.pdf [4] Becker, G., & Pera Espinoza, C. (3 de Enero de 2019). UNINTERlingua. Obtenido de https://www.transformacion-educativa.com/index.php/articulos-sobre-educacion/54-importancia-de-la- investigacion-educativa [4] Becker, G., & Pera Espinoza, C. (3 de Enero de 2019). UNINTERlingua. Obtenido de https://www.transformacion-educativa.com/index.php/articulos-sobre-educacion/54-importancia-de-la- investigacion-educativa [5] Bijarro, Francisco (2007): "Desarrollo estratégico para la investigación científica", Cancún, <www.eumed.net/libros-gratis/2007c/306/306.zip> [15/08/2016]. [5] Bijarro, Francisco (2007): "Desarrollo estratégico para la investigación científica", Cancún, <www.eumed.net/libros-gratis/2007c/306/306.zip> [15/08/2016]. [6] Borsotti, Carlos. 1989. La situación problemática. El problema de investigación, Fichas de trabajo Universidad de Luján, Buenos Aires, Argentina. http://www.transformacion-educativa.com/biblioteca-virtual-de-educacion/18-publicaciones-periodicas- y-suplementos/54-insurgencia-magisterial-ano-1-no- 4#:~:text=investigar%20es%20un%20proceso%20por,de%20informaci%C3%B3n%20v%C3%A1lida%20 y%20confiable.%E2%80%9D [6] Borsotti, Carlos. 1989. La situación problemática. El problema de investigación, Fichas de trabajo Universidad de Luján, Buenos Aires, Argentina. http://www.transformacion-educativa.com/biblioteca-virtual-de-educacion/18-publicaciones-periodicas- y-suplementos/54-insurgencia-magisterial-ano-1-no- 4#:~:text=investigar%20es%20un%20proceso%20por,de%20informaci%C3%B3n%20v%C3%A1lida%20 y%20confiable.%E2%80%9D [7] Coelho, F. (17 de Mayo de 2019). Significado de Investigación. Significados.com. Obtenido de: https://www.significados.com/investigacion/ [7] Coelho, F. (17 de Mayo de 2019). Significado de Investigación. Significados.com. Obtenido de: https://www.significados.com/investigacion/ [8] GOMEZ, J.; DIEGUEZ, P.; GOMEZ 2014 Motivando el interés por la investigación científica en estudiantes de educación media superior Congreso Iberoamericano de Ciencia, Tecnología, Innovación y Educación D ISBN: 978-84-7666-210-6 – Artículo 353 Buenos Aires Argentina Benemérita Universidad Autónoma de Puebla. México. file:///C:/Users/uf/Downloads/353.pdf [8] GOMEZ, J.; DIEGUEZ, P.; GOMEZ 2014 Motivando el interés por la investigación científica en estudiantes de educación media superior Congreso Iberoamericano de Ciencia, Tecnología, Innovación y Educación D ISBN: 978-84-7666-210-6 – Artículo 353 Buenos Aires Argentina Benemérita Universidad Autónoma de Puebla. México. file:///C:/Users/uf/Downloads/353.pdf [9] Herrera Miranda, G. L. (Enero-Febrero 2015). ¿Por qué potenciar la formación en investigación para la integralidad del profesional de la Medicina? Revista de Ciencias Médicas de Pinar del Río 19 (1), pp. 9-12. Recuperado de: http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S1561-31942015000100004 [9] Herrera Miranda, G. L. (Enero-Febrero 2015). ¿Por qué potenciar la formación en investigación para la integralidad del profesional de la Medicina? REFERENCES Revista de Ciencias Médicas de Pinar del Río 19 (1), pp. 9-12. Recuperado de: http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S1561-31942015000100004 47 Research and Education in The Medical Area Research and Education in The Medical Area [10] Martínez, G. R. A. 2007. La investigación en la práctica educativa: guía metodológica de la investigación para el diagnóstico y evaluación en los centros docentes. Ministerio de educación y ciencia dirección general de educación, formación profesional e innovación educativa Centro de Investigación y Documentación Educativa (CIDE). Colección investigamos No 5. Edita: © Secretaría General Técnica. Madrid. [10] Martínez, G. R. A. 2007. La investigación en la práctica educativa: guía metodológica de la investigación para el diagnóstico y evaluación en los centros docentes. Ministerio de educación y ciencia dirección general de educación, formación profesional e innovación educativa Centro de Investigación y Documentación Educativa (CIDE). Colección investigamos No 5. Edita: © Secretaría General Técnica. Madrid. [10] Martínez, G. R. A. 2007. La investigación en la práctica educativa: guía metodológica de la investigación para el diagnóstico y evaluación en los centros docentes. Ministerio de educación y ciencia dirección general de educación, formación profesional e innovación educativa Centro de Investigación y Documentación Educativa (CIDE). Colección investigamos No 5. Edita: © Secretaría General Técnica. Madrid. [11] Mazuera Arias, Rina La investigación y las revistas científicas Revista Virtual Universidad Católica del Norte, núm. 47, febrero-mayo, 2016, pp. 1-3 Fundación Universitaria Católica del Norte Medellín, Colombia. http://www.redalyc.org/pdf/1942/194244221001.pdf [11] Mazuera Arias, Rina La investigación y las revistas científicas Revista Virtual Universidad Católica del Norte, núm. 47, febrero-mayo, 2016, pp. 1-3 Fundación Universitaria Católica del Norte Medellín, Colombia. http://www.redalyc.org/pdf/1942/194244221001.pdf [12] Norero V., Colomba. (2000). Medicina y ciencia: una relación indispensable. Revista chilena de pediatría, 71(2), 81-82. https://dx.doi.org/10.4067/S0370-41062000000200001 [12] Norero V., Colomba. (2000). Medicina y ciencia: una relación indispensable. Revista chilena de pediatría, 71(2), 81-82. https://dx.doi.org/10.4067/S0370-41062000000200001 https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0370-41062000000200001 [12] Norero V., Colomba. (2000). Medicina y ciencia: una relación indispensable. Revista chilena de pediatría, 71(2), 81-82. https://dx.doi.org/10.4067/S0370-41062000000200001 https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0370-41062000000200001 p // y / p p p _ p [13] Universia México. (2016). La importancia de la investigación en las tareas universitarias. Recuperado de https://noticias.universia.net.mx/cultura/noticia/2016/11/04/1145206/importanciainvestigacion-tareas- universitarias.html Padrón, J. 2007. Tendencias Epistemológicas de la Investigación Científica en el Siglo XXI p // y / p p p p [13] Universia México. (2016). La importancia de la investigación en las tareas universitarias. Recuperado de https://noticias.universia.net.mx/cultura/noticia/2016/11/04/1145206/importanciainvestigacion-tareas- universitarias.html Padrón, J. 2007. Tendencias Epistemológicas de la Investigación Científica en el Siglo XXI https://noticias.universia.net.mx/cultura/noticia/2016/11/04/1145206/importanciainvestigacion-tareas- universitarias.html Padrón, J. 2007. REFERENCES Tendencias Epistemológicas de la Investigación Científica en el Siglo XXI [14] Valdés Cuervo, A., & Estévez Nenninger, E., & Vera Noriega, J. (2013). Desarrollo de competencias científicas en estudiantes de posgrado desde la perspectiva del docente. Educere, 17 (58), 419- 430.http://www.redalyc.org/pdf/356/35630404005.pdf [14] Valdés Cuervo, A., & Estévez Nenninger, E., & Vera Noriega, J. (2013). Desarrollo de competencias científicas en estudiantes de posgrado desde la perspectiva del docente. Educere, 17 (58), 419- 430.http://www.redalyc.org/pdf/356/35630404005.pdf [14] Valdés Cuervo, A., & Estévez Nenninger, E., & Vera Noriega, J. (2013). Desarrollo de competencias científicas en estudiantes de posgrado desde la perspectiva del docente. Educere, 17 (58), 419- 430.http://www.redalyc.org/pdf/356/35630404005.pdf [15] Vallejo López, A. (2020). El papel del docente universitario en la formación de estudiantes investigadores desde la etapa inicial. Educación Médica Superior, 34(2). Recuperado de http://www.ems.sld.cu/index.php/ems/article/view/1579 [15] Vallejo López, A. (2020). El papel del docente universitario en la formación de estudiantes investigadores desde la etapa inicial. Educación Médica Superior, 34(2). Recuperado de http://www.ems.sld.cu/index.php/ems/article/view/1579 [16] Vallejo López, A., Daher Nader, J., & Rincón Rios, T. (2020). Investigación y creatividad para el desarrollo de competencias científicas en estudiantes universitarios de la salud. EducacióN MéDica Superior, 34(3). Recuperado de http://www.ems.sld.cu/index.php/ems/article/view/1606/1033 [16] Vallejo López, A., Daher Nader, J., & Rincón Rios, T. (2020). Investigación y creatividad para el desarrollo de competencias científicas en estudiantes universitarios de la salud. EducacióN MéDica Superior, 34(3). Recuperado de http://www.ems.sld.cu/index.php/ems/article/view/1606/1033 48 International Journal of Research -GRANTHAALAYAH
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Community Radio for cohesion and integration during Kenya’s 2017 post-election violence: A study of Migori County
African social science and humanities journal
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African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj *Corresponding author *Corresponding author Abstract: Kenya’s elections are mostly marred with violence, especially after announcements of presidential results. Peace and media experts on the other hand have blamed Kenyan media for fueling cases of post-poll conflicts. It is in this understanding that this study assessed the contents of community radios in Migori County to investigate the role they played in promotingcohesionandintegrationduringthe2017Kenya’spost-election violence. The research, which was conducted in Migori town, studied five radio stations namely; Milambo, Onagi, Anyuola, Rameny and Tarumbeta which are located within Migori County. The specific objectives were: to assess the extent to which the 2017 post-election violence affected residents of Migori town; to establish if the five stations developed contents that promoted cohesion and integration during the violence and to assess the extent to which the contents aired on the radio stations promoted cohesion and integration among residents of Migori town. Study was a survey that utlilised mixed research approach where qualitative and quantitative methods of data collection were used. Systematic random sampling was employed to administer 399 questionnaires. For qualitative data, 21 key informants were purposeively selected and interviewed. The quantitative data collected was analysed using SPSS while qualitative data was analyzed using content analysis. From the study it was found and concluded that Community Radio, if professionally used, promotes cohesion and integration during post-poll conflicts. The study has recommended that in order to reduce incessant post-poll violence in Kenya, the relevant stakeholders should work together to air contents that enhance cohesion and integration in order to curb violence recurrence. Policies should be developed and funding availed to help facilitate matters media and peace. ) Article detail Received: 27 September 2021 Accepted: 29 December 2021 Published: 12 January 2022 Conflict of Interest: The author/s declared no conflict of interest. Community Radio for cohesion and integration during Kenya’s 2017 post-election violence: A study of Migori County This article is published by JFP Publishers in the African Social Science and Humanities Journal (ASSHJ). Volume 3, Issue 1, 2022. ISSN: 2709-1309 (Print) Nicholas Anyuor1*, Rosechieng’2 1&2Communication, Journalism and Media Studies, Rongo University, Kenya, nanyuor@gmail.com1, achijebet@gmail.com2 This article is distributed under a Creative Common Attribution (CC BY-SA 4.0) International License. Article detail Received: 27 September 2021 Accepted: 29 December 2021 Published: 12 January 2022 Conflict of Interest: The author/s declared no conflict of interest. Conflict of Interest: The author/s declared no conflict of interest. Keywords – Community Radio, Integration and cohesion, Post-election violence, Vernacular radio African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj 1. INTRODUCTION Media are powerful influencers that play crucial role in shaping opinions of members of the public and their world views (Mcombs, 1994); ‘the media therefore through various forms of programmes provide a space where societies can dialogue and articulate solutions to problems that easily trigger conflicts among them and promote peaceful co-existence’(Mwangi,2017: 1). African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |43 African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj https://journals.jfppublishers.com/asshj Role of media, particularly community radio in promoting peace, has been witnessed globally. In Columbia, for example, community radio was included in peace-building initiatives after the signing of peace agreement between the Colombian State and the FARC EP guerrillas (Vega et al 2019) Role of media, particularly community radio in promoting peace, has been witnessed globally. In Columbia, for example, community radio was included in peace-building initiatives after the signing of peace agreement between the Colombian State and the FARC-EP guerrillas (Vega et al., 2019). Since 1997, with Decree 1447, the Colombian State had said that community radio should be a space for community reflection, consultation, and conflict resolution in the country. As a result, during the last 20 years, community radio stations emerged and generated popular and citizen reflections on peace and conflict, which translated into communicative processes with the capacity to foster diversity, intercultural dialogue, and tolerance. As a consequence, community media became privileged partners in conflict resolution and reconciliation (Pereira & Cadavid, 2011 in Vega et al., 2019: 1392). In Africa, Mwangi (2017) argues, the influence of radio has continued to grow and has been utilised in peace building process and in conflict resolution. Community radios have been used in peace building in some war-torn countries. This has led to attainment of certain level of cohesion and integration among the people in the regions. In countries like Burundi, Rwanda, Kenya, among others, community radios have been accused of being destructive,butatcertainpointtreasuredfortheirconstructivecontents.In,urundi,forinstance,Ijambostudio’s role in promoting cohesion among the people cannot be gainsaid. It brought together journalists from different tribes who developed peace-building programmes to restore order and integration in this country (Gilboa, 2002 in Mwangi, 2017). 1. INTRODUCTION The setting up of studio Ijambo in 1995 saw a team of journalists drawn from Hutu and Tutsi ethnic groups initiate programs aimed at promoting dialogue, reconciliation and peaceful co-existence…this enabled the two tribes reach a common ground in the conflict that afflicted the region (Gilboa, 2002 in Mwangi, 2017: 1). In Kenya, the need to have community and vernacular radios made the government to liberate airwaves, offering invaluable opportunities for the opening of many radio stations-the commercial radios, faith-based radios, among others. Some of these stations have also been accused of fueling violence while others have been promoting peace among conflicting parties. For example, KASS FM, a vernacular radio station in the Rift Valley region, was accused of fueling 2007/08 post-poll violence after by introducing partisan programming (Waki et al., 2008). On the other hand, some radios developed peace-builiding programmes in efforts to restore cohesion and integration in Kenya after the 2007/08 post-election violence. Community-cum-faith-based Radio Amani, based in Nakuru County, developed peace-building programme that ‘provided a platform for peace building through community participation’(Wakietal.,2008:2). Therefore, in asmuchas community radios can be destructive, they can also be used to air contents that promote peace as has been witnessed in different parts of the world. It is in this background that this study assessed the role community radio played in promoting cohesion and integration during the 2017 post-election violence in Kenya. 2. LITERATURE SURVEY 2. LITERATURE SURVEY 2.1. Kenya’s 2017 post-election violence 2.1. Kenya’s 2017 post-election violence 2.2. The impact of Community Radio in the society Communication has been critical in the society as it is an ingredient that fuels development in all aspects. To communicate to wider audience, mass media such as radio, are invaluable. Radio communication ensures that information reaches the right and the relevant audience and offers opportunity for the people to exchange ideas. Community radio is important in the society as it offers communication services beyond commercial; not-for profit and acts as a watchdog (Seidu, 2011). In some countries such as Australia, community media have changed the societies into positive thinking about themselves and community at large. This was evident in the response of ustralia’sindigenouscommunities to the contents of their community media-as viewed by the Aboriginal and Torres Strait Islander people who trusted community media as a player of central role in organizing lives of the local people (Romano, 2010). These indigenous audiences, accordingtoRomano(2010),describetheircommunitymediaas‘aprimarysource of news and information that helps to sustain social networks, promote social self-esteem, nourish community identities and kinship ties, maintain language and culture, educate people and support cultural dialogue’ (Meadows, et al., 2007: 51-68 in Romano, 2010: 23). It is through community radio stations that in some countries such as South Africa, public journalism has been promoted, even though licenses here are given with stringent rules (Broadcasting Act No. 4 of 1999 in Davidson, 2020). This kind of framework makes community radios have their missions and identities that are different from public and commercial broadcastings. In Nigeria, community radios that have been established have spurred development and understanding among the people towards the good of the communities. It is argued that even though the Nigeria’s population is over 150 million, newspaper circulation is low and the papers majorly use English, a language which is not understood by many in this West Africa State; leading to increasing influence of radio stations where, for example, Radio Nigeria claims more than 100 million listeners while Raypower FM claims to reach 23 million listeners (UNESCO, 2008 in Awoshakin, 2020). This kind of proliferation of community radio outlets allowed journalists to play closer attention to community issues where they communicate these in their local languages and therefore, effectively engaging local communities (Awoshakin, 2020). 2.2. The impact of Community Radio in the society Awoshakin (2020) argues that this scenario has been possible for the reporting of health concerns and political engagements; that through community radios, journalists are in positiontoreportthe‘issues that the local communities were wrestling with, brought local people into the studios to have conversations with politicians, and broadcast these conversations in local languages’ (woshakin, 2010: 57). By adhering to this globally, the community radios have improved development initiatives at grassroot levels where they serve (Seidu, 2011). In East Africa, even though community radios have been sources of violence in some countries such as Rwanda, according to Ogenga (2020), they can also be used to mobilise and educate people on how to share resources in efforts to avoid conflicts. For example, a successful peace journalism strategy thatendedorratherreducedtheLord’sResistancermy conflict has been witnessed in Uganda sometime. Ugandan military resorted to use of radio after failed military interventions. The military established Radio Freedom for peace. This initiative drove away fighters, including child soldiers, from LRA back to their homes and handing over the firearms to the government (Laker, 2020). This means community radios are powerful and can be used to influence and shape communities. Therefore, it is on this note that this study was conducted to assess the impact of community radio in cohesion and integration during the 2017 post-election in Migori, Kenya. 2.1. Kenya’s 2017 post-election violence ThevotingprocessofKenya’s2017electionswaspeacefuluntilUhuruKenyattawasdeclaredwinnerby3lectoral body (Mutahi, 2019). Opposition supporters from different regions including Migori prtested Uhuru’s victory. Police then acted to quell the violence (Humann Rights Watch, 2017); however, they were using force and live ammunitions and many people in these regions became victims (Mutahi, 2019). A lot of crimes such as sexual violence were committed during this poll violence not only by locals but also by State agents such as police officers (Reliefweb, 2021). According to Muhumuza (2017), 92 people were killed during the months of election violence in 2017 while 86 cases of sexual or gender-based violence were reported. African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |44 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj 2.2. The impact of Community Radio in the society 2.4. Impact of Community Radio in cohesion and integration TheconceptofmediaandpeaceorratherpeacejournalismwasdevelopedbyascholarJohan7altung’in1960s (Youngblood, 2020). Youngblood (2020) argues that journalism that promotes cohesion should focus on the importance of language that does not victimize other parties. This focus of using media in promoting peace would address conflicts that have been witnessed across the globe. Youngblood (2020) accuses Kenyan media of playing negative role when it comes to political conflict; giving an example with 2008/2007 election violence, arguing that media was ill prepared to cover it. Ogenga (2020), however, argues that community radio can be good media outlets or platforms for peace building and cohesion. He asserts that community radios can perform this act of enhancing cohesion in the society since they ‘are owned by the community and serve the interest of the community’(Ogenga,2020: 23). It is in this backdrop that community radio can be used to promote cohesion and integration among the people. But Ogenga (2020) avers that for community radio to succeed in matters cohesion and integration, members of the public must be involved in discussing matters peace using language that is understood well by the community. It is through these radios that locals can easily be mobilized and educated on the importance of peace and on sharing of natural resources to avoid conflicts. 2.3. Community Radio in Kenya In Kenya, community radio can also be referred to as ‘rural radio’, ‘people’s radio’, (Ochichi, 2013). Kenya produced the first Community Radio (CR), the Homa Bay CR in May 1982 in the current Homa Bay County, then Nyanza Province (Quarmyne, 2006). Currently, community based radio stations in Kenya have increased in African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |45 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj number from 10 in 1999 to over 120 in 2015 (Oluoch, 2020). Oluoch (2020) argues that such stations give invaluable local broadcasting where matters development are addressed. African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj number from 10 in 1999 to over 120 in 2015 (Oluoch, 2020). Oluoch (2020) argues that such stations give invaluable local broadcasting where matters development are addressed. 3. PROBLEM STATEMENT Political violence has not only been witnessed in Kenya, but it has been a global concern. Media on the other hand, regardless of the outlet, have been accused of fueling such violence. For example, in Rwanda, media outlets-radio and newspapers-were blamed for fueling the 1994 Genocide that left almost one million people dead in a span of 90 days.Kenya’s2007/2008post-election violence never went without a blame on the media especially community radios. On the other hand, vernacular and community radios’ contents have been utilised in promoting peace among warring communities worldover. This was witnessed in Uganda to curb the Lord Resistance rmy’s incessant conflict. In 2017, Kenyans witnessed post-election violence where some people were killed and others displaced, and counties such as Migori were classified as hotspots. But eventually peace and harmony were restored. Was the community radio one of the actors used to promote peace, especially in Migori County? There is no single study that has been conducted to find out the role community radios played in peace-building process during 2017 post-electionviolenceinKenya’sMigoriounty.Thisisthegapthestudyhasfilledafterinvestigating the role five community radios based in Migori County played in promoting cohesion and integration in Migori during the 2017 post-poll violence. Key findings y g The first research question was to find out the extent to which the 2017 post-election violence affected residents of Migori town. To answer this, we compared percentage of responses from 399 questionnaires and from 21 key informants. The tables below show various results. y g The first research question was to find out the extent to which the 2017 post-election violence affected residents of Migori town. To answer this, we compared percentage of responses from 399 questionnaires and from 21 key informants. The tables below show various results. Table 1: Are you a resident of Migori town? Responses Frequencies Valid Percent (%) Yes 310 80.9 No 73 19.1 Total 383 100.0 Table 2: Did you experience the effects of 2017 post-election violence in Migori town? Responses Frequancies Valid Percent (%) Yes 266 69.5 No 117 30.5 Total 383 100 Table 1: Are you a resident of Migori town? Responses Frequencies Valid Percent (%) Yes 310 80.9 No 73 19.1 Total 383 100.0 Table 2: Did you experience the effects of 2017 post-election violence in Migori town? Responses Frequancies Valid Percent (%) Yes 266 69.5 No 117 30.5 Total 383 100 5. DATA ANALYSIS AND DISCUSSIONS 5.1. The key findings and analysis This study used interview schedules and questionnaires as tools of data collection. A total of 399 questionnaires were administered to residents of Migori town. Out of the 399 questionnaires, 383 were filled and returned. This is 96% of the respondents, which is enough population for a study. 21 interviews were conducted where media, cohesion and integration, and human rights stakeholders were selected as the key informants. Out of this, 19 key informants responded; giving 95%, which is good response for research. This study looked at the contents of five community radio stations in Migori County, namely; Milambo, Onagi, Anyuola, Rameny and Tarumbeta. From the study, the quantitative data results were as follow: 4. RESEARCH METHODOLOGY 4. RESEARCH METHODOLOGY The study used mixed research approach where both quantitative and qualitative data collection methods were used. It employed systematic random sampling to administer 399 questionnaires from a population of 71,660 residentsofMigoritown.SamplesizewascalculatedusingYamane’sformulatoget399respondentsfrom71,660 residents of Migori town, according to Census Report of 2019. For qualitative data, 21 key informants, purposively selected from relevant stakeholders, were interviewed. They were drawn from the newsrooms of the community radio stations and peace stakeholders in Migori County. Migori County was selected for the study since it had been identifiedasoneofthehotspotsduringthe2017pollclashes,accordingtoreportpublishedbyhina’sXinhuain 2018; and is also a cosmopolitan county (Star, April 14, 2017) that is comprising different tribes and races such as Asia, Somali, Luhyia, Kuria, Kikuyu, Kisii, among others and therefore, call for peace, cohesion and integration during this period was crucial. The investigated the contents of five radio stations namely; Milambo, Onagi, Anyuola, African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |46 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj Rameny and Tarumbeta, because they are the main community radios in Migori County with wider coverage of the county and Migori town. Rameny and Tarumbeta, because they are the main community radios in Migori County with wider coverage of the county and Migori town. First analysis y Based on the responses from the participants, 69.5% (266 respondents) were affected by the post-election violence in Migori town while 117 respondents (30.5%) disagreed. From qualitative data, 19 key informants responded from a total of 20. This is a good response of 95% for research. From the 19 key informants, 17 (89.5%) agreed that indeed they were extremely affected by the 2017 post-election violence in Migori town. However, 10.5 % (Two key informants) said people were affected but mildly. During the interviews, participant A, a journalist in Migori with one of the community radio stations under study, averred that the violence indeed affected residents of Migori town. He gave an example with the People Living with Disabilities (PLWDs), women and children who were ‘badlyaffectedbythesituation.’ I did news stories about PLWDs to highlight their plight. The blind and the crippled were extremely affected. The blind could not see, the crippled could not run away from the danger. We also ran stories about children and women who were most vulnerable during the violence. It was sad that even some children were shot by police, and women and girls gang raped by State agents. The situation was bad here (Participant A, March 14, 2021). I did news stories about PLWDs to highlight their plight. The blind and the crippled were extremely affected. The blind could not see, the crippled could not run away from the danger. We also ran stories about children and women who were most vulnerable during the violence. It was sad that even some children were shot by police, and women and girls gang raped by State agents. The situation was bad here (Participant A, March 14, 2021). The comment concurs with literature by many scholars and reports by several agencies about the 2017 post-election violence in Kenya. Mutahi (2019) argues that people from Kisumu, Migori, Homa Bay, Mathare, Kibra, Kisii were heavilyaffectedbytheprotestsafterUhuruKenyattawasdeclaredwinneroftheugust8,2017election,‘with24 The comment concurs with literature by many scholars and reports by several agencies about the 2017 post-election violence in Kenya. Mutahi (2019) argues that people from Kisumu, Migori, Homa Bay, Mathare, Kibra, Kisii were heavilyaffectedbytheprotestsafterUhuruKenyattawasdeclaredwinneroftheugust8,2017election,‘with24 ican Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online |47 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj fatal police shootings confirmed by Human Rights organisations during thosefatefulday’(Mutahi,2019: 5). First analysis In a report by International Federation for Human Rights (FIDH) and the Kenya Human Rights Commission (KHRC) in Reliefweb (2021), sexual violence was witnessed in Migori during the 2017 violence. According to a rape victims interviewed by FIDH in Migori town, police officers were also rape agents. Therefore, the 2017 post- election violence in Kenya affected people of Migori town differently. y p p g y The second research question sought to establish if the five radio stations in the study developed contents that promoted cohesion and integration among residents of Migori town during the 2017 Post-Election Violence. Table 3: Did you listen to any of the following radio stations based in Migori County, during the 2017 post-election vio Anyuola, Rameny and Tarumbeta? Table 3: Did you listen to any of the following radio stations based in Migori County, during the 2017 post-election violence? Milambo, Onagi, Anyuola, Rameny and Tarumbeta? Responses Frequencies Percentage (%) Yes 300 78.3 No 83 21.7 Total 383 100 From the results, 78% (300 respondents) listened to at least one of the named radio stations during the 2017 post- election violence. The listenership was as indicated below: Name of the station Percentage (%)/100% Milambo FM 50 Onagi Radio 31 Anyuola FM 8 Tarumbeta 6 Rameny 5 The third research question was on how the contents aired on the five community radio stations promoted cohesion and integration among residents of Migori town during 2017 Post-Election violence. The results from the questionnaires and interviews conducted during the study were as follow: _____________________________________________________________________________________________ Table 4 (i): Did this station (identified in b above) have programmes and news items on matters peace? Responses Frequencies Percentage (%) Yes 280 73 No 103 27 Total 383 100 Table 4 (ii): If yes, in 4 (i), were the contents geared towards cohesion and integration among residents of Migori town? Responses Frequencies Percentage (%) Yes 250 65 No 133 35 Total 383 100 Table 4 (iii): If yes in 4 (ii) above, to what extent if you were to rate between 1 (Unsatisfied) and 10 (Very Satisfied), did the contents aired impact peace, cohesion and integration on residents of Migori? Responses Frequancies Percentage (%) Unsatisfied 23 6 @on’tknow 32 8.4 Satisfied 256 66.8 Very Satisfied 72 18.8 Total 383 100 Table 4 (i): Did this station (identified in b above) have programmes and news items on matters peace? Second analysis These were busy days for us and we also gave bonus/extra airtime for the paid shows as way of partnering with communities and peace agencies (Participant C, April 18, 2021). Some communities such as Luhya, Kuria, Luo, which are the majority in Migori County, also organized peace programmes that were aired on our radio almost on a daily basis. Morning hours we invited religious leaders to pray and offer spiritual guidance to people on matters peace. These were busy days for us and we also gave bonus/extra airtime for the paid shows as way of partnering with communities and peace agencies (Participant C, April 18, 2021). These versions agree with the opinions of Media and Peace scholars who argue that it is the role of the community radios to promote peace in the society and to enhance cohesion and integration during times of violence. Ogenga (2020) observes that community radio can play imperative role in conflict resolution and peace-building in the society. He avers that for community radio to effectively perform its role, members of the said community should be engaged in discussing matters peace. Through this, these members find forum to engage and educate themselves on the importance of peace within the community and society at large. In table 4 (ii), the result shows that the contents aired were majorly aimed at promoting cohesion and integration. From the data collected by use of questionnaires, 250 respondents (65%) agree that the stations aired contents that were geared towards cohesion and integration, while 133 respondents (35%) disagreed. This again concurs with the results of qualitative data from key informants where all the 19 respondents (100%) agreed that the stations aired peace contents during the 2017 post-poll violence in Kenya. Participants D & E who work with peace organisations within Migori County argued that as organisations, they were invited by the community radio stations during talk shows to discuss matters living together; co-exitence among residents of Migori. Even though they were invited by the stations, at times they would pay for airtime to address members of the public on issues cohesion and integration. Yes we partnered with these radio stations. They invited us to specifically engage people on matters cohesions and integration. First analysis Responses Frequencies Percentage (%) Yes 280 73 No 103 27 Total 383 100 Table 4 (iii): If yes in 4 (ii) above, to what extent if you were to rate between 1 (Unsatisfied) and 10 (Very Satisfied), did the contents aired impact peace, cohesion and integration on residents of Migori? Responses Frequancies Percentage (%) Unsatisfied 23 6 @on’tknow 32 8.4 Satisfied 256 66.8 Very Satisfied 72 18.8 Total 383 100 African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |48 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj Second analysis Table 4 (i) shows that 73% of the respondents (280 respondents) agreed that the five radio stations aired contents that were promoting peace in Migori town during the post-poll violence of 2017 while 103 respondents (27%) denied. This corroborates what was found during the key informants interviews where 15 key informants (78.9%) agreed that the contents aired promoted peace. 2 key informants (10.5%) disagreed. Participants B&C, whoareprogrammes’editorsinoneofthefiveradiostations studied agree that during the post-election violence of 2017, the radio station that they work for aired many programmes and ran several news stories about the violence and peace. They remarked that they developed initiative where political, community and religious leaders were invited to free talk-shows to address local communities on the need for peace. g p We had many programmes on cohesion and integration for the people of Migori. We invited political, religious and community leaders during talk-shows to talk to people about the need for peace, cohesion and integration among themselves. It actually worked well for us. I hosted a number of talk-shows; both free and paid ones (Participant B, April 18, 2021). We had many programmes on cohesion and integration for the people of Migori. We invited political, religious and community leaders during talk-shows to talk to people about the need for peace, cohesion and integration among themselves. It actually worked well for us. I hosted a number of talk-shows; both free and paid ones (Participant B, April 18, 2021). Even though these stations hosted free talk-shows and ensured those invited were from different communities and backgrounds, some peace organisations paid for the airtime during the talk-shows to have discussions with people about peace. However, these radio stations still offered extra airtime for free for such programmes. Even though these stations hosted free talk-shows and ensured those invited were from different communities and backgrounds, some peace organisations paid for the airtime during the talk-shows to have discussions with people b t H th di t ti till ff d t i ti f f f h about peace. However, these radio stations still offered extra airtime for free for such programmes. Some communities such as Luhya, Kuria, Luo, which are the majority in Migori County, also organized peace programmes that were aired on our radio almost on a daily basis. Morning hours we invited religious leaders to pray and offer spiritual guidance to people on matters peace. African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) Second analysis However, sometimes we as the organisations would buy airtime to have our own related programmes run on air; though this also sometimes proved difficult when there were had no enough funding (Participant D, April 17, 2021). Participant E argued that the conflict situation was so extreme that at times they would set an agenda of consoling the victims. This means they would send messages to particular families they knew had been affected by the violence and educate them, and other vulnerable ones on what to do in such scenarios. We went to the extent of consoling the victims we knew. During our talk-shows and while on air we would also educate them on how to handle their specific situations and caution them. We also reached out to the relatives of the victims to inform them about the loss because some were not aware (Participant E, April 17, 2021). African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |49 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj This kind of spreading messages of cohesion and integration during violence is one of the key roles played by the media, moreso community radios. For example, such strategy was witnessed in Uganda sometime back during the Lord’sResistancermyconflicts. Military intervention failed and so a community radio, Radio Freedom, was started in7ulutownin1998,andlater,a‘numberoffightersslowlybeganescapingfromtheLRcamps-a positive step in the search forpeace’(Laker,2020:16-17). In table 4 (iii), 328 respondents (85.6%) were either satisfied or were very satisfied with the kind of cohesion and integration messages that were passed through the five radio stations, while 55 respondents (14.4%) were not satisfied (6%) or didn’tknow(8.4%). From the qualitative data, 17 key informants (89.5%) were satisfied with the contents while 2 (10.5%) said they felt not satisfied; that more would have been done to enhance cohesion and integration. Participant F, for example, a programme host in one of the stations under study, said that during their evaluations through in-calls, they realized many locals were satisfied with the kind of cohesion and integration messages the particular radio station was passing across. Some confessed that locals were changing their minds into embracing each other in the villages after listening to the programmes on the radios: This violence went far. People resorted to settling their political scores around. 7. CONTRIBUTIONS TO SCIENTIFIC, COMMUNITY AND FUTURE RESEARCH This is a social science paper whose findings are key in finding peaceful way of life of a community as a result of effective and proper use of community radio in promoting cohesion and integration. It reveals the destructiveness and constructiveness of community radios and gives scientific recommendations that when properly and effciently implemented will be of great significance to communities and the society at large. By doing this, this research spurs majorly Sustsianable Development Goal 16 that talks about need for Peace, Justice and Strong Instituions. More related study can be conducted to find out if social media can be utilised in a communal way by members of communities on matters peace, cohesions and integration. Second analysis But through our programmes and talks about the need to live together peacefully, many confessed to us that they were engaging in political strategies for peace. They were changing their mindsets (Participant F, April 17, 2021). This is similar to what was evident with the military radio, the Radio Freedom in Gulu, Uganda whose impact was felt when the LRA conflict ended (Laker, 2020). Therefore, community radio stations in Migori County in one way or the other promoted peace, cohesion and integration among residents of Migori town during the 2017 post- election violence in Kenya. 6. RESEARCH IMPLICATIONS 6. RESEARCH IMPLICATIONS States and global peace agencies should fund establishment of peace community radios in conflict-prone areas to air contents that aim at promoting peace, cohesion and integration. Policies and guidelines should be developed on how community radios worldover can engage communities they are serving on issues of cohesion and integration. Journalists and other media practitioners should be trained on peace journalism at all costs. Political, religious and community leaders should be actively engaged in peace initiatives and trained on how to handle media on matters cohesion and integration. Community radios can also have contents on resource allocation and good governance in efforts to enhance harmony and integration among the community members they are serving. 9. FUNDING 9. FUNDING This research paper didn’treceiveanyinternal or external funding. References Awoshakin, T. (2010). Civic Journalism Initiatives in Nigeria. In Romano, A. (Ed). International Journalism and Democracy: Civic Engagement Models from Around the World. New York: Routledge Tylor & Francis Group. Colin, F. & Sonia, R. (2002). Community Radio for Change and Development. New Delhi: SAGE Publications. Davidson, B. (2010). Public Journalism in South Africa: Experiences and Experiments with Local and Community Media. In Romano, A. (Ed). International Journalism and Democracy: Civic Engagement Models from Around the World. New York: Routledge Tylor & Francis Group. Forges, A. D. (2007). Call to Genocide: Radio in Rwanda, 1994. In Thompson, A. (Ed). The Media and Rwanda Genocide. Kampala: Fountain Publishers. Forges, A. D. (2007). Call to Genocide: Radio in Rwanda, 1994. In Thompson, A. (Ed). The Media and Rwanda Genocide. Kampala: Fountain Publishers. Human Rights Watch. (2017). Kenya: Post-Election Killings, Abuse: Investigate Police Use of Excessive Force: Uphold Right to Peaceful Protest. Available at: https://www.hrw.org/news/201708/27/kenya-post-election- killings-abuse. Laker, G. (2020). Peace Journalism in the LRA Conflict. In Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual for Media Practitioners. London & New York: Routledge Tylor & Francis Group. Maweu, J. (2020). The Conversation. Nairobi. Available at: https://the conversation.com/how-community-radio- has-contributed-to-building-peace-a-kenyan-case-study-416222. Muhumuza, R. (2017). Kenya Watchdog says 92 people killed in Election Violence. Available at: https://apnews.com/article/6c6862192c48c16992653a4972a3c3 Muhumuza, R. (2017). Kenya Watchdog says 92 people killed in Election Violence. Available at: https://apnews.com/article/6c6862192c48c16992653a4972a3c3 Mutahi, P. (2019). Violence, SecurityandthePolicingofKenya’s20173lections.Journalof3asternfricanStudies 13(2). 1-19. Available at: https://www.researchgate.net/publication/331793528-violence-security-and-the- policing-of-kenya’s-2017-elections p g y Ochichi, J. P. (2013). Assessment of Community Radio in the Context of Rural Development in Kenya. Nairobi: University of Nairobi. Ogenga, F. (2020). Thinking About Community Radio and Beyond for Conflict Management in the North Rift. In Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual for Media Practitioners. London & New York: Routledge Tylor & Francis Group. Oluoch, J. (2020). Re-Situating Local Mass media: A Tool for Peacebuilding among the Abakuria in Kenya. In Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual for Media Practitioners. London & New York: Routledge Tylor & Francis Group. g y p Quarmyne, A.T. (2006). From Homa Bay to Ada: Lessons on Community Radio, Access and Participation. A paper presentedattheWorldPressCreedom@ayonference,“Media,@evelopmentandPoverty3radication.”Sri Lanka. Reliefweb. (2021). Kenya: Sexual Violence Committed during 2017 Elections must be Accounted for in the Run-up to 2022 Elections. A Report by International Federation for Human Rights & Kenya Human Rights Commission. Available at: https://reliefweb.int/report/kenya/kenya-sexual-violence-committed-during-2017- Romano, A. (2010). 8. CONCLUSION The research findings indicate how important community radios are in promoting peace, cohesion and integration. Through these radios, programmes and talk-shows can be organized to discuss need to peaceful co-existence with each other. Again, it is through community radios that local people can be engaged to address each other, and engage one another in enhancing peace, cohesion and integration. Apart from such messages and engagements, community radios can inform and educate people on how to handle conflicts, resource allocations and sharing, and African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |50 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj how to channel information to the relevant authorities. Therefore, community radios are key in promoting cohesion and integration for a better governance. Milambo, Anyuola, Tarumbeta, Onagi and Rameny community radios played role in promoting cohesion and integration in Migori County during 2017 post-election violence. African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj how to channel information to the relevant authorities. Therefore, community radios are key in promoting cohesion and integration for a better governance. Milambo, Anyuola, Tarumbeta, Onagi and Rameny community radios played role in promoting cohesion and integration in Migori County during 2017 post-election violence. how to channel information to the relevant authorities. Therefore, community radios are key in promoting cohesion and integration for a better governance. Milambo, Anyuola, Tarumbeta, Onagi and Rameny community radios played role in promoting cohesion and integration in Migori County during 2017 post-election violence. African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) References Deliberation and Journalism. In Romano, A. (Ed). International Journalism and Democracy: African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |51 African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj Civic Engagement Models from Around the World. New York: Routledge Tylor & Francis Group. Seidu, A., Alhassan, A., & Abdulai A. (2011). The Role of Community Radio in Livelihood Improvement: The Case of Simli Radio. The Journals of field actions. 5, 2011. Available at: https://journals.openedition.org/factsreporting/869 The Star. (April 14, 2017). Migori is Cosmopolitan County. The Xinhua. (2017). Over 150 Kenyans injured in Post-Election Violence Chaos: Agencies. Available at: https://www.xinhuanet.com/english/2017-08/13/c-136522704.htmhinhuaNet Waki, P., & McFadyen, G. (2008). Report from the Commission of Inquiry into Post-Election Violence. Nairobi. Youngblood, S. (2020). Peace Journalism Approach. In Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual for Media Practitioners. London & New York: Routledge Tylor & Francis Group. African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj Civic Engagement Models from Around the World. New York: Routledge Tylor & Francis Group. Seidu, A., Alhassan, A., & Abdulai A. (2011). The Role of Community Radio in Livelihood Improvement: The Case of Simli Radio. The Journals of field actions. 5, 2011. Available at: https://journals.openedition.org/factsreporting/869 The Star. (April 14, 2017). Migori is Cosmopolitan County. The Xinhua. (2017). Over 150 Kenyans injured in Post-Election Violence Chaos: Agencies. Available at: https://www.xinhuanet.com/english/2017-08/13/c-136522704.htmhinhuaNet Waki, P., & McFadyen, G. (2008). Report from the Commission of Inquiry into Post-Election Violence. Nairobi. Youngblood, S. (2020). Peace Journalism Approach. In Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual for Media Practitioners. London & New York: Routledge Tylor & Francis Group. African Social Science and Humanities Journal (ASSHJ) https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |52
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Cross-sectional associations between multisensory impairment and brain volumes in older adults: Baltimore Longitudinal Study of Aging
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www.nature.com/scientificreports www.nature.com/scientificreports Cross‑sectional associations between multisensory impairment and brain volumes in older adults: Baltimore Longitudinal Study of Aging OPEN Sensory impairment and brain atrophy is common among older adults, increasing the risk of dementia. Yet, the degree to which multiple co-occurring sensory impairments (MSI across vision, proprioception, vestibular function, olfactory, and hearing) are associated with brain morphometry remain unexplored. Data were from 208 cognitively unimpaired participants (mean age 72 ± 10 years; 59% women) enrolled in the Baltimore Longitudinal Study of Aging. Multiple linear regression models were used to estimate cross-sectional associations between MSI and regional brain imaging volumes. For each additional sensory impairment, there were associated lower orbitofrontal gyrus and entorhinal cortex volumes but higher caudate and putamen volumes. Participants with MSI had lower mean volumes in the superior frontal gyrus, orbitofrontal gyrus, superior parietal lobe, and precuneus compared to participants with < 2 impairments. While MSI was largely associated with lower brain volumes, our results suggest the possibility that MSI was associated with higher basal ganglia volumes. Longitudinal analyses are needed to evaluate the temporality and directionality of these associations. Keywords  Sensory, Neuroimaging, Brain aging, Olfactory impairment Keywords  Sensory, Neuroimaging, Brain aging, Olfactory impairment Sensory impairment in the form of hearing and/or vision loss, proprioceptive impairment, lower vestibular function, and impaired olfaction is prevalent among older ­adults1,2. Among older Americans, an estimated 33% have hearing ­impairment3, 24% have olfactory ­impairment4, and 18% have vision ­impairment3,5. Importantly, prior research suggests that sensory impairments might be an early sign of cognitive ­decline6,7 and ­dementia8. p gg y p g y g g Studies have linked multiple sensory impairments (multisensory impairment [MSI]) to cognitive and physi- cal ­decline9,10 and increased risk of mortality among older ­adults11. Emerging evidence show an association between single sensory impairments and altered brain ­structure12,13. Yet, whether the co-occurrence of these sensory impairments leads to lower volumes in each brain region or additional surrounding brain regions remains unclear. With neuroimaging evidence linking brain atrophy and cognitive ­change14, identifying sen- sory impairments or combinations of MSI associated with brain structure could provide novel mechanistic insights and modifiable or treatable targets involved between multiple sensory loss and cognitive impairment and dementia risk.h This study examined whether single and multiple sensory impairments are associated with relative brain volumes across several regions among cognitively unimpaired older adults. Though this study is exploratory, we hypothesized that a greater number of sensory impairments would be associated with smaller brain volumes and as a corollary larger ventricular space. 1Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. 2Center on Aging and Health, Johns Hopkins University, 2024 E. Monument Street, Suite 2‑700, Rm 2‑726, Baltimore, MD 21205, USA. 3Intramural Research Program, National Institute on Aging, Baltimore, MD, USA. 4Department of Health and Community Systems, University of Pittsburgh School of Nursing, Pittsburgh, PA, USA. 5Department of Otolaryngology, Johns Hopkins School of Medicine, Baltimore, MD, USA. *email: awaniga1@jhu.edu | https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 www.nature.com/scientificreports/ Methods Participants Participants were enrolled in the Baltimore Longitudinal Study of Aging (BLSA), an ongoing longitudinal cohort study conducted by the National Institute on Aging Intramural Research ­Program15. The BLSA recruits par- ticipants aged 20 years and older with no major physical or cognitive impairments or chronic diseases, except controlled hypertension. At each visit, participants undergo comprehensive physical, cognitive, sensory assess- ments, along with neuroimaging exams. Visits are scheduled every 1–4 years, depending on age (participants under age 60 visited every 4 years, those aged 60–79 years old visited every 2 years, and those aged 80 and above visited annually). y Eligibility criteria for this cross-sectional study include: (1) aged 50 years or older and (2) free of mild cogni- tive impairment or dementia based on a two-step assessment of neurocognitive testing. First, neurocognitive data of participants were analyzed, and cognitive impairment was defined if their Clinical Dementia Rating score were ≥ 0.516 or if they had > 3 errors on the Blessed Information-Memory-Concentration ­test17. Second, the diagnosis of dementia and Alzheimer’s Disease were based on the Diagnostic and Statistical Manual of Mental Disorders, 3rd ed., Revised18 and the National Institute of Neurological and Communication Disorder and Stroke-Alzheimer’s Disease and Related ­Disorders19. Mild cognitive impairment was defined according to the Petersen ­criteria20.i A total of 775 participants had at least one of five sensory measures collected from December 2015 to Decem- ber 2018 (Fig. 1). Approximately 54% had all five sensory measures collected at their most recent visit (n = 420). Along the 420 participants, 241 participants also had a brain MRI scan during the same visit. Thirteen partici- pants were excluded due to cognitive impairments and twenty participants were excluded because they were younger than 50 years. The final analytic sample was 208 participants ≥ 50 years old who were cognitively unim- paired with complete data collected for sensory measures and brain MRI. All participants provide written informed consent at each study visit, and the study protocol was approved by the Institutional Review Board of the National Institutes of Health Intramural Research ­Program15. All experi- ments were performed in accordance with U.S. Common Rule, 45 CFR 46. Sensory impairment Vision Vision Vision was assessed in four ways: visual acuity, visual fields, contrast sensitivity, and stereo acuity. A particip was considered visually impaired if they had impairment in any of these features. Visual acuity. Presenting visual acuity was based on better eye acuity from an ETDRS (Early Treatment of Diabetic Retinopathy Study) eye chart. Participants were given the ETDRS eye chart and asked to correctly read at least 3 letters out of 5 letters. Scores were calculated using the average of the logarithm 10 transfer of the minimum angle of resolution of the better eye. A score larger than 0.30 was defined as impaired visual ­acuity24,25. Visual fields. Visual fields were measured with the area of vision that the participant can see using a single intensity visual field test. The test was given to each eye separately but combined to generate binocular fields using an algorithm. The visual fields were separated into the central field (56 points), the upper peripheral field (18 points), and the lower peripheral field (22 points). Scores were calculated by the total number of points missed (out of a total of 96 points) on the binocular visual field test, and visual field impairment was defined as a score greater than one standard deviation from the population ­mean26. Contrast sensitivity. Contrast sensitivity was measured by the ability to discern between shades using a Pelli– Roboson chart positioned 1 m away, which is a 90 × 60 cm (36 × 24 inches) wall chart with 8 lines of letters with different contrasts. Each letter was 4.9 × 4.9 cm (2 × 2 inches) large, and there were 6 letters per line. The left 3 letters had more contrast than the right triplet. The contrast of the letters declined from the top to the bottom and from the left to the right of the chart. The top left had the highest contrast, 100%, and the bottom right had the lowest contrast, 0.6%. Scores were recorded as the logarithmic contrast sensitivity (1/contrast) of the line that at least 2 letters of the right triplet were correctly seen. Impairment was defined as the log of contrast sensitivity < 1.55, based on published population-based studies of older adults aged 60 years and ­older24,25. Stereo acuity. Stereo acuity measures depth perception which was categorized using the Randot Stereo Vision ­Test27,28. Sensory impairment The test booklet was presented upright in front of the participant who was asked to identify stereo images of decreasing depth differentials. The minimum depth differential in seconds of arcs was recorded (pos- sible values: 800, 400, 200, 140, 100, 80, 60, 50, or 40), and if the value was less than or equal to 80, the participant was defined as impaired for stereo ­acuity24,25. Vestibular function b l f f Vestibular function was assessed by saccular function and semicircular canal function. A participant was con- sidered impaired in vestibular function if they had impairment in any of these features. Saccular function. Saccular function was measured using the cervical vestibular-evoked myogenic potential (cVEMP). During the test, participants sat on a chair and had electromyographic (EMG) electrodes placed on their sternocleidomastoid muscle and sternoclavicular junction bilaterally. The ground electrode was placed on the manubrium. Sound stimuli (500 Hz and 125 dB tone bursts) were delivered to evoke cervical myo- genic potentials, which were recorded and normalized for background EMG. Saccular function impairment was defined if cVEMP was absent ­bilaterally12. Semicircular canal function. Semicircular canal function was measured by vestibulo-ocular reflex (VOR) gain. VOR was determined using the Video Head Impulse Testing (VHIT), and VOR gain, the ratio of eye velocity to head velocity was determined using the EyeSeeCam system (Interacoustics, Eden Prarie, MN) by placing it at the same plane of the horizontal semicircular canals. During VHIT, the participant’s head was placed 30-degree down from the horizontal axis initially. Then, the participant was asked to fix their gaze on a 1.5-m away wall while the head was moved randomly 150–250 degrees per second in the horizontal plant toward the right or left. Each side was completed at least 10 times. The EyeSeeCam captured the eye and head velocity to calculate VOR gain. Semicircular canal function impairment was defined if VOR gain was smaller than 0.712. Proprioception Proprioception was defined by a threshold for perception of passive ankle movement. Participants’ bare feet were placed on two pedals at a neutral position. The right pedal has a motor that directs its movement, while the left pedal is moved by the participant. While blindfolded, the participant was asked to move their left foot to match the rotation angle of the right foot driven by the motor ­pedal29. There were four trials with a sequential pedal direction: plantarflexion, dorsiflexion, dorsiflexion, and plantarflexion. Proprioception impairment was defined as the average of best plantarflexion and best dorsiflexion was > 2.2 degrees, based on established thresholds for older ­adults30. MRI‑measured brain volumesh The primary study outcome is regional brain volumes measured by MRI. Brain MRI data were acquired using a 3T Philips Achieva scanner, with a T-1 volumetric scan magnetization prepared rapid acquisition with gradient echo scan (MPRAGE: repetition time = 6.8 ms, echo time = 3.2 ms, flip angle = 8°, image matrix = 256 × 256, 170 slices, pixel size = 1 × 1 mm, slice thickness = 1.2 mm; sagittal acquisition). Multi-atlas region Segmentation using Ensembles (MUSE) software was used to obtain anatomical labels and regional brain ­volumes21,22. There are 48 regional volumes of interest that broadly include total brain; gray and white matter; cerebellum; ventricular space; frontal, temporal, parietal, and occipital lobes; and specific regions within. Intracranial volume (ICV) was approximated using a template warping algorithm by estimating ICV residuals based on centering at age 70 years in the larger BLSA ­sample23. Figure 1. Baltimore longitudinal study of aging (BLSA) participant flowchart (n = 208). Figure 1. Baltimore longitudinal study of aging (BLSA) participant flowchart (n = 208). https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Olfaction Olfaction was measured with the number of correctly identified odors on the 16-item Sniffin’ Sticks Odor Iden- tification test. Participants were randomly assigned to one of the two odor identification tests, A or B. Olfactory impairment was defined if the score was below the 10th percentile (8 for test A and 7 for test B) in the current ­sample29,31. https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Covariates Covariates in this study include age (years), sex, race, body mass index (BMI; kg/m2), usual gait speed (m/s), intracranial volume, and years of education. Age, sex, race, and years of educations collected via staff adminis- tered surveys. Usual gait speed (m/s) was measured during an 6 m overground walking test. BMI was calculated as weight (measured via digital scale) divided by height squared (measured via stadiometer). Intracranial volume ­(cm3) was also added as a covariate and estimated at age 70 years using linear mixed effects models. Each covari- ate has been separately associated with sensory impairments and brain ­volumes15,23,34–37. Statistical analysis Independent t-tests or chi-square tests were performed to compare the demographic characteristics of the 208 study participants. Sensory impairments were analyzed dichotomously and included five types: vision, proprio- ceptive, vestibular, olfactory, and hearing impairment. Multisensory impairment (MSI) was defined when the individual had two or more co-occurring sensory impairments. Each regional brain volume outcome was treated as a continuous variable using multivariable linear regres- sion models. We analyzed cross-sectional associations of sensory impairments with brain volumes in four ways, differentiated based on how sensory impairments (SI) were operationalized. First, each sensory impairment (yes/no) was included as a predictor. Second, MSI was analyzed as a count (number of sensory impairments, ranging from 0 to 5). Third, MSI was analyzed as a categorical variable (MSI ≥ 2 sensory impairments vs. MSI < 2 sensory impairments) to capture co-occurring sensory impairments.f For each model comparing differences in brain volumes by categorical impairment variables, sensitivity analyses were conducted by redefining the reference of “no impairment” to those with no sensory impairment across any of the five sensory functions (n = 68). Though this study is exploratory and multiple comparisons were conducted, we added a correction for multiple comparison testing (n = 336 comparisons) using Benjamin–Hoch- berg false discovery rate (FDR)38. g y All models were adjusted for age (years), sex, race, body mass index (BMI; kg/m2), years of education, usual gait speed (m/s), and intracranial volume. Two-tailed hypothesis testing with an alpha level = 0.05 was used to determine statistical significance. All statistical analysis was performed using Stata version 16.0 (Stata Corpora- tion, College Station, TX; https://​www.​stata.​com/​stata​16/). Hearing g Hearing was measured with a speech frequency pure-tone average (PTA) at thresholds of 0.5, 1, 2, and 4 kHz. Participants were seated in a soundproof chamber under unaided conditions with an automated testing device (Virtual Equipment Co., Audiometer Model 320). The hearing score was both a categorical and continuous vari- able, with the former defined by the common-used WHO defined categories: normal (PTA < 25 dB), mild loss (25–40 dB), moderate loss (41–70 dB), and severe loss (> 70 dB). Participants were defined as hearing impaired if their PTA was ≥ 25 ­dBHL32,33. Results Demographic characteristics of the final analytic sample (n = 208) by sensory impairment are summarized in Table 1. The overall mean age was 72.1 years with a standard deviation of 10.1 years (range 50–95 years) and 59% were women. Participants were well-educated (mean years of education 17.7 years, SD = 2.6 years) and functioning with a mean usual gait speed of 1.17 m/s (SD = 0.22 m/s). Participants with vestibular impairment tended to be older on average than other groups (mean age 80.1 years, SD = 7.3 years), while participants with proprioceptive impairment tended to be younger than other groups on average (mean age 74.3 years, SD = 12.5 years). For all sensory impairments except proprioception, the mean age was higher for those who were impaired than the unimpaired. Those with hearing impairments tended to be men (p = 0.006) and White (p = 0.007). Participants with impaired vision and vestibular function had significantly slower usual gait speed than participants without these impairments.h The distribution of sensory impairments by number and type are shown in Table 2. Among 208 participants, 138 participants (66.5%) had no or only one sensory impairment. The most prevalent sensory impairment was hearing (44.7%) whereas 9.6% had proprioceptive impairment. Forty-four participants (21.1%) had two sensory impairments with vision and hearing the most common pair (n = 20, 9.6%), followed by vestibular and hearing (n = 8, 3.8%). For multiple co-occurring impairments, nineteen participants (9.1%) had three, five participants (2.4%) had four and two participants had five. Single sensory impairment and brain volumes Participants with vision impairment had lower frontal gray matter volume ( β = − 3.00 ­cm3, SE = 1.45, p = 0.041) and inferior temporal gyrus volume ( β = − 0.89 ­cm3, SE = 0.24, p < 0.001) compared to participants without vision impairment in fully adjusted models, the latter association remained statistically significant after the FDR analysis (Table 3). Participants with proprioceptive impairment had higher ventricular space ( β = 10.10 ­cm3, SE = 3.84, p = 0.009) and frontal lobe white matter ( β = 5.10 ­cm3, SE = 2.53, p = 0.046), and caudate volumes ( β = 0.38 ­cm3, SE = 0.16, p = 0.019), and lower entorhinal cortex volume ( β = − 0.26 ­cm3, SE = 0.10, p = 0.008) than partici- pants without proprioceptive impairment. Participants with vestibular impairment had lower superior frontal gyrus volume ( β = − 0.94 ­cm3, SE = 0.38, p = 0.015), lower orbitofrontal gyrus volume ( β = − 0.91 ­cm3, SE = 0.43, p = 0.033), lower superior parietal lobe volume ( β = − 1.00 ­cm3, SE = 0.29, p = 0.001), lower superior occipital gyrus volume ( β = − 0.35 ­cm3, SE = 0.17, p = 0.047), and lower inferior occipital gyrus volume ( β = − 0.57 ­cm3, SE = 0.23, p = 0.016) than those without vestibular impairment. The lower superior parietal lobe volume associa- tion remained statistically significant after the FDR analysis. Participants with olfactory impairment had lower https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Table 1. Characteristics of study population by composite sensory impairments (n = 208). Independent t-test was performed for age, body mass index, years of education, and usual gait speed. Chi-square test was performed for sex, race, and APOE e4 allele. Total Vision impairment Proprioception impairment (n = 208) Yes No p-value Yes No p-value Participant, no. (%) 208 66 (31.7) 142 (68.3) 20 (9.6) 188 (90.4) Age (years), mean (sd) 72.1 (10.1) 76.1 (8.7) 70.2 (10.1) < 0.001 74.3 (12.5) 71.9 (9.8) 0.312 Women, no. (%) 122 (58.7) 38 (57.6) 84 (59.2) 0.830 10 (50.0) 112 (59.6) 0.408 Body Mass Index (kg/m2), mean (sd) 27.3 (4.4) 26.3 (3.8) 27.8 (4.5) 0.028 27.4 (3.6) 27.3 (4.5) 0.935 White, no. Single sensory impairment and brain volumes (%) 128 (61.5) 43 (65.2) 85 (59.9) 0.718 12 (60.0) 116 (61.7) 0.325 Years of education, mean (sd) 17.7 (2.6) 18.2 (2.6) 17.5 (2.6) 0.106 17.8 (2.1) 17.7 (2.6) 0.914 Usual pace gait speed (m/s), mean (sd) 1.17 (0.22) 1.11 (0.23) 1.20 (0.21) 0.005 1.09 (0.21) 1.18 (0.22) 0.069 APOE e4 allele, no. (%) 50 (24.0) 13 (19.7) 37 (26.1) 0.318 3 (15.0) 47 (25.0) 0.320 Vestibular impairment Olfaction impairment Hearing impairment Yes No p-value Yes No p-value Yes No p-value Participant, no. (%) 38 (18.3) 170 (81.7) 28 (13.5) 180 (86.5) 93 (44.7) 115 (55.3) Age (years), mean (sd) 80.1 (7.3) 70.3 (9.7) < 0.001 78.4 (6.9) 71.1 (10.1) < 0.001 77.5 (8.3) 67.7 (9.1) < 0.001 Women, no. (%) 20 (52.6) 102 (60.0) 0.404 13 (46.4) 109 (60.6) 0.158 45 (48.4) 77 (67.0) 0.007 Body Mass Index (kg/m2), mean (sd) 26.4 (4.4) 27.5 (4.4) 0.157 26.2 (4.0) 27.5 (4.4) 0.164 26.9 (4.1) 27.7 (4.6) 0.185 White, no. (%) 24 (63.2) 104 (61.2) 0.376 13 (46.4) 115 (63.9) 0.051 69 (74.2) 59 (51.3) 0.006 Years of education, mean (sd) 18.2 (2.4) 17.6 (2.7) 0.274 18.5 (2.0) 17.6 (2.7) 0.081 18.1 (2.7) 17.5 (2.5) 0.094 Usual pace gait speed (m/s), mean (sd) 1.08 (0.23) 1.20 (0.21) 0.003 1.18 (0.26) 1.17 (0.21) 0.838 1.14 (0.21) 1.20 (0.22) 0.054 APOE e4 allele, no. (%) 6 (15.8) 44 (25.9) 0.188 8 (28.6) 42 (23.3) 0.546 21 (22.6) 29 (25.2) 0.658 Table 1. Characteristics of study population by composite sensory impairments (n = 208). Independent t-test was performed for age, body mass index, years of education, and usual gait speed. Chi-square test was performed for sex, race, and APOE e4 allele. orbitofrontal gyrus volume ( β = − 0.94 ­cm3, SE = 0.50, p = 0.047) and lower posterior cingulate gyrus volume ( β = − 0.34 ­cm3, = 0.16, p = 0.035).h 3 The associations between vision impairment and inferior temporal gyrus ( β = − 1.33 ­cm3, SE = 0.41, p = 0.002) and proprioception impairment and entorhinal cortex ( β = − 0.40 ­cm3, SE = 0.18, p = 0.035) were robust to sensi- tivity analyses comparing each sensory impairment with participants who had no sensory impairments (n = 68). The associations between proprioception impairment with ventricular space (p > 0.05) and olfactory impairment with posterior cingulate gyrus (p > 0.05) were not robust to sensitivity analyses. MSI and brain volumes In fully adjusted models, participants with two or more co-occurring sensory impairments (n = 70, 34%) had lower mean volume in the frontal gray matter ( β = − 3.20 ­cm3, SE = 1.62, p = 0.05), parietal lobe gray matter ( β = − 2.51 ­cm3, SE = 0.97, p = 0.01), superior frontal gyrus ( β = − 1.01 ­cm3, SE = 0.34, p = 0.003), orbitofrontal gyrus ( β = − 0.91 ­cm3, SE = 0.38, p = 0.018), superior parietal lobe ( β = − 0.68 ­cm3, SE = 0.27, p = 0.011), and precuneus ( β = − 0.74 ­cm3, SE = 0.35, p = 0.038) compared to those with less than two sensory impairments (Table 4, second column). None of the results remained statistically significant after FDR correction.h y git These results were not robust when changing the reference to participants with no sensory impairments. However, the sensitivity analyses showed that those with two or more sensory impairments had lower amygdala volume ( β = − 0.04 ­cm3, SE = 0.02, p = 0.04) compared to those with no sensory impairments. Count of sensory impairments and brain volumes h dd l d Each additional sensory impairment was associated with lower mean volume of the orbitofrontal gyrus ( β = − 0.35 ­cm3, SE = 0.17, p = 0.04) and in the entorhinal cortex ( β = − 0.09 ­cm3, SE = 0.03, p = 0.006) in fully adjusted models (Table 4, first column). Only the association with entorhinal cortex remained statistically sig- nificant after FDR correction. There was also a higher mean volume in the caudate ( β = 0.14 ­cm3, SE = 0.05, p = 0.006) and in the putamen ( β = 0.13 ­cm3, SE = 0.06, p = 0.043) of the basal ganglia though none of the results remained statistically significant after FDR correction. Single sensory impairment and brain volumes However, the other associations had similar magnitude and directionality as reported above but did not reach statistical significance (p > 0.05). Sensory impairment patterns with brain volumes For participants with two co-occurring sensory impairments (MSI = 2), there were 8 observed combinations; for participants with three sensory impairments (MSI = 3), there were 8 observed combinations; for participants with four types of sensory impairments (MSI = 4), there were 2 observed combinations (Table 2). Scientific Reports | (2024) 14:9339 | https://doi.org/10.1038/s41598-024-59965-w www.nature.com/scientificreports/ Table 2. Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VES vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Sensory impairment patterns with brain volumes Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VES vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Table 2. Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VES vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Scientific Reports | (2024) 14:9339 | Sensory impairment patterns with brain volumes Numbers of participants Numbers of sensory impairment  0 68 (33.0%)  1 70 (33.5%)  2 44 (21.1%)  3 19 (9.1%)  4 5 (2.4%)  5 2 (1.0%) Combinations of two impairments  VS + H 20 (9.6%)  VES + H 8 (3.8%)  SML + H 4 (1.9%)  VS + SML 3 (1.4%)  VS + VES 3 (1.4%)  VS + PROP 2 (1.0%)  VES + SML 2 (1.0%)  PROP + H 2 (1.0%)  VES + PROP 0 (0.0%)  PROP + SML 0 (0.0%) Combinations of three impairments  VS + SML + H 6 (2.9%)  VS + VES + H 5 (2.4%)  VS + PROP + H 2 (1.0%)  VES + SML + H 2 (1.0%)  VS + VES + PROP 1 (0.5%)  VS + VES + SML 1 (0.5%)  VES + PROP + H 1 (0.5%)  PROP + SML + H 1 (0.5%)  VES + PROP + SML 0 (0.0%)  VS + PROP + SML 0 (0.0%) Combinations of four impairments  VS + VES + SML + H 3 (1.4%)  VS + VES + PROP + H 2 (1.0%)  VS + VES + PROP + SML 0 (0.0%)  VS + PROP + SML + H 0 (0.0%)  VES + PROP + SML + H 0 (0.0%) Numbers of participants Numbers of sensory impairment  0 68 (33.0%)  1 70 (33.5%)  2 44 (21.1%)  3 19 (9.1%)  4 5 (2.4%)  5 2 (1.0%) Combinations of two impairments  VS + H 20 (9.6%)  VES + H 8 (3.8%)  SML + H 4 (1.9%)  VS + SML 3 (1.4%)  VS + VES 3 (1.4%)  VS + PROP 2 (1.0%)  VES + SML 2 (1.0%)  PROP + H 2 (1.0%)  VES + PROP 0 (0.0%)  PROP + SML 0 (0.0%) Combinations of three impairments  VS + SML + H 6 (2.9%)  VS + VES + H 5 (2.4%)  VS + PROP + H 2 (1.0%)  VES + SML + H 2 (1.0%)  VS + VES + PROP 1 (0.5%)  VS + VES + SML 1 (0.5%)  VES + PROP + H 1 (0.5%)  PROP + SML + H 1 (0.5%)  VES + PROP + SML 0 (0.0%)  VS + PROP + SML 0 (0.0%) Combinations of four impairments  VS + VES + SML + H 3 (1.4%)  VS + VES + PROP + H 2 (1.0%)  VS + VES + PROP + SML 0 (0.0%)  VS + PROP + SML + H 0 (0.0%)  VES + PROP + SML + H 0 (0.0%) Table 2. Table 2.   Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VE vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Discussion Th d h This study characterized cross-sectional associations between co-occurring sensory impairments and brain volumes in cognitively unimpaired adults aged 50 years and older. Combinations of MSI that include vision, proprioceptive, and/or vestibular impairments were associated with lower brain volumes in the frontal gray matter, superior frontal gyrus, orbitofrontal gyrus, superior parietal lobe, and entorhinal cortex regions. Further, there is a possibly that MSI is uniquely associated with the lower amygdala volume. Oppositely, MSI was associ- ated with higher volumes in the basal ganglia region, suggesting a possible compensatory mechanism within this region as the brain attempts to adapt to brain atrophy in other regions. Collectively, these results highlight potential associations between MSI and brain structure so that future longitudinal research focusing on these regions may be performed to understand the temporality of these associations.i g y p p y It is unclear the specific sensory impairments that drive the detected association between MSI and lower volume in the orbitofrontal gyrus. However, in the single sensory impairment analyses, vestibular and olfactory impairment separately were significantly associated with lower orbitofrontal gyrus volume. This suggests that a combination of vestibular and/or olfactory impairments may reflect or result from lower orbitofrontal gyrus volume, but the sample with both vestibular and olfactory impairment is too small to produce stable estimates. This is supported by animal studies showing that neural pathways between sensory cortices and the orbitofrontal gyrus exhibit diminished numbers of neural connections in animals with sensory ­impairments39,40. It is important to acknowledge that this finding was not robust when accounting for multiple testing.h gi g g p g The observed association between MSI and lower volume in the entorhinal cortex is consistent with kno inputs from the vestibular system into the entorhinal cortex, which contains a neuronal population called Scientific Reports | (2024) 14:9339 | https://doi.org/10.1038/s41598-024-59965-w www.nature.com/scientificreports/ Table 3. Differences in mean regional brain volumes ­(cm3) for each separate sensory impairment^. Table 3.   Differences in mean regional brain volumes ­(cm3) for each separate sensory impairment^. All are multivariable linear regression models adjusted for age, sex, race, BMI (body mass index), icv70 (intracranial volume at age 70 years old), and years of education. ^The reference group for each column is no impairment in that specific sensory function. *p < 0.05; **p < 0.01; ***p < 0.001. # The association remained statistically significant after the FDR analysis. Discussion Th d h All are multivariable linear regression models adjusted for age, sex, race, BMI (body mass index), icv70 (intracranial h Vision impairment Proprioceptive impairment Vestibular impairment Olfactory impairment Hearing impairment Beta coefficient (SE) Cerebellum 1.867 (1.593) − 4.054 (2.290) − 0.475 (1.960) 1.579 (2.273) − 0.162 (1.677) Total brain 0.940 (6.412) 16.157 (9.613) − 10.873 (7.914) 6.196 (8.725) 2.909 (6.763) Ventricular space 0.473 (2.584) 10.095 (3.836)** − 1.685 (3.203) 3.900 (3.510) 0.634 (2.726) Gray matter − 4.846 (3.846) − 1.238 (5.829) − 6.684 (4.765) 0.235 (5.260) 0.628 (4.074) White matter 5.145 (3.676) 7.734 (5.549) − 2.478 (4.577) 1.873 (5.030) 1.500 (3.896) Frontal lobe − 0.604 (2.544) 7.129 (3.808) − 2.322 (3.151) − 2.155 (3.463) 1.809 (2.681) Temporal lobe − 0.932 (1.378) − 0.377 (2.083) − 1.798 (1.706) 0.872 (1.879) 1.173 (1.454) Parietal lobe − 0.033 (1.463) 0.720 (2.208) − 2.657 (1.805) 0.910 (1.992) − 0.107 (1.544) Occipital lobe − 0.136 (1.168) 1.977 (1.758) − 1.520 (1.445) 0.691 (1.591) − 1.024 (1.230) Frontal gray matter − 2.997 (1.454)* 2.030 (2.213) − 2.194 (1.815) − 1.839 (1.997) 0.723 (1.549) Temporal gray matter − 1.710 (0.902) − 1.390 (1.370) − 0.886 (1.126) − 0.177 (1.239) 1.042 (0.957) Parietal gray matter − 1.018 (0.881) − 0.333 (1.334) − 2.057 (1.087) 0.361 (1.204) − 0.400 (0.932) Occipital gray matter − 0.571 (0.801) 0.574 (1.210) − 0.946 (0.992) 0.250 (1.093) − 0.657 (0.845) Frontal white matter 2.393 (1.687) 5.098 (2.534)* − 0.128 (2.103) − 0.316 (2.310) 1.086 (1.787) Temporal white matter 0.778 (0.877) 1.014 (1.325) − 0.912 (1.088) 1.049 (1.195) 0.131 (0.927) Parietal white matter 0.985 (0.874) 1.054 (1.322) − 0.600 (1.087) 0.549 (1.194) 0.294 (0.925) Occipital white matter 0.435 (0.535) 1.403 (0.803) − 0.574 (0.663) 0.441 (0.729) − 0.366 (0.565) Superior frontal gyrus − 0.578 (0.309) 0.381 (0.470) − 0.938 (0.381)** − 0.629 (0.422) 0.408 (0.327) Middle frontal gyrus − 0.397 (0.393) 0.454 (0.594) 0.044 (0.488) 0.152 (0.536) 0.350 (0.415) Inferior frontal gyrus − 0.199 (0.218) 0.307 (0.329) − 0.091 (0.271) 0.070 (0.298) 0.018 (0.231) Medial frontal cortex − 0.017 (0.069) 0.023 (0.105) − 0.113 (0.086) − 0.003 (0.095) − 0.010 (0.073) Orbitofrontal gyrus − 0.618 (0.345) 0.338 (0.524) − 0.914 (0.426)* − 0.936 (0.469)* 0.139 (0.367) Precentral gyrus − 0.298 (0.258) 0.187 (0.391) 0.338 (0.320) − 0.228 (0.353) 0.021 (0.273) Postcentral gyrus − 0.030 (0.266) − 0.355 (0.401) − 0.412 (0.329) 0.127 (0.363) 0.069 (0.281) Superior parietal lobe − 0.239 (0.241) − 0.046 (0.365) − 0.999 (0.291)**# − 0.205 (0.329) 0.160 (0.255) Supramarginal gyrus − 0.169 (0.224) 0.280 (0.337) − 0.394 (0.276) 0.111 (0.305) 0.038 (0.236) Angular gyrus − 0.255 (0.276) − 0.526 (0.416) 0.316 (0.342) 0.196 (0.376) − 0.399 (0.290) Precuneus − 0.346 (0.320) 0.233 (0.485) − 0.529 (0.397) 0.202 (0.438) − 0.300 (0.338) Superior temporal gyrus 0.125 (0.194) 0.159 (0.293) − 0.233 (0.240) − 0.190 (0.264) 0.289 (0.204) Middle temporal gyrus − 0.328 (0.340) − 0.923 (0.511) − 0.242 (0.423) − 0.122 (0.465) − 0.250 (0.359) Inferior temporal gyrus − 0.889 (0.240)***# − 0.199 (0.374) − 0.040 (0.308) − 0.109 (0.338) 0.410 (0.260) Hippocampus − 0.086 (0.092) 0.059 (0.139) − 0.118 (0.114) -0.046 (0.125) − 0.162 (0.096) Parahippocampus − 0.069 (0.091) − 0.060 (0.137) − 0.132 (0.112) 0.010 (0.124) − 0.044 (0.096) Entorhinal cortex − 0.089 (0.066) − 0.264 (0.098)** − 0.104 (0.082) − 0.115 (0.090) − 0.034 (0.070) Amygdala − 0.018 (0.030) − 0.034 (0.045) − 0.020 (0.037) − 0.014 (0.041) − 0.050 (0.031) Fusiform gyrus − 0.151 (0.218) − 0.177 (0.329) 0.029 (0.271) 0.145 (0.297) 0.294 (0.229) Superior occipital gyrus 0.089 (0.142) 0.030 (0.214) − 0.348 (0.174)* 0.051 (0.193) 0.141 (0.149) Middle occipital gyrus − 0.306 (0.179) 0.156 (0.272) 0.052 (0.224) − 0.083 (0.246) − 0.156 (0.190) Inferior occipital gyrus − 0.108 (0.192) − 0.116 (0.289) − 0.567 (0.234)* − 0.014 (0.261) − 0.234 (0.202) Occipital pole − 0.108 (0.142) 0.282 (0.214) 0.190 (0.176) − 0.071 (0.194) 0.002 (0.150) Cuneus 0.146 (0.178) 0.018 (0.269) 0.147 (0.221) 0.120 (0.243) -0.058 (0.188) Anterior cingulate gyrus − 0.064 (0.160) 0.470 (0.240) 0.024 (0.199) − 0.176 (0.218) 0.222 (0.168) Posterior cingulate gyrus − 0.143 (0.117) 0.150 (0.177) 0.024 (0.146) − 0.336 (0.158)* -0.089 (0.124) Middle cingulate gyrus − 0.090 (0.137) 0.205 (0.206) − 0.093 (0.169) 0.055 (0.186) 0.163 (0.144) Caudate 0.170 (0.107) 0.378 (0.160)* 0.063 (0.133) 0.254 (0.145) 0.084 (0.113) Globus pallidus 0.024 (0.040) 0.0130 (0.060) − 0.022 (0.050) − 0.020 (0.055) 0.065 (0.042) Putamen 0.119 (0.128) 0.189 (0.193) 0.159 (0.159) 0.267 (0.174) 0.085 (0.135) Thalamus − 0.108 (0.144) 0.321 (0.217) 0.091 (0.179) 0.186 (0.196) − 0.026 (0.152) https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Table 4. Discussion Th d h Associations between MSI (as a count and in categories) and mean regional b (n = 208). SI sensory impairment, MSI multisensory impairment. All are multivariable l dj t d f BMI (b d i d ) i 70 (i t i l l t 70 Continuous SI ranging from 0 to 5 impairments Categorical MSI (MSI ≥ 2 vs. Table 4.   Associations between MSI (as a count and in categories) and mean regional brain volumes ­(cm3) (n = 208). SI sensory impairment, MSI multisensory impairment. All are multivariable linear regression models adjusted for age, sex, race, BMI (body mass index), icv70 (intracranial volume at age 70 years old), and years of education. *p < 0.05; **p < 0.01; ***p < 0.001. # The association remained statistically significant after the FDR analysis. www.nature.com/scientificreports/ y gg g g g y g g p y g Two or more co-occurring sensory impairments were associated with lower volumes in the gray matter of the frontal and parietal lobes, in the superior frontal gyrus and superior parietal lobe, and the precuneus. Our findings suggest that these gray matter associations may be driven by vision impairment and the frontal and parietal lobe associations driven by vestibular impairment. However, caution in interpreting these exploratory findings should be noted since the prevalence of various patterns was low and subject to type 1 error due to multiple testing and did not remain statistically significant after accounting for multiple testing.hhii g y git g g There are limitations to acknowledge. The first limitation is the small sample size, that was defined with par- ticipants who had all five sensory measures plus complete brain MRI scans. Second, the generalizability of the findings is narrow because BLSA participants tend to have higher education and better health than the general older adult population. Third, the prevalence of MSI with three or more sensory impairments observed was low, yielding low statistical power. Fourth, the cross-sectional design does not account for temporality and thus the direction of association is unclear. Fifth, multiple statistical tests were performed, increasing the likelihood of type 1 error. Though this was an exploratory study, we performed FDR analyses in the sensitivity analyses to address issues with multiple comparison testing. Yet, when doing so might increase type II error that reduces the detection of true associations.h This study has multiple strengths. First, sensory impairments were comprehensively measured across a large sample of older adults. Second, brain volumes were quantified via brain MRI scans. Third, this study was able to describe specific patterns of MSI and explore their relationships to brain volumetric measures. pi p p p In conclusion, this study found that higher numbers of sensory impairments were linked to higher ventricular volumes and lower brain volumes, primarily in the superior frontal gyrus, orbitofrontal gyrus, and precuneus. In contrast, those living with MSI had higher volumes in the basal ganglia regions. Future research is needed to explore longitudinal associations to evaluate whether multiple sensory impairments lead to accelerated brain atrophy and whether there are some brain regions may be preserved or activated (i.e., the basal ganglia) in response, and assess whether these relationships are linked to cognitive outcomes. www.nature.com/scientificreports/ cells which are involved in spatial ­navigation41. Indeed, vestibular impairment has been associated with entorhinal cortex atrophy in prior ­work42. These findings are also consistent with a previous study in rats, which performed deafferentation injury surgeries on the entorhinal cortex of rats’ brains and assessed the rats’ performance using sensory integration testing. By analyzing histological brain slides and performance scores, researchers concluded that rats with lesions in the entorhinal cortex showed sensory integration deficits and behavioral ­change43. This suggests that the entorhinal cortex plays a role in multisensory function. Future research into the biological mechanisms behind the link between MSI and both the orbitofrontal gyrus and entorhinal cortex in humans is warranted. In sensitivity analyses comparing MSI versus no sensory impairment, a possible unique association was revealed between two or more sensory impairments and lower amygdala volumes. If this association is true, this is consistent with the amygdala’s role in the processing of sensory ­information44. However, this finding did not remain statistically significant when accounting for multiple testing. Without replication of this finding in larger studies, this relationship is likely spurious since it was not detected in the main analysis. MSI and proprioceptive impairment alone were associated with a higher mean volume in the basal ganglia region in the main analysis but did not remain statistically significant after multiple testing correction. Still, a possible explanation for this potential finding is that the basal ganglia region may play a compensatory role in how the brain adapts to MSI. The basal ganglia are involved in both motor control and cognitive ­function45 and are thought to work with cortical regions in executing stereotyped motor and cognitive actions under cortical volitional control. Conceivably, with the loss of sensory-driven cortical control, “implicit” behaviors and actions stored in the basal ganglia are increasingly activated or are lost more slowly than other brain regions. A previous study using a smaller sample of BLSA participants also demonstrated that worse vestibular function was related to higher volumes of the basal ganglia region, the caudate and putamen, ­specifically42. Also, this finding might coincide with the basal ganglia tending to be last to atrophy and lose function according to the “first in, last out” ­principle46. Collectively, these findings suggest the possibility that proprioceptive and/or vestibular dysfunction may trigger a higher utilization of the basal ganglia, which indirectly mitigates this region’s atrophy with age. www.nature.com/scientificreports/ Also, leveraging machine learning approaches (e.g., random forest) using MSI information to detect or predict changes in brain volumes are warranted. Such work may help provide mechanistic insights linking sensory impairments with aging brain and detection of cognitive decline. Replication is needed given the exploratory nature of the findings and the possibility of false discovery. Data availability y Because of the sensitive nature of the data collected for this study, requests to access the data set from qualified researchers trained in human subject confidentiality protocols may be sent to the Intramural Research Program of the National Institute on Aging at https://​blsa.​nih.​gov. Received: 29 November 2023; Accepted: 17 April 2024 Received: 29 November 2023; Accepted: 17 April 2024 Discussion Th d h MSI < 2) Beta coefficient (SE) Cerebellum 0.055 (0.762) − 1.382 (1.762) Total brain 1.679 (3.158) − 5.286 (7.140) Ventricular space 1.574 (1.269) 1.747 (2.879) Gray matter − 2.197 (1.897) − 8.332 (4.265) White matter 2.255 (1.813) 1.605 (4.117) Frontal lobe 0.359 (1.254) − 1.429 (2.835) Temporal lobe − 0.180 (0.680) − 1.053 (1.537) Parietal lobe − 0.255 (0.721) − 2.722 (1.620) Occipital lobe − 0.195 (0.576) − 0.216 (1.302) Frontal gray matter − 0.941 (0.721) − 3.197 (1.622)* Temporal gray matter − 0.499 (0.447) − 1.334 (1.010) Parietal gray matter − 0.648 (0.433) − 2.510 (0.970)* Occipital gray matter − 0.338 (0.395) − 0.579 (0.893) Frontal white matter 1.300 (0.831) 1.768 (1.886) Temporal white matter 0.319 (0.433) 0.282 (0.980) Parietal white matter 0.393 (0.431) − 0.211 (0.978) Occipital white matter 0.142 (0.264) 0.363 (0.597) Superior frontal gyrus − 0.241 (0.153) − 1.014 (0.340)** Middle frontal gyrus 0.055 (0.194) − 0.15 (0.439) Inferior frontal gyrus − 0.017 (0.108) − 0.118 (0.243) Medial frontal cortex − 0.022 (0.034) 0.015 (0.077) Orbitofrontal gyrus − 0.351 (0.170)* − 0.914 (0.382)** Precentral gyrus − 0.024 (0.128) − 0.129 (0.289) Postcentral gyrus − 0.078 (0.131) − 0.356 (0.296) Superior parietal lobe − 0.213 (0.118) − 0.679 (0.265)** Supramarginal gyrus − 0.051 (0.110) − 0.456 (0.248) Angular gyrus − 0.130 (0.136) − 0.302 (0.307) Precuneus − 0.182 (0.158) − 0.740 (0.354)* Superior temporal gyrus 0.049 (0.096) − 0.0001 (0.216) Middle temporal gyrus − 0.287 (0.167) − 0.548 (0.378) Inferior temporal gyrus − 0.168 (0.122) − 0.367 (0.276) Hippocampus − 0.075 (0.045) − 0.114 (0.102) Parahippocampus − 0.052 (0.045) − 0.119 (0.101) Entorhinal cortex − 0.089 (0.032)**# − 0.081 (0.074) Amygdala − 0.024 (0.015) − 0.043 (0.033) Fusiform gyrus 0.032 (0.108) − 0.175 (0.243) Superior occipital gyrus 0.007 (0.070) − 0.038 (0.158) Middle occipital gyrus − 0.094 (0.089) − 0.183 (0.201) Inferior occipital gyrus − 0.181 (0.094) − 0.211 (0.213) Occipital pole 0.025 (0.070) − 0.065 (0.159) Cuneus 0.064 (0.088) 0.197 (0.198) Anterior cingulate gyrus 0.064 (0.079) 0.106 (0.179) Posterior cingulate gyrus − 0.078 (0.058) − 0.173 (0.131) Middle cingulate gyrus 0.028 (0.067) 0.006 (0.152) Caudate 0.143 (0.052)** 0.213 (0.119) Globus pallidus 0.015 (0.020) 0.017 (0.045) Putamen 0.128 (0.063)* 0.173 (0.143) Thalamus 0.041 (0.071) 0.045 (0.161) https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ 2. 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Association of gait speed with regional brain volumes and risk of dementia in older Japanese: The Hisayama study. Arch. Gerontol. Geriatr. 106, 104883 (2023). 8. Benjamini, Y. & Hochberg, Y. References 1. Schneider, J. et al. Prevalence and 5-year incidence of dual sensory impairment in an older Australian population. Ann. Epidemiol 22, 295–301 (2012). 2. Correia, C. et al. Global sensory impairment in older adults in the United States. J. Am. Geriatr. Soc. 64, 306–313 (2016). 3. Crews, J. E. & Campbell, V. A. Vision impairment and hearing loss among community-dwelling older Americans: Implications for health and functioning. Am. J. Public Health 94, 823–829 (2004). g J , ( ) 4. Murphy, C. Prevalence of olfactory impairment in older adults. JAMA 288, 2307 (2002). https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | Acknowledgementsh g The authors of this manuscript thank the participants and staff in the Baltimore Longitudinal Study of Aging for their contributions. https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Competing interests  Th h d l The authors declare no competing interests. Author contributions JAS, YA, AAW, and CT conceptualized the project. All authors participated in the analysis, interpretation and in drafting and critically reviewing the manuscript for intellectual content and developed the final version for submission. All authors read and approved this manuscript. Funding YA, ALG, JAS and AAW were supported by Grant Number R01AG061786 from the National Institute on Aging, National Institutes of Health. AAW was supported by Grant Number K01AG076967 from the National Institute on Aging, National Institutes of Health. The Baltimore Longitudinal Study of Aging was supported by the Intra- mural Research Program of the National Institute on Aging (ZIAAG000015 and ZIAAG000191). © The Author(s) 2024 Additional information Correspondence and requests for materials should be addressed to A.A.W. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Diversification of Colonization Factors in a Multidrug-Resistant Escherichia coli Lineage Evolving under Negative Frequency-Dependent Selection
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Diversification of colonization factors in a multidrug-resistant Escherichia coli lineage evolving under negative frequency-dependent selection License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): McNally, A, Kallonen, T, Connor, C, Abudahab, K, Aanensen, DM, Horner, C, Peacock, SJ, Parkhill, J, Croucher, NJ & Corander, J 2019, 'Diversification of colonization factors in a multidrug-resistant Escherichia coli lineage evolving under negative frequency-dependent selection', mBio, vol. 10, no. 2, e00644-19. https://doi.org/10.1128/mBio.00644-19 Link to publication on Research at Birmingham portal Link to publication on Research at Birmingham portal Publisher Rights Statement: Checked for eligibility: 08/05/2019 Publisher Rights Statement: Checked for eligibility: 08/05/2019 Publisher Rights Statement: Checked for eligibility: 08/05/2019 Citation for published version (Harvard): McNally, A, Kallonen, T, Connor, C, Abudahab, K, Aanensen, DM, Horner, C, Peacock, SJ, Parkhill, J, Croucher, NJ & Corander, J 2019, 'Diversification of colonization factors in a multidrug-resistant Escherichia coli lineage evolving under negative frequency-dependent selection', mBio, vol. 10, no. 2, e00644-19. https://doi.org/10.1128/mBio.00644-19 Download date: 24. Oct. 2024 Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 RESEARCH ARTICLE Ecological and Evolutionary Science RESEARCH ARTICLE Ecological and Evolutionary Science crossm RESEARCH ARTICLE Ecological and Evolutionary Science crossm Citation McNally A, Kallonen T, Connor C, Abudahab K, Aanensen DM, Horner C, Peacock SJ, Parkhill J, Croucher NJ, Corander J. 2019. Diversification of colonization factors in a multidrug-resistant Escherichia coli lineage evolving under negative frequency-dependent selection. mBio 10:e00644-19. https://doi.org/ 10.1128/mBio.00644-19. Citation McNally A, Kallonen T, Connor C, Abudahab K, Aanensen DM, Horner C, Peacock SJ, Parkhill J, Croucher NJ, Corander J. 2019. Diversification of colonization factors in a multidrug-resistant Escherichia coli lineage evolving under negative frequency-dependent selection. mBio 10:e00644-19. https://doi.org/ 10.1128/mBio.00644-19. Diversification of Colonization Factors in a Multidrug-Resistant Escherichia coli Lineage Evolving under Negative Frequency- Dependent Selection Alan McNally,a Teemu Kallonen,b,c Christopher Connor,a Khalil Abudahab,b David M. Aanensen,b Carolyne Horner,d Sharon J. Peacock,b,e,f Julian Parkhill,b Nicholas J. Croucher,g Jukka Coranderb,c,h aInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom bInfection Genomics, Wellcome Sanger Institute, Cambridge, United Kingdom cDepartment of Biostatistics, University of Oslo, Oslo, Norway dBritish Society of Antimicrobial Chemotherapy, Birmingham, United Kingdom eDepartment of Medicine, University of Cambridge, Cambridge, United Kingdom fLondon School of Hygiene and Tropical Medicine, London, United Kingdom gFaculty of Medicine, School of Public Health, Imperial College, London, United Kingdom hDepartment of Mathematics and Statistics, University of Helsinki, Helsinki, Finland on May 8, 2019 by guest http://mbio.asm.org/ aded from ABSTRACT Escherichia coli is a major cause of bloodstream and urinary tract infec- tions globally. The wide dissemination of multidrug-resistant (MDR) strains of ex- traintestinal pathogenic E. coli (ExPEC) poses a rapidly increasing public health burden due to narrowed treatment options and increased risk of failure to clear an infection. Here, we present a detailed population genomic analysis of the ExPEC ST131 clone, in which we seek explanations for its success as an emerging patho- genic strain beyond the acquisition of antimicrobial resistance (AMR) genes. We show evidence for evolution toward separate ecological niches for the main clades of ST131 and differential evolution of anaerobic metabolism, key colonization, and virulence factors. We further demonstrate that negative frequency-dependent selec- tion acting across accessory loci is a major mechanism that has shaped the popula- tion evolution of this pathogen. IMPORTANCE Infections with multidrug-resistant (MDR) strains of Escherichia coli are a significant global public health concern. To combat these pathogens, we need a deeper understanding of how they evolved from their background populations. By understanding the processes that underpin their emergence, we can design new strategies to limit evolution of new clones and combat existing clones. By combin- ing population genomics with modelling approaches, we show that dominant MDR clones of E. coli are under the influence of negative frequency-dependent selection, preventing them from rising to fixation in a population. Furthermore, we show that this selection acts on genes involved in anaerobic metabolism, suggesting that this key trait, and the ability to colonize human intestinal tracts, is a key step in the evo- lution of MDR clones of E. coli. Editor Julian E. Davies, University of British Columbia Copyright © 2019 McNally et al. Diversification of Colonization Factors in a Multidrug-Resistant Escherichia coli Lineage Evolving under Negative Frequency- Dependent Selection The lack of phylogeographic signal and phylogenetic structure based on host source suggests rapid global dispersal and frequent host transitions within clade C (14). Research has suggested that the acquisition of fluoro- quinolone resistance via point mutations in DNA gyrase and DNA topoisomerase genes was the primary driver in the rapid emergence of clade C, alongside the predated acquisition of well-defined ExPEC virulence factors (11, 12). Later work also suggested that clade C E. coli ST131 may dominate as a successful MDR clade due to the ability to offset the fitness cost of MDR plasmid acquisition and maintenance via compensatory mutations in gene regulatory regions (14). Genome-wide association studies (GWAS) have been used to identify loci and lineage-specific alleles significantly associated with clade C E. coli ST131, which suggested a secondary flagellum locus encoding lateral flagella (Flag-2 [15]), and a number of hypothetical proteins and promoter regions as being clade C E. coli ST131-associated loci (14). Recent work on E. coli causing bacteremia provided compelling evidence that resistance to antimicrobials has not been the major driver of the success of ST131 (16). An analysis of a large 11-year population survey across the United Kingdom showed that ST131 rapidly stabilized at a level of approximately 20% after its emergence around 2002 in the United Kingdom. This was far in excess of already-resident MDR clones, such as ST88 or ST405. Nevertheless, the overall prevalence of resistance phenotypes re- mained approximately constant in the population. Furthermore, most currently known major ExPEC clones (primarily ST12, ST73, ST95, and ST69, the last of which also rapidly emerged in 2002) show a similar stable population frequency across the 10 years following the introduction of ST131, despite exhibiting far less extensive resistance profiles. These observations suggested the distribution of ExPEC strains was shaped by negative frequency-dependent selection (NFDS) (16). NFDS describes the situation in which a given phenotype is most beneficial to a population when it is rare, such as the emergence of a new antigen or resource-use strategy. This is because as the phenotype becomes common it either becomes costly, such as when antigens are recognized by an increased proportion of hosts, or less beneficial, such as when strains compete more fiercely for the same resources. Recently, a multilocus NFDS model of postvaccination Streptococcus pneumoniae population dynamics was described (17). Diversification of Colonization Factors in a Multidrug-Resistant Escherichia coli Lineage Evolving under Negative Frequency- Dependent Selection This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. KEYWORDS AMR, Escherichia coli, evolutionary genomics, negative frequency- dependent selection E scherichia coli is now the most common cause of bloodstream infections in the developed world, outnumbering cases of Staphylococcus aureus bacteremia by 2:1 (1). E. coli is also the most common cause of urinary tract infections (UTI), which in turn are among the most common bacterial infections in the world (2). Bacteremia and UTI are caused by a subset of E. coli termed extraintestinal pathogenic E. coli (ExPEC). ExPEC E s d March/April 2019 Volume 10 Issue 2 e00644-19 ® ® mbio.asm.org 1 mbio.asm.org 1 ® McNally et al. are not a phylogenetically distinct group of E. coli but rather represent strains which have acquired virulence-associated genes that confer the ability to invade and cause disease in extraintestinal sites (3). Genes associated with virulence that confer the ability to adhere to extraintestinal tissues, to sequester extracellular iron, and to evade the nonspecific immune response and toxins resulting in localized tissue destruction have all been described as essential in the process of ExPEC pathogenesis (4). The problem presented by the scale of ExPEC infections is exacerbated by the number of cases involving multidrug-resistant (MDR) strains (1, 5, 6). Epidemiological surveys report as many as 60% of UTI ExPEC isolates as being resistant to three or more classes of antibiotics and as many as 50% of bacteremia isolates (5, 6). The increase in MDR ExPEC prevalence has been rapid and primarily attributable to a small number of ExPEC lineages (5). The most common of these is the E. coli ST131 lineage, which has rapidly become a dominant cause of ExPEC UTI and bacteremia globally (5–7). E. coli ST131 is particularly associated with carriage of the CTX-M class of extended-spectrum -lactamase (ESBL) which confers resistance to 3rd-generation cephalosporins (7), and there have been a small number of reports of E. coli ST131 isolates carrying metallo- -lactamases conferring resistance to carbapenems (8). The carriage of these resistance genes is driven by the acquisition and stable maintenance of large MDR plasmids (9). The phylogenetic structure of E. coli ST131 is well characterized (10–14) and shows the emergence of a globally disseminated MDR-associated clade C from primarily drug-susceptible clades A and B. March/April 2019 Volume 10 Issue 2 e00644-19 RESULTS Species-wide distribution of the E. coli accessory genome. The multilocus NFDS model devised to explain the impact of vaccination on the S. pneumoniae population was applied to the 1,094 systematically sampled E. coli isolates from the BSAC collection (16). Collected between 2001 and 2011, this collection revealed that the sequence clusters, or strains, corresponding primarily to sequence types ST69 and ST131 rose rapidly at the start of the sampling period, prior to stabilizing at frequencies of 8% and 18%, respectively. The rise of ST131 was primarily driven by increases in MDR clade C isolates, although isolates from ST69 and ST131 clade B rose in frequency despite them lacking such a resistance profile. It is possible this represents drift in a neutral popu- lation. However, this seemed unlikely, given that the invading genotypes rose quickly and displaced some strains, such as ST10, ST14, and ST144, while leaving the common ST73 and ST95 at quite stable frequencies. Alternatively, these strains could each be filling one distinct separate niche. However, these two strains displace multiple other genotypes, suggesting no strict demarcation between niches. Additionally, there was little evidence of any strain being associated with a range of unique traits. Using the previous analysis of gene content with Roary, the 18 strains with at least ten repre- sentatives in the population had a mean of 16.7 private genes (range, 1 to 49), defined as those loci present at 95% in one strain and 5% in all others. However, this does not mean the association between sequence clusters and accessory loci was random. The same Roary analysis identified 8,311 intermediate- frequency genes, present in between 5% and 95% of the overall population analyzed by Kallonen et al. (16) (see Fig. S2 in the supplemental material). Their distribution exhibited a strong association with individual sequence clusters, as defined by an analysis of the core genome (see Materials and Methods), in agreement with recent k-mer-based analyses of this population that showed divergence in the core and accessory genome was strongly correlated (19). Hence, as in S. pneumoniae, strains appear to be defined by a characteristic combination of common accessory loci, rather than distinctive private sequences (14, 20). This suggests sequence clusters may be ecologically distinct but with overlapping niches. We therefore tested whether the population-level frequencies of these intermediate frequency genes were conserved, as following perturbation by vaccination in S. pneu- moniae. Diversification of Colonization Factors in a Multidrug-Resistant Escherichia coli Lineage Evolving under Negative Frequency- Dependent Selection Frequencies of accessory genes were found to be highly conserved across multiple populations on different continents, despite these populations themselves being composed of different strains, as defined by core ge- nome sequences. Detailed modeling and functional analysis indicated changes in strain prevalence could be explained by NFDS driving accessory loci toward equilibrium mbio.asm.org 2 ® Colonization Factors in MDR E. coli frequencies, through mechanisms involving interactions with other bacteria, hosts, or mobile elements (17). The levels of the selective force were estimated to be similar across the populations and manifested in the maintenance of stable population frequencies of accessory loci, despite a substantial perturbation of the population by the introduction of the pneumococcal vaccine (17). Here, we synthesized different genomic analyses to provide a detailed view of the ecology and evolution of ST131. We provide evidence of NFDS shaping the species- wide carried E. coli population using 1,094 systematically sampled bacteremia isolates from the British Society for Antimicrobial Chemotherapy (BSAC) collection. These genomes, and those collated from previous large-scale phylogenomic studies (11–14, 16, 18), allowed a high-resolution analysis of E. coli ST131 using a total of 862 genomes, revealing the steps in the clades’ diversification. This found clade C to have accumu- lated significantly elevated allelic diversity, particularly enriched for genes involved in anaerobic metabolism and other loci important for colonization of the human host by ExPEC. Our data suggest the evolution of the MDR phenotype is part of a wider ongoing adaptation toward prolonged human colonization that permits expansion despite NFDS due to an adaptive radiation through diversification of metabolic and antigenic loci. March/April 2019 Volume 10 Issue 2 e00644-19 RESULTS This shows the changing correlation of gene and Spearman methods, in each year relative to those observed in 2001. Both measures indicate a divergence in until 2010, at which point there is a reversion to the frequencies seen in the original population. (C) Emergence d). The frequencies of the subclades of ST131 are shown by the red dashed lines. 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 Gene frequencies in 2001 Gene frequency ranges post−2001 A 0.80 0.85 0.90 0.95 2002.5 2005.0 2007.5 2010.0 Year Correlation with 2001 frequencies Method Pearson Spearman B B A Method Pearson Spearman on May 8, 2019 by guest http://mbio.asm.org/ Downloaded from C 0.00 0.05 0.10 0.15 0.20 2002.5 2005.0 2007.5 2010.0 Year Frequency in population Strain SC9 CladeA CladeB CladeC SC13 C 0 0 0 Frequency in population Strain SC9 CladeA CladeB CladeC SC13 ay 8, 2019 by guest FIG 1 Summarizing the population dynamics of the British Society for Antimicrobial Chemotherapy extraintestinal pathogenic E. coli collection. These isolates were collected from bacteremia cases around the United Kingdom between 2001 and 2011. (A) Conservation of gene frequencies. Each point corresponds to one of the 6,824 genes identified by Roary in the BSAC collection with mean frequencies between 0.05 and 0.95 across all years. Error bars indicate the full range observed across annual samples. (B) Correlation of gene frequencies with those observed in 2001. This shows the changing correlation of gene frequencies, calculated by both the Pearson and Spearman methods, in each year relative to those observed in 2001. Both measures indicate a divergence in gene frequencies as ST69 and ST131 emerge, until 2010, at which point there is a reversion to the frequencies seen in the original population. (C) Emergence of ST69 (SC9, in orange) and ST131 (SC13, red). The frequencies of the subclades of ST131 are shown by the red dashed lines. samples from until 2008, as might be expected with neutral drift (Fig. 1B), as ST131 and ST69 became more prevalent (Fig. 1C). However, the gene frequencies “bounced back” toward their original frequencies in 2008 (21), as indicated by the increased correlation with the 2001 frequencies (Fig. 1C). This elevated correlation was sustained in subse- quent years, despite the persistently high frequencies of ST131 or ST69, suggesting a reconfiguration of other lineages in the population. Multilocus NFDS modeling of the E. coli population. RESULTS Comparisons between the pre-ST131 2001 samples and subsequent data from up to 2011 found strong linear correlations between the prevalences of their intermediate-frequency genes (Fig. 1A, see also Fig. S3A). This is consistent with these loci existing at “equilibrium” frequencies, determined by their costs and frequency- dependent benefits. These correlations with the first sample, in 2001, weakened with mbio.asm.org 3 March/April 2019 Volume 10 Issue 2 e00644-19 ® McNally et al. 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 Gene frequencies in 2001 Gene frequency ranges post−2001 A 0.80 0.85 0.90 0.95 2002.5 2005.0 2007.5 2010.0 Year Correlation with 2001 frequencies Method Pearson Spearman B 0.00 0.05 0.10 0.15 0.20 2002.5 2005.0 2007.5 2010.0 Year Frequency in population Strain SC9 CladeA CladeB CladeC SC13 C FIG 1 Summarizing the population dynamics of the British Society for Antimicrobial Chemotherapy extraintestinal pathogenic E. coli collection. These isolates were collected from bacteremia cases around the United Kingdom between 2001 and 2011. (A) Conservation of gene frequencies. Each point corresponds to one of the 6,824 genes identified by Roary in the BSAC collection with mean frequencies between 0.05 and 0.95 across all years. Error bars indicate the full range observed across annual samples. (B) Correlation of gene frequencies with those observed in 2001. This shows the changing correlation of gene frequencies, calculated by both the Pearson and Spearman methods, in each year relative to those observed in 2001. Both measures indicate a divergence in gene frequencies as ST69 and ST131 emerge, until 2010, at which point there is a reversion to the frequencies seen in the original population. (C) Emergence of ST69 (SC9, in orange) and ST131 (SC13, red). The frequencies of the subclades of ST131 are shown by the red dashed lines. 0.75 1.00 001 0.80 0.85 0.90 0.95 2002.5 2005.0 2007.5 2010.0 Year Correlation with 2001 frequencies Method Pearson Spearman B 0.00 0.05 0.10 0.15 0.20 2002.5 2005.0 2007.5 2010.0 Year Frequency in population Strain SC9 CladeA CladeB CladeC SC13 C s of the British Society for Antimicrobial Chemotherapy extraintestinal pathogenic E. coli collection. These isolates d the United Kingdom between 2001 and 2011. (A) Conservation of gene frequencies. Each point corresponds to n the BSAC collection with mean frequencies between 0.05 and 0.95 across all years. Error bars indicate the full Correlation of gene frequencies with those observed in 2001. March/April 2019 Volume 10 Issue 2 e00644-19 mbio.asm.org 4 RESULTS To obtain a population-wide view of these dynamics, the previously described multilocus NFDS model was applied to this data set to test whether these strain dynamics were consistent with selection of the phenotypes encoded by a set of accessory loci, L. L was primarily composed of the 7,204 genes, identified by Roary, present at intermediate frequencies (between 5% and 95%) in the 2001 sample. These prevalences were assumed to represent the equilibrium frequencies, el, determined by NFDS in an unperturbed population (17). As only a single sample was available that predated the emergence of ST69 and ST131, these el estimates are likely to be associated with considerable noise; however, the bounce back mbio.asm.org 4 March/April 2019 Volume 10 Issue 2 e00644-19 ® Colonization Factors in MDR E. coli of accessory locus frequencies toward these el suggests they are likely to be accurate for a high proportion of the modeled loci. Additionally, L included seven antibiotic resistance phenotypes present in the same intermediate frequency range. Although mechanistic models disagree as to whether antibiotic resistance is under NFDS directly (22) or linked to other loci under balancing selection (23), the stability of individual phenotype frequencies in this population (16) suggests this simplifying assumption is reasonable. These were then simulated as evolving under NFDS; a fraction pf evolved under strong NFDS, determined by the parameter f, while the rest evolved under weak NFDS, according to parameter w (see Materials and Methods). g p w The model was initialized with the 2001 population, which was seeded with genotypes observed in later years at a low level, representing the possibility they were present in the population but unsampled. Subsequent simulation with a Wright-Fisher framework included these post-2001 genotypes migrating into the population at a rate m. Each isolate was assigned to a sequence cluster, or strain, through identifying clusters in the previously published hierBAPS analysis that approximately corresponded to a multilocus sequence typing clonal complex (see Materials and Methods). The invasion of the resident population by the sequence clusters corresponding to ST131 and ST69 was driven by an increased reproductive fitness relative to the rest of the population, parameterized in the model as r. Fitting this model through approximate Bayesian computation (ABC) using BOLFI estimated the parameters listed in Table S2a, which identified significant evidence for NFDS (f and pf greater than their respective lower bounds used in fitting). RESULTS The 95% credibility interval w was not higher than the lower bound used in fitting, which might represent a genuine absence of NFDS acting on these loci or a lack of power to detect NFDS in this population over this interval. The conclusion that NFDS acting on at least a subset of accessory loci accounts for the observed population dynamics better than equivalent simulations in which NFDS is negligible provides a quantitative gene-level model that explains the previous strain- level observations of Kallonen et al. (16). These simulations successfully reproduced several aspects of the observed data (Fig. 2 and S3B). Both ST131 and ST69 rapidly spread through the population, before stabilizing at an equilibrium frequency of 20%. This does not occur at the expense of the established common clones, such as ST73 and ST95. Instead, in accordance with the genomic data, the displaced sequence clusters include ST10, ST14, ST144, and ST405. These patterns are qualitatively distinct from an equivalent neutral model fit (Fig. 2, bottom). In these simulations lacking NFDS, both ST131 and ST69 are predicted to exponentially increase in frequency rather than rapidly stabilize at a frequency that permits coexistence with the already-resident strains. All other strains commensurately decrease at accelerating rates, proportionate to their original prevalence, meaning the biggest falls are observed for ST73 and ST95, which were actually unperturbed by the invading strains. The greater invasion rate of ST131 relative to ST69 is an artifact of its higher prevalence in the overall data set, meaning it is seeded at a higher level, rather than a true ecological difference. Factors affecting the emergence of ST131. The estimate of the fitness advantage driving the invasion of ST69 and ST131, r, is contingent on aspects of the model structure. The absence of population genomic data predating 2001, or from other locations, makes it difficult to model the initial invasion of ST131. Therefore, isolates from 2002 onwards were seeded in the initial population at a frequency of 103 to replicate the likely scenario in which these genotypes were already present in the United Kingdom or Ireland in 2001 but not captured by the first sample. Increasing the level at which these strains were seeded by 10-fold resulted in a slightly increased final frequency (see Fig. S4). This frequency should represent an approximate upper bound, beyond which it would be unlikely that the strains would have remained unsampled in 2001. mbio.asm.org 6 RESULTS Decreasing the seeding level had an almost negligible effect on the simulated dynamics. Hence, the inferred parameters are not markedly sensitive to the seeding level used in the first time step of the simulations. mbio.asm.org 5 March/April 2019 Volume 10 Issue 2 e00644-19 ® McNally et al. FIG 2 Simulations of changes in the BSAC extraintestinal pathogenic E. coli population evolving under multilocus NFDS. Genomic data (top) and median frequencies (middle) observed from 100 simulations run with the best-matching parameter set identified by fitting the model with BOLFI. This corresponded to f  0.029, r  0.179, m  0.001, pf  0.425, and w  0.0048. Each column corresponds to a sequence cluster identified by hierBAPS (see Materials and Methods) and is annotated with the predominant sequence type with which it is associated. Each bar indicates the frequency of the sequence cluster in (Continued on next page) March/April 2019 Volume 10 Issue 2 e00644-19 mbio.asm.org 6 on May 8, 2019 by guest http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ Downloaded from on May 8, http://mbio.asm.org/ d from FIG 2 Simulations of changes in the BSAC extraintestinal pathogenic E. coli population evolving under multilocus NFDS. Genomic data (top) and median frequencies (middle) observed from 100 simulations run with the best-matching parameter set identified by fitting the model with BOLFI. This corresponded to f  0.029, r  0.179, m  0.001, pf  0.425, and w  0.0048. Each column corresponds to a sequence cluster identified by hierBAPS (see Materials and Methods) and is annotated with the predominant sequence type with which it is associated. Each bar indicates the frequency of the sequence cluster in (Continued on next page) March/April 2019 Volume 10 Issue 2 e00644-19 ® Colonization Factors in MDR E. coli The estimate of r was also affected by the simulated population size. To overcome the greater stochasticity inherent to smaller populations, r must be higher for ST69 and ST131 for them to consistently rise from their low initial frequencies in the early phases of the simulations. Hence, the same r with a 10-fold smaller population than was computationally feasible for fitting (5  104) results in lower median frequencies for ST69 and ST131, whereas maintaining r with a 20-fold increased population size results in the emerging strains reaching a higher prevalence by the end of the simulations (see Fig. S5A). RESULTS Hence, as the effective population size of E. coli across the United Kingdom and Ireland is likely to be greater than 5  104, the fitness advantage driving these emerging strains to invade is probably lower than the r estimated here. The emergence driven by r is limited by NFDS, which generally constrains the invasion of new clusters of genetically coherent isolates, as the traits with which they are associated have a smaller net benefit as they increase in frequency. Simulations in which ST69 and ST131 have the same fitness benefit, r, in the absence of NFDS confirm this advantage would be sufficient to dominate the population in a neutrally evolving population (Fig. S5B). This inhibition would be expected to most strongly affect lineages with very similar sets of accessory loci. As the MDR isolates of clade C share the most recent common ancestor of the three clades, around 25 years ago (11), their expansion would be expected to be subject to the strongest inhibition by NFDS. Their rapid rise suggests the effects of NFDS may have been ameliorated by the diversification of accessory loci, such that the per locus constraint of the equilibrium frequencies does not limit the strain’s overall prevalence. Extensive variation suggestive of this diversity has previously been noted with the capsules expressed within clade C (23). Therefore, the distributions of intermediate frequency loci within each sequence cluster and the clades of ST131 were analyzed by estimating the overall gene content through a Heap’s law analysis of pangenome curves (24) and through pairwise analysis of gene content similarity by estimating genomic fluidity (25). Comparing the metrics across the sets of isolates revealed that ST131 was one of a small number of clades that had an open pangenome but low genomic fluidity (Fig. S4B). This implies each pair of isolates is similar, reflecting their recent shared ancestry; but, many isolates have undergone different diversifying recombinations at one or more loci present at an intermediate frequency in the wider population. This can account for the overall clade containing many loci in total, despite its recent origin. This is consistent with the adaptive radiation of a successful strain expanding in its population size. g consecutive time periods, from left to right. The bars are colored according to the number of antibiotic resistance phenotypes associated with the isolates within the sequence cluster at different time points. (Bottom) The equivalent best fit in the absence of NFDS. Only sequence clusters reaching a frequency of at least 2.5% at one time point in the genomic sample are shown; the full results of the simulation, including measures of between-simulation variation, are shown in Fig. S3. March/April 2019 Volume 10 Issue 2 e00644-19 mbio.asm.org 7 RESULTS The taxa are color coded by BAPS grouping (Table S1) and show clade A (green, BAPS-3), clade B (red, yellow, and purple, BAPS-2, -4, and -5, respectively), and clade C (blue, BAPS-1). FIG 3 (A) Maximum likelihood phylogeny of 862 E. coli ST131 strains. The phylogeny was inferred using RAxML with a GTR Gamma model of substitution, on an alignment of concatenated core CDS as determined by Roary. (B) PANINI plot of the accessory genome content of all 862 strains based on a tSNE plot. The plot is a diagrammatical representation of the relatedness of each strain based on the presence/absence of accessory genes and is presented as a two dimensional representation. The taxa are color coded by BAPS grouping (Table S1) and show clade A (green, BAPS-3), clade B (red, yellow, and purple, BAPS-2, -4, and -5, respectively), and clade C (blue, BAPS-1). discernible substructuring into distinct groups. This concurs with the previous analysis of the gene content of clade C and the previous finding of multiple accessory genome subclusters within this set of isolates (14). Low-frequency accessory genes suggest clade A and clade B/C E. coli ST131 rarely cohabit the same ecological spaces. To identify which aspects of the accessory genome differed between the clades of ST131, the distributions of the 32,631 sets of orthologous genes identified by Roary were analyzed. Characterizing the full set of loci present at intermediate frequencies was not feasible, as even focusing on the 3,354 present at between 5% and 95% frequency revealed that the majority of these were present at a frequency 20% (see Fig. S1). Therefore, the search was refined to clade-specific genes occurring at a frequency of 95% in one clade but at 5% in the other two clades. In both clade A and clade B/C, the overwhelming majority of low-frequency acces- sory genes encode hypothetical proteins (64.4% clade A, 58% clade B/C). Excluding the hypothetical proteins from the analysis showed an unexpected bias in functional gene categories differentially observed in the lineages (Fig. 4). The most common gene types were functional phage, plasmid, and other mobile genetic element (MGE) genes, with more private phage genes present in clade B/C than in clade A. Conversely, there were more private plasmid genes in clade A than in clade B/C, despite the presence of a diverse number of MDR plasmids within clade C (14). RESULTS To better understand this diversification and the basis for the high fitness of ST131 represented by r, a comprehensive genomic data set encompassing all known ST131 genome sequences was created to understand the unique characteristics of the ST131 lineage, with particular focus on the successful clades B and C. Core and accessory genomic structure of the ST131 population. A maximum likelihood phylogeny generated from an alignment of concatenated core coding DNA sequences (CDS) from all 862 genomes confirmed the earlier consensus three-clade structure of the lineage (Fig. 3A), and in agreement with previous studies, there was no strong phylogeographic signal or host source clustering evident in the phylogeny (https://microreact.org/project/BJKoeBt2b). To confirm that the collation of the 862 genomes was consistent with previous descriptions of the accessory genome distribu- tion in ST131, isolate relatedness based on shared accessory gene content was visual- ized as a two-dimensional projection using PANINI (Fig. 3B) (26). Clades A and B largely resided in dense clusters at the periphery of the projection. In contrast, clade C isolates were more diffuse, overlapping with some clade B isolates, forming a cloud with March/April 2019 Volume 10 Issue 2 e00644-19 mbio.asm.org 7 ® McNally et al. FIG 3 (A) Maximum likelihood phylogeny of 862 E. coli ST131 strains. The phylogeny was inferred using RAxML with a GTR Gamma model of substitution, on an alignment of concatenated core CDS as determined by Roary. (B) PANINI plot of the accessory genome content of all 862 strains based on a tSNE plot. The plot is a diagrammatical representation of the relatedness of each strain based on the presence/absence of accessory genes and is presented as a two dimensional representation. The taxa are color coded by BAPS grouping (Table S1) and show clade A (green, BAPS-3), clade B (red, yellow, and purple, BAPS-2, -4, and -5, respectively), and clade C (blue, BAPS-1). McNally et al. FIG 3 (A) Maximum likelihood phylogeny of 862 E. coli ST131 strains. The phylogeny was inferred using RAxML with a GTR Gamma model of substitution, on an alignment of concatenated core CDS as determined by Roary. (B) PANINI plot of the accessory genome content of all 862 strains based on a tSNE plot. The plot is a diagrammatical representation of the relatedness of each strain based on the presence/absence of accessory genes and is presented as a two dimensional representation. March/April 2019 Volume 10 Issue 2 e00644-19 RESULTS Together, this suggests that clade A strains of E. coli ST131 and clade B/C strains of E. coli ST131 are exposed to different plasmid and phage pools, an observation which is most parsimoniously explained by them rarely sharing the same ecological habitats at the same time. Clade A contained the highest number of loci exclusive to a lineage (27) despite constituting the least-sampled clade. This is likely explained by the fact that clade A is on the longest branch of the ST131 phylogeny, and as such, has had most evolutionary time to accumulate differences in the gene repertoire. Clade B had only 2 exclusive loci and clade C had 18. When clades B and C were combined against clade A, there were 60 loci exclusively present in the B/C combination. The majority of clade A private genes encode hypothetical proteins, while those private to clade C encode DNA mbio.asm.org 8 FIG 4 Bar chart depicting functional classes of accessory genes differentially present in clade A and clade B/C E. coli ST131. Functional classes are based on GO classes as described in Materials and Methods. *, significant difference exists between clade A and clade C as determined by t test. Colonization Factors in MDR E. coli ® Colonization Factors in MDR E. coli on May 8, 2019 by http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ aded from FIG 4 Bar chart depicting functional classes of accessory genes differentially present in clade A and clade B/C E. coli ST131. Functional classes are based on GO classes as described in Materials and Methods. *, significant difference exists between clade A and clade C as determined by t test. modification proteins and metabolic functions. The genes private to clade B/C com- bined also encode hypothetical proteins and metabolic functions, notably, five dehy- drogenase enzymes involved in anaerobic metabolism labeled yihV, garR_3, fadJ, fdhD, and gnd in our data set. A BLAST analysis against the NCBI nonredundant database suggested that the dehydrogenase enzyme gene annotated as pdxA in our Roary data set was confined to clade C ST131 strains. These dehydrogenase enzyme genes were found to be present across phylogroup B2 E. coli strains (of which ST131 is a member) through blastn searches of the NCBI nonredundant database. March/April 2019 Volume 10 Issue 2 e00644-19 RESULTS coli ST131, based on concatenated core CDS, with the presence of alternative alleles of 64 loci involved in anaerobic metabolism. Each blue box along the top of the tree annotation represents an individual anaerobic metabolism gene, and its presence in the ST131 population is indicated by a blue line. The inset is a bar chart displaying the proportions of the accessory pangenome that are occupied by genes involved in anaerobic metabolism for ST131 clade A, clade B, subsampled clade C versus clade A, and subsampled clade C versus clade B. P  0.042 for clade C versus clade A and P  0.086 for clade C versus clade B. Error bars represent standard errors of the means. Significance was determined using the median value P value from chi-square tests performed on random subsamples of the C clade. sensor kinase for activation of the cobalamin biosynthesis operon. These anaerobic metabolism genes would normally be considered essential to E. coli. Interrogation of the pangenome matrix for the data set showed that versions of these genes existed as core genome loci. Comparison of the nucleotide sequences of the clade-specific loci with their core genome counterparts showed that these were not genes private to clade B/C per se but rather represented multiple unique alleles of genes that are core to the ST131 population, which differ at the nucleotide sequence level by more than 5%. This suggests a unique selection pressure is acting on these core genes in clade B/C compared to those in clade A. The discovery of these core genes that display allelic variation is explained by the fact that the percent nucleotide identity cutoff applied in the formation of our pangenome matrix is 95%, meaning that any alternative alleles of core genes differing by 5% or more will appear in our matrix as accessory genes. Further scrutiny of low-frequency loci in clade B/C also identified alternative alleles of a large number of well-characterized extraintestinal pathogenic E. March/April 2019 Volume 10 Issue 2 e00644-19 RESULTS Therefore, these loci are not unique to clade C ST131 and were either acquired by an ancestral clade B/C strain or have been lost by clade A. High diversity in core anaerobic metabolism genes unique to clade B/C. An analysis of accessory loci private to clade B/C (present in 95% of that population) identified two separate loci encoding 3-hydroxyisobutyrate dehydrogenase enzymes and loci encoding 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-CoA) dehydrogenase, 6-phosphogluconate dehydrogenase, and formate dehydrogenase. An analysis of clade B/C loci circulating at a low frequency of 20% also identified a significant overrep- resentation of genes encoding dehydrogenase enzymes involved in anaerobic metab- olism (a total of 64 loci), including seven variants of formate dehydrogenase. There were also seven variants of the eutA gene found in the ethanolamine utilization pathway (the eut operon) and a distinct version the cobW gene, which encodes the mbio.asm.org 9 ® McNally et al. Clade A Clade C vs. Clade A Clade B Clade C vs. Clade B 0.000 0.005 0.010 0.015 0.020 0.025 Proportion of Anaerobic Metabolic Genes in Acessory Pangenome * FIG 5 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of 64 loci involved in anaerobic metabolism. Each blue box along the top of the tree annotation represents an individual anaerobic metabolism gene, and its presence in the ST131 population is indicated by a blue line. The inset is a bar chart displaying the proportions of the accessory pangenome that are occupied by genes involved in anaerobic metabolism for ST131 clade A, clade B, subsampled clade C versus clade A, and subsampled clade C versus clade B. P  0.042 for clade C versus clade A and P  0.086 for clade C versus clade B. Error bars represent standard errors of the means. Significance was determined using the median value P value from chi-square tests performed on random subsamples of the C clade. Clade A Clade C vs. Clade A Clade B Clade C vs. Clade B 0.000 0.005 0.010 0.015 0.020 0.025 Proportion of Anaerobic Metabolic Genes in Acessory Pangenome * on May 8, 2019 by http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ aded from FIG 5 Annotation of a maximum likelihood phylogeny of E. mbio.asm.org 10 RESULTS Cell division Capsule on May 8, 2019 by http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ aded from FIG 6 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of loci involved in capsule production, cell division, iron acquisition, pili/fimbriae production, flagella, and MDR efflux pumps. Each box represents an individual gene, and its presence in the ST131 population is indicated by an appropriately colored line. from other sources. Given that these alleles differ from the normal conserved versions of genes by 5% at the nucleotide level, it is implausible that these alleles would be arising repeatedly and independently via mutation. Instead, the most parsimonious explanation is that the minor frequency alternative alleles are being distributed through the population via recombination. This conclusion is supported by the fact that every one of the allele variants identified in our analysis has 100% nucleotide identity matches with genes present in other E. coli in the NCBI nonredundant database. g Given that our data set is biased toward clade C genomes, we performed compar- ative analyses of the frequency with which allelic diversity occurs in anaerobic metab- olism genes. We randomly subsampled clade C 100 times and compared equal num- bers of clade A, B, and C genomes for allelic diversity. Our data show that even when randomly subsampling clade C, the levels of diversity observed in anaerobic metabo- lism genes is significantly higher than in clade A, providing evidence that the accumu- lation of sequence diversity is specific to the MDR clade C, despite this clade having the most recent common ancestor of the three (Fig. 5). Finally, we sought to exclude the possibility that the presence of these allelic variants was skewed by some form of geographically localized expansion of variants. To do this, we compared the relative frequencies of all accessory genes, highlight- ing the allele variants in anaerobic metabolism, capsule, cell division, MDR efflux, iron acquisition, fimbriae, and flagella present in U.K. versus non-U.K. isolate genomes (see Fig. S6). March/April 2019 Volume 10 Issue 2 e00644-19 RESULTS coli virulence- associated genes: antigen 43 (7 alternative alleles); heavy metal resistance, such as arsenic (5 loci), copper (4 loci), and mercury (5 loci); capsule biosynthesis (20 loci); cell division and septation (14 loci); antibiotic resistance to chloramphenicol (3 loci), mac- rolides (2 loci), rifampin (1 locus), and MDR efflux pumps (21 loci); iron acquisition (39 loci); curli and type I fimbriae and P pili (42 loci); lateral and classical flagella (26 loci); and LPS synthesis (9 loci). These variant loci represent alternative alleles of genes found widely across the E. coli phylogeny, indicating there are multiple allelic variants of important genes that are confined to clade B/C of the E. coli ST131 lineage. We sought to determine the distribution of this allelic diversity across the E. coli ST131 phylogeny by annotating the tips of the phylogenetic tree with the presence/ absence of each of the anaerobic metabolism (Fig. 5) and capsule, cell division, MDR efflux, iron acquisition, pili, and flagellum divergent loci (Fig. 6). Our analysis shows that each alternative allele occurs at very low frequency, but that alleles are randomly distributed throughout the phylogeny of the C clade and are exclusive to clade C. An analysis of the source attribution of isolates displaying this allelic diversity showed that 80% of isolates displaying allelic diversity were human isolates (32% of those blood- stream isolates, 20% UTI isolates, 2% commensal, and the remainder of an unknown source), 5% were avian isolates, 10% companion animal isolates, and the remainder mbio.asm.org 10 ® Colonization Factors in MDR E. coli Capsule Cell division Iron acquisition Pili/fimbriae Flagella MDR efflux FIG 6 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of loci involved in capsule production, cell division, iron acquisition, pili/fimbriae production, flagella, and MDR efflux pumps. Each box represents an individual gene, and its presence in the ST131 population is indicated by an appropriately colored line. Capsule Cell division Iron acquisition Pili/fimbriae Flagella MDR efflux FIG 6 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of loci involved in capsule production, cell division, iron acquisition, pili/fimbriae production, flagella, and MDR efflux pumps. Each box represents an individual gene, and its presence in the ST131 population is indicated by an appropriately colored line. RESULTS Iterative chi-square tests were performed to assess significance, as described in Materials and Methods. *, P  0.05; **, P  0.01; ***, P  0.001. 0.00 0.05 0.10 0.15 0.20 0.25 Phage Genetic Processing Mobile Genetic Elements Metabolism Protein of Unknown Function Plasmid Molecular Transport Virulence Environmental Interaction Conjugal Transfer Miscellaneous Adhesion Anaerobic Metabolism Antimicrobial Resistance Cell Division Outer Membrane Proteins Iron Acquisition Inner Membrane Proteins Cell Wall Flagella Ribosome Proportion of accessory pangenome Subsampled ST131 ST73 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * A 0.00 0.05 0.10 0.15 0.20 0.25 Phage Genetic Processing Mobile Genetic Elements Metabolism Protein of Unknown Function Plasmid Molecular Transport Virulence Environmental Interaction Conjugal Transfer Miscellaneous Adhesion Anaerobic Metabolism Antimicrobial Resistance Cell Division Outer Membrane Proteins Iron Acquisition Inner Membrane Proteins Cell Wall Flagella Ribosome Proportion of accessory pangenome Subsampled ST131 ST95 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * B B A on May 8, 2019 by http://mbio.asm.org/ Downloaded from on May 8, 2019 by guest http://mbio.asm.org/ Downloaded from Proportion of accessory pangenome on May 8, 2019 by guest http://mbio.asm.org/ aded from FIG 7 Bar charts depicting the compositions of the accessory genomes of ST73 (A) and ST95 (B) compared to a repetitively sampled clade C ST131. The proportions of the accessory genome are plotted against manually assigned functional categories. Hypothetical proteins are responsible for the majority of the accessory pangenome and are omitted from the graphs. Error bars are standard errors of the means. Iterative chi-square tests were performed to assess significance, as described in Materials and Methods. *, P  0.05; **, P  0.01; ***, P  0.001. of the most dominant lineages associated with clinical extraintestinal disease alongside ST131 (5, 16) but are predominantly non-MDR lineages and rarely associated with MDR plasmids (16). As with our interclade comparisons, we randomly subsampled clade C ST131 100 times to allow equal numbers of genomes per lineage to be compared. RESULTS Our data showed a strong linear relationship between the frequency of genes in the two populations, indicating that the data were not biased by expansion of alleles in a given geographical location, and that this accumulated diversity was equally as likely to happen in any given strain independent of its geographical origin. Allelic diversity of anaerobic metabolism genes in clade C ST131 is not ob- served in other dominant ExPEC lineages. The possibility exists that the above observations made for clade C of E. coli ST131 simply reflect the general evolutionary path of a successful commensal and extraintestinal pathogen. To test this, we per- formed an identical analysis on the pangenome of 261 ST73 isolates and of 160 ST95 isolates from the U.K. BSAC population survey (16). E. coli ST73 and ST95 represent two mbio.asm.org 11 March/April 2019 Volume 10 Issue 2 e00644-19 ® McNally et al. 0.00 0.05 0.10 0.15 0.20 0.25 Phage Genetic Processing Mobile Genetic Elements Metabolism Protein of Unknown Function Plasmid Molecular Transport Virulence Environmental Interaction Conjugal Transfer Miscellaneous Adhesion Anaerobic Metabolism Antimicrobial Resistance Cell Division Outer Membrane Proteins Iron Acquisition Inner Membrane Proteins Cell Wall Flagella Ribosome Proportion of accessory pangenome Subsampled ST131 ST73 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 0.00 0.05 0.10 0.15 0.20 0.25 Phage Genetic Processing Mobile Genetic Elements Metabolism Protein of Unknown Function Plasmid Molecular Transport Virulence Environmental Interaction Conjugal Transfer Miscellaneous Adhesion Anaerobic Metabolism Antimicrobial Resistance Cell Division Outer Membrane Proteins Iron Acquisition Inner Membrane Proteins Cell Wall Flagella Ribosome Proportion of accessory pangenome Subsampled ST131 ST95 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * A B FIG 7 Bar charts depicting the compositions of the accessory genomes of ST73 (A) and ST95 (B) compared to a repetitively sampled clade C ST131. The proportions of the accessory genome are plotted against manually assigned functional categories. Hypothetical proteins are responsible for the majority of the accessory pangenome and are omitted from the graphs. Error bars are standard errors of the means. March/April 2019 Volume 10 Issue 2 e00644-19 DISCUSSION The evolutionary events that led to the emergence of E. coli ST131 have been an intense focus of research, with consensus opinion suggesting that following the acquisition of key ExPEC virulence factors, the acquisition of fluoroquinolone resistance in the 1980s by the clade C sublineage of ST131 was a key event in that emergence (11, 12). However, a recent nationwide U.K. population survey rejected this hypothesis and suggested that success of the major ExPEC clones is not dictated by resistance traits (16). Here, we combined an analysis of population frequency dynamics with an analysis of the pangenome of the ST131 lineage, strongly suggesting that this species’ popu- lation structure and dynamics are shaped by NFDS acting on genomic islands. Such multilocus NFDS is able to account for how an otherwise stable population was disrupted by the invasion of ST131 and ST69, displacing some lineages while leaving other, largely antibiotic-susceptible, genotypes at almost untouched prevalence. Previous work has suggested that clade C strains of E. coli ST131 undergo reduced levels of detectable core genome recombination compared to those of other phylo- group B2 E. coli (30) or ST131 clade A strains (14). We have previously postulated that this may be a result of ecological separation between clade C strains and other common ExPEC (14, 30). Our analysis of nearly 900 genomes has allowed us to interrogate accessory gene movement to a far greater resolution than previously possible. From the analysis of the accessory genome, we identified thousands of plasmid, phage, and other mobile genetic element genes which are private to clade A and the combined clade B/C. Such an observation is a classic signature of ecological separation of the two populations (31, 32), particularly given that the genetic distance between clade A and clade B/C is much smaller than between other lineages and species from which the circulating genes are also found in the NCBI nonredundant database. Our analysis also identified a significantly increased level of sequence diversity in genes involved in key host colonization processes in clade C. This diversity was uncovered through our pangenome analysis as allelic variants of core genes. Primary among these is a large number of genes involved in anaerobic metabolism, including seven allelic variants of the formate dehydrogenase gene, as well as allelic variants of genes involved in ethanolamine utilization and cobalamin biosynthesis. RESULTS Our analysis showed higher levels of plasmid, phage, and hypothetical proteins in the accessory genomes of both ST73 and ST95 than in ST131 (Fig. 7). ST73 and ST95 displayed similar ratios of alternative alleles in adhesins, cell division, and septation genes and multiple iron acquisition genes as observed in ST131 but a higher diversity in virulence-associated genes. However, enrichment in allelic variation in anaerobic metabolism genes was significantly higher in any given subsampled set of clade C ST131 genomes than in both lineages. This supports the hypothesis that the observa- tion of increased diversity accumulating in anaerobic metabolism genes is not a more general extraintestinal pathogenic E. coli trait but is particularly enriched in the ST131 lineage. It also suggests that this is independent of diversification of adhesins or virulence factors, which was enhanced in the ST73 and ST95 lineages. The accumulation of nucleotide diversity in a given set of loci can often be interpreted as a signature of some form of selection occurring on those genes. However, the low levels of frequency of any given allele across clade C strains contradict a hypothesis for positive selection, where one would expect successful or beneficial alleles to sweep to a high frequency or fixation. Indeed, a comparison of the sequences of each of anaerobic metabolism loci in which three or more alleles were observed identified just three loci which showed a mixture of weakly positive and highly negative Tajima’s D scores, indicating no common selective mechanism in these loci (Table S2b). However, these results can be reconciled with a lineage evolving under NFDS. Different resource use strategies can facilitate coexistence between competing strains, such those cocolonizing a host, resulting in frequency-dependent selection (28, 29). This would explain the sustained intermediate frequencies of genes encoding dehy- drogenases over multiple years (see Fig. S7). Hence, this diversification of metabolic loci could represent the adaptive radiation of particular traits within a successful genetic background, able to efficiently compete with the resident E. coli population through a diverse panel of metabolic capacities suited to exploiting resources under anaerobic conditions. mbio.asm.org 12 ® Colonization Factors in MDR E. coli March/April 2019 Volume 10 Issue 2 e00644-19 DISCUSSION The pivotal role of ethanolamine production and cobalamin biosynthesis in the ability of Gram-negative pathogens to outcompete bacteria in the human intestine is well documented (33, 34), and this phenomenon only occurs when supported by an increased ability to perform anaerobic respiration in the presence of inflammation (33). It has been shown that MDR E. coli ST131 is able to colonize the gastrointestinal tract of humans for months or years in the absence of antibiotic selection (35, 36) and that this colonization results in a displacement of the E. coli colonizing the host prior to exposure to the MDR strain (35). While this diversity in anaerobic metabolism genes was unique to clade C ST131, the allelic variation observed in other human colonization and virulence factors, such as iron acquisition, fimbriae, and cell division, was also observed in two of the other most commonly isolated lineages of E. coli from extraintestinal infections, ST73 and ST95. This diversity likely reflects selection occurring on genes important for ExPEC patho- genesis. Iron acquisition is well characterized as a key virulence determinant in ExPEC, with the ability to initiate successful UTI completely abrogated in the absence of functional iron acquisition systems (37). Recent experimental vaccine work exploiting siderophore production by ExPEC has shown it to be highly effective in rodent models on ExPEC UTI (38). The importance of iron acquisition can also explain many of the MDR efflux allele variants seen in this data set, with half occurring in the acrD gene, which has been experimentally shown to play a role in iron acquisition in E. coli (39). We identified multiple alleles of genes in the type 1 fimbria operon and in genes in the P pilus operon, which are classical virulence determinants in UTI (40), and multiple genes involved in capsule biosynthesis, which we have previously reported as being a hot spot for recombination in E. coli ST131 (13, 41). We also identified multiple alleles of mbio.asm.org 13 March/April 2019 Volume 10 Issue 2 e00644-19 ® McNally et al. genes involved in controlling incomplete septation and filamentous growth, which is a crucial process in the formation of the filamentous intracellular bacterial communities (IBCs) which are thought to be fundamental in the ability of ExPEC to survive inside bladder epithelial cells and cause UTI (42). DISCUSSION There are a small number of allelic variants in anaerobic metabolism genes also present in ST73 and ST95, possibly reflecting recent experimental studies suggesting a crucial role for the cytochrome bd oxidase system in the ability to cause urinary tract infection (43). Also, previous studies using saturated mutagenesis techniques and studying global transcriptional patterns during urinary tract infection of ExPEC strains have suggested a key role for dehydrogenase enzymes involved in anaerobic metabolism in the ability to cause pathology in the mammalian urinary tract (27, 44, 45). y Recent modeling data on why drug-resistant and drug-susceptible populations of bacteria coexist highlighted that any factors which increase the duration of colonization in a human host will also increase the selective pressure for them to evolve antibiotic resistance (23). Hence, both the success of ST131 in invading the population and the association of many isolates in this lineage with an MDR phenotype would be consis- tent with the accumulating diversity in anaerobic metabolism loci facilitating enhanced persistence within its host, perhaps through an improved ability to outcompete resident commensal E. coli strains. The fact that this selection is only seen in clade C of ST131 suggests that this occurred around the time of the emergence of the lineage as a human clinical threat (13) alongside the development of fluoroquinolone resistance. Subsequent acquisition of MDR plasmids, and the consequent selection for an ability to offset the fitness costs of long-term MDR plasmid maintenance (14), is likely to have occurred as a result of prolonged exposure to selective antibiotic environments during colonization of humans. Nevertheless, neither anaerobic metabolism genes nor antibi- otic resistance loci have swept to fixation in ST131, reflecting their fluctuating but stable prevalence in the broader E. coli population. This diversification can instead be explained by NFDS, under which these genes are beneficial when rare, because they provide an advantage over cocolonizing strains which will typically lack the same metabolic capacities. However, as these traits become more common as ST131 expands, representatives of this lineage will more commonly encounter one another, therefore necessitating further diversification for different clade C represen- tatives to sustain their advantage over competitors. DISCUSSION Similarly, the capsule locus diversifica- tion previously observed within clade C, resulting in the capsule synthesis locus corre- sponding to a “hot spot” of recombination (23), could result from NFDS on variable antigens (46), with the host immune system selecting for a diversity of capsule structures as the dominant type becomes more common following ST131’s emergence (16). yp g g This study presents evidence for two complementary aspects of diversification within the E. coli ST131 lineage. The first is ecological niche separation across a lineage, whereby the different clades of ST131 find themselves competing for similar niches, resulting in them very rarely cohabiting the same habitat. This then results in the formation of distinct subclades within ST131. The second is NFDS across a species within the specific environment of the human gut and the diversification of key genes involved in the colonization of the human gut niche to escape the constraints imposed by NFDS. Further studies are required to fully determine the extent to which niche separation and NFDS are either separate or linked processes. Determining whether loci subject to NFDS are also those that determine niche adaptation will be integral to this process. Understanding the processes that govern the epidemiological dynamics of dominant E. coli lineages, and those of similar pathogens causing bloodstream infec- tions, is critical for addressing the public health threat of antibiotic resistance. March/April 2019 Volume 10 Issue 2 e00644-19 MATERIALS AND METHODS Genome data. For the NFDS modeling study, the 1,094 isolates from the BSAC resistance surveillance bacteremia collection from the United Kingdom and Ireland between 2001 and 2011 were used. These were described by Kallonen et al. (16). For the ST131-specific study, a collection of 862 E. coli ST131 genomes (see Table S1 in the supplemental material), of which 684 were previously sequenced as part of phylogenomic investigations mbio.asm.org 14 March/April 2019 Volume 10 Issue 2 e00644-19 ® Colonization Factors in MDR E. coli of the ST131 lineage (10, 11, 13, 14, 16, 47), was analyzed. An additional 184 ST131 isolates from the BSAC bacteremia resistance surveillance project conducted in the United Kingdom and Ireland between 2001 and 2011 (indicated in Table S1) were included. Together, this collection represents bacteria isolated from invasive disease (bloodstream infections, 504 isolates; 58.5%), human asymptomatic carriage (21 isolates, 2.4%), disease resulting from intestinal carriage (UTI, 163 isolates; 18.9%), and bacteria isolated from a range of veterinary livestock, pets, wild birds, and the wider environment (67 isolates, 7.8%). This provides a data set isolated between 1967 and 2013, providing as broad a view of isolate origin as possible within existing data sets. In an attempt to avoid any issues arising from different assembly or annotation metrics employed in the previous projects, only raw sequence data were downloaded in fastq format using the previously published accession data. De novo assembly was then performed on all the genomes using Velvet (48) and annotation using Prokka (49) as previously described (16). A pangenome of the entire data set was constructed using Roary with 95% identity cutoff (50). A concatenated core CDS alignment was made from the Roary output, and a maximum likelihood phylogenetic tree was constructed from the alignment using RaxML version 8.2.8 (51) and the GTR model with Gamma rate heterogeneity. For comparative lineage analysis, 264 ST73 genomes and 162 ST95 genomes that were sequenced and fully characterized as part of the U.K. BSAC genome study (16) were utilized. Multilocus NFDS modeling. Multilocus NFDS modeling used genomic data from the previous publication analyzing the population dynamic of bloodstream infection E. coli isolates in the United Kingdom (16). This first required splitting the population into genetically coherent clusters of isolates. MATERIALS AND METHODS This is necessary, as the sequence cluster (or “strain”) frequencies are used for model fitting; therefore, the clusters must be coherent enough to behave reproducibly but large enough to overcome the stochasticity of the model. The previous hierBAPS analysis of the core genome provided three levels of hierarchical clustering (52). The highest-level clusters corresponded to individual common strains in some cases but polyphyletic collections of diverse genotypes in others. Therefore, to ensure the sequence clusters used in the model were of similar levels of diversity, available sequence typing data were used to identify the level of clustering that corresponded to clonal complexes. Each of the highest-level hierBAPS clusters was tested by constructing links between single- and double-locus variants. If the isolates formed a single clonal complex, the cluster was defined as a sequence cluster. Otherwise, the second level of clustering was tested in the same way; if these groups represented a clonal complex, they were used as a sequence cluster. Otherwise, the third most fine-grained level of clustering was used to define a sequence cluster. This identified 56 sequence clusters across the population. The multilocus NFDS model also requires the definition of a set of L loci, each of which is associated with an equilibrium population-wide frequency, el. The majority of these correspond to accessory genetic loci, identified as sets of orthologous sequences by a previous Roary analysis (16). Only the 7,204 present at between 5% and 95% frequency in the first sample, from 2001, were included in L and therefore modelled as evolving toward el under NFDS. The set of L loci was supplemented with seven loci associated with resistance phenotypes if they were present within this frequency range in 2001: amoxicillin, clavulanic acid, ciprofloxacin, cefuroxime, gentamicin, piperacillin-tazobactam, and trimethoprim. The first six of these were directly inferred from the previously published analysis. Trimethoprim was instead inferred from the sul and dfrA alleles identified by Roary; data from the Cambridge University Hospitals collection (16) was used to train a model constructed with the randomForest R library (https://cran.r-project.org/web/packages/ randomForest/), which had 93% accuracy when applied back to the training data set. This was used to infer resistance phenotypes for the BSAC collection. Analysis used the heterogeneous multilocus NFDS model described previously (17), modified to treat a vaccine cost, v, as a fitness advantage, r. March/April 2019 Volume 10 Issue 2 e00644-19 MATERIALS AND METHODS This represents the likely scenario in which they were present in the sampled region in 2001 but were not observed in the necessarily finite set of sequenced isolates. The parameter m represented the rate at which all isolates entered the population through migration; this was biased to import all sequence clusters at the same rate, to avoid any fits in which high rates of migration would artifactually replicate the population observed in the later years of the collection (17). Model fitting to genomic data. As in Corander et al. (17), the simulation model was fitted through approximate Bayesian computation (ABC) using the BOLFI algorithm, which has been shown to accel- erate ABC inference 1,000 to 10,000 times without loss of accuracy (53). The prior constraints placed on the parameter values were as follows: the lower bound on all parameters was set to 0.0009; the upper bounds were 0.99 for ri, 0.2 for m, 0.99 for pf, 0.03 for f, and 0.005 for w. We used 500 iterations of the BOLFI algorithm to minimize the Jensen-Shannon divergence of the sequence cluster frequencies in the genomic data and in the simulations, as ascertained through randomly sampling discrete sets of isolates in accordance with the size and timings of the genomes selected for sequencing from the original collection. Convergence of BOLFI was monitored after every 100 iterations and the approximate likelihood estimate was assessed to have been stabilized after 500 iterations (53). The 95% posterior credible intervals for the parameters were obtained using three generations of sequential Monte Carlo sampling with the same default settings as used in Corander et al. (17). The neutral model was fitted by fixing pf, f, and w at zero and estimating r and m through 500 iterations of the BOLFI algorithm, followed by sequential Monte Carlo sampling, as with the full model. Accessory genome analysis. The pangenome matrix from Roary was utilized to investigate the presence of clade-specific loci. The PANINI tool was used with the default setting to visualize the accessory gene-sharing patterns in the population available at https://microreact.org/project/BJKoeBt2b (26). PANINI has been demonstrated to provide efficient complementary visual means to phylogenetic trees to accurately extract both distinct lineages present in a population-wide genomic data set and to highlight clusters within lineages, which are explained by rapidly occurring, homoplasic alterations, such as phage infection. MATERIALS AND METHODS To simulate the invasion of the resident population by ST131 and ST69, all individuals, i, of the two sequence clusters containing ST131 (and related sequence types ST5640, ST5387, ST5494, ST5432, and ST5630) and ST69 (and related sequence type ST106) were assigned the same fitness advantage, ri  r. For all other individuals in the population, ri  0. Hence, the function defining the number of progeny, Xi,t, produced by i at time t was Xi,t  Pois  Nt1  ri1  m1  f i,t  1  w i,t In this formula, density-dependent competition is parameterized by the carrying capacity , set at 50,000 to represent a large population that is still computationally feasible, and the total number of cells in the simulated population at t, Nt. The strength of NFDS was determined by the parameters pf, f, and w. As previously, the set of L accessory loci and resistance phenotypes were ordered according to the statistic Δl: l fl,t 0  el 2 1  el1  el where fl,t0 is the mean post-2001 locus frequency. If the L loci and phenotypes considered to be under NFDS were ordered by ascending values of Δl, then lf was the highest ranking locus meeting the criterion l lf L pf. This determined the strength of NFDS acting on each locus. Therefore, the effect of NFDS on the reproductive fitness of individual i depended on which loci were encoded in its genome, as represented by the binary variable gi,l, and on the deviation of their simulated frequencies at time t, fl,t, from their corresponding equilibrium frequencies: mbio.asm.org 15 March/April 2019 Volume 10 Issue 2 e00644-19 ® McNally et al. McNally et al. and i,t  l lf 1 L gi,l el  fl,t These summed deviations served as the exponents for the NFDS terms of the reproductive fitness, with i,t and f corresponding to those loci under stronger NFDS, and i,t and w corresponding to those loci under weaker NFDS. The simulations were initialized with a random selection of  genotypes from the genomic data, which were biased such that those isolates observed in 2001 were represented at 1,000-fold greater frequency than genotypes collected in later years. This was necessary to “seed” the initial population with ST131 and ST69, to facilitate their expansion in a realistic manner in subsequent years. March/April 2019 Volume 10 Issue 2 e00644-19 MATERIALS AND METHODS Roary was run on the entire data set using the default 95% sequence identity threshold to cluster genes, allowing the separation of genes based on allelic as well functional differences. Based on a frequency distribution histogram (Fig. S1), a locus as assigned as being clade specific if it occurred at a frequency 95% in one clade and at 5% in the other two clades. Loci identified as clade specific were functionally annotated by performing a tblastn analysis of the nucleotide sequence of the loci against the NCBI nonredundant database. Functional categorization of pangenomes. To assess the functional composition of the accessory pangenome, Gene Ontology (GO) terms were assigned to gene sequences from the pangenome. Briefly, representative sequences from the pan genome of ST131 were mapped to orthologous groups in the bactNOG database using the eggNOG emapper utility (54) Mapping was performed using the diamond search algorithm. The output from eggNOG was filtered to remove orthologous groups with no GO terms. The number of genes mapping to an orthologous group was used as a score for GO term enrichment analysis. y Comparisons of lineage and clade-specific loci. To compare lineage pangenomes while account- ing for differences in the number of genomes, a sampling approach was utilized. Specifically, a subset with a size equal either to the number of ST73 or ST95 genomes was selected at random from the ST131 clade C, while ensuring a cross representation of the phylogenetic diversity of the clade. The functional enrichment of genes in the subset was quantified and statistically compared to the ST73 or ST95 pangenome using a chi-square test. This process was repeated 100 times to produce 100 P values, from which the median P value was calculated. Utilizing the same subsampling approach, the pangenome composition of clade C ST131 genomes was compared to both the clade A and clade B pangenomes. Chi-square statistical tests were performed to assess the significance of the observed differences in functional enrichment. Briefly, with each iteration of the sampling procedure, a chi-square test was performed using the functional proportion of the subsampled pangenome as the observed value and the proportion for ST73 or ST95 as the expected value. This generated 100 P values from which one can use the average, maximum, or median to assess significance of the observed differences. In addition, proportional Z statistic tests were also performed to assess the significance of the observed difference. REFERENCES 1-producing Escherichia coli isolates that belong to the successful and virulent clone ST131. 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MATERIALS AND METHODS The measurements from the 100 replicates of the subsampling procedure were used to generate an average for the proportions as well as to estimate the variance. The tests were conducted using the proportional measurements from ST73 and ST95 as the “true” means and quantifying how distinct the ST131 subsamples were from these reference values. Comparisons of lineage and clade-specific loci. To compare lineage pangenomes while account- ing for differences in the number of genomes, a sampling approach was utilized. Specifically, a subset with a size equal either to the number of ST73 or ST95 genomes was selected at random from the ST131 clade C, while ensuring a cross representation of the phylogenetic diversity of the clade. The functional enrichment of genes in the subset was quantified and statistically compared to the ST73 or ST95 pangenome using a chi-square test. This process was repeated 100 times to produce 100 P values, from which the median P value was calculated. Utilizing the same subsampling approach, the pangenome composition of clade C ST131 genomes was compared to both the clade A and clade B pangenomes. p g p p g Chi-square statistical tests were performed to assess the significance of the observed differences in functional enrichment. Briefly, with each iteration of the sampling procedure, a chi-square test was performed using the functional proportion of the subsampled pangenome as the observed value and the proportion for ST73 or ST95 as the expected value. This generated 100 P values from which one can use the average, maximum, or median to assess significance of the observed differences. In addition, proportional Z statistic tests were also performed to assess the significance of the observed difference. The measurements from the 100 replicates of the subsampling procedure were used to generate an average for the proportions as well as to estimate the variance. The tests were conducted using the proportional measurements from ST73 and ST95 as the “true” means and quantifying how distinct the ST131 subsamples were from these reference values. mbio.asm.org 16 ® Colonization Factors in MDR E. coli Analysis of diversity observed in genes involved in anaerobic metabolism. The sequences of 64 anaerobic metabolic genes in which allelic diversity was observed were extracted from individual genomes. The nucleotide sequences were then clustered at 80% identity and 80% length using CD-HIT, which was run using the accurate flag and “word size” of 5 (55). MATERIALS AND METHODS An additional CD-HIT script was used to extract gene sequences for clusters with more than 3 genes, the minimum required by MEGA-CC for analysis. The sequences were then aligned using Muscle with default settings (56). The resulting alignment files were analyzed in MEGA-CC to produce measurements of Tajima’s D (57). ST131 clade-specific SNPs. To visualize the ST131 clades A, B, C, C1, and C2 within the ML tree and the PANINI clustering we identified clade-specific single-nucleotide polymorphisms (SNPs) (Table S1) as previously described (16). Data availability. Accession numbers for the reads used in this study are listed in Table S1 with information of year and place of isolation and the results of the in silico PCR for clade-specific SNPs. ACKNOWLEDGMENTS This work was supported by the Health Innovation Challenge Fund (HICF-T5-342 and WT098600), a parallel funding partnership between the U.K. Department of Health and Wellcome. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health, Public Health England, or Wellcome. T.K. was funded by the Norwegian Research Council JPIAMR (grant number 144501). J.C. was funded by the ERC (grant number 742158). N.J.C. is funded by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and Royal Society (grant number 104169/Z/14/Z). C.C. is funded by the Wellcome MIDAS doctoral training program at UoB. May 8, 2019 by guest Supplemental material for this article may be found at https://doi.org/10.1128/mBio .00644-19. Supplemental material for this article may be found at https://doi.org/10.1128/mBio 00644-19. FIG S1, PDF file, 0.3 MB. FIG S2, PDF file, 14.5 MB. FIG S3, PDF file, 0.3 MB. on May 8, 2019 by guest http://mbio.asm.org/ aded from FIG S4, PDF file, 0.1 MB. FIG S5, PDF file, 0.1 MB. FIG S6, JPG file, 0.1 MB. FIG S7, PDF file, 0.1 MB. TABLE S1, CSV file, 0.1 MB. 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A multilayer perceptron-based model applied to histopathology image classification of lung adenocarcinoma subtypes
Frontiers in oncology
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OPEN ACCESS 1Key Laboratory of Geophysical Exploration Equipment, Ministry of Education, College of Instrumentation and Electrical Engineering, Jilin University, Changchun, China, 2Department of Thoracic Surgery, The First Hospital of Jilin University, Changchun, China, 3Department of Pathology, Central Hospital Affiliated to Shenyang Medical College, Shenyang, China, 4Center for Reproductive Medicine and Center for Prenatal Diagnosis, The First Hospital of Jilin University, Changchun, China CITATION Liu M, Li L, Wang H, Guo X, Liu Y, Li Y, Song K, Shao Y, Wu F, Zhang J, Sun N, Zhang T and Luan L (2023) A multilayer perceptron-based model applied to histopathology image classification of lung adenocarcinoma subtypes. Front. Oncol. 13:1172234. doi: 10.3389/fonc.2023.1172234 Objective: Lung cancer is one of the most common malignant tumors in humans. Adenocarcinoma of the lung is another of the most common types of lung cancer. In clinical medicine, physicians rely on the information provided by pathology tests as an important reference for the fifinal diagnosis of many diseases. Thus, pathological diagnosis is known as the gold standard for disease diagnosis. However, the complexity of the information contained in pathology images and the increase in the number of patients far exceeds the number of pathologists, especially in the treatment of lung cancer in less-developed countries. COPYRIGHT © 2023 Liu, Li, Wang, Guo, Liu, Li, Song, Shao, Wu, Zhang, Sun, Zhang and Luan. 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. Methods: This paper proposes a multilayer perceptron model for lung cancer histopathology image detection, which enables the automatic detection of the degree of lung adenocarcinoma infifiltration. For the large amount of local information present in lung cancer histopathology images, MLP IN MLP (MIM) uses a dual data stream input method to achieve a modeling approach that combines global and local information to improve the classifification performance of the model. In our experiments, we collected 780 lung cancer histopathological images and prepared a lung histopathology image dataset to verify the effectiveness of MIM. TYPE Original Research PUBLISHED 18 May 2023 DOI 10.3389/fonc.2023.1172234 Frontiers in Oncology KEYWORDS A multilayer perceptron-based model applied to histopathology image classification of lung adenocarcinoma subtypes OPEN ACCESS EDITED BY Marco Rengo, Sapienza University of Rome, Italy REVIEWED BY James C. L. Chow, University of Toronto, Canada Yee Kai Tee, Tunku Abdul Rahman University, Malaysia *CORRESPONDENCE Tianyu Zhang zty@jlu.edu.cn Lan Luan luanlan33@symc.edu.cn RECEIVED 02 March 2023 ACCEPTED 05 May 2023 PUBLISHED 18 May 2023 CITATION Liu M, Li L, Wang H, Guo X, Liu Y, Li Y, Song K, Shao Y, Wu F, Zhang J, Sun N, Zhang T and Luan L (2023) A multilayer perceptron-based model applied to histopathology image classification of lung adenocarcinoma subtypes. Front. Oncol. 13:1172234. doi: 10.3389/fonc.2023.1172234 1 Introduction Morphological observation of tumor cells under a microscope revealed that different types of tumor cells have different nuclei, cell size, and morphology (5). However, sometimes the microscope cannot clearly identify tumor type; hence, tumor type has to be further determined by the indexes obtained by immunohistochemistry. If the tumor is small, then immunohistochemistry cannot also give a good judgment. Current treatment methods for lung cancer are limited, irreversible, and harmful to the human body. Therefore, accurate diagnosis prior to lung cancer treatment is necessary to avoid unnecessary harm to patients. Deep learning has become widely used in different fields with the rapid development of artificial intelligence (6). and increasing studies are showing the reliability of deep learning algorithms in medical image analysis (7). Some examples are as follows: Hua et al. (8) applied the DBN to classify lung nodules as malignant or benign with a sensitivity rate of 73.40% and a specificity rate of 82.20%.Roy et al. (9) studied a system based on fuzzy inference system for the image classification of cancerous and non-cancerous lung tumors. In a paper published in 2018 at New York University School of Medicine (10), a deep learning-based triple classification task for non-small cell lung cancer histopathology slices was explored, and verified the reliability of CNNs applied to pathology tasks. In 2019, X. Wang et al. (11) proposed a new method based on weakly supervised learning to solve the classification problem of pathological sections of lung cancer. W. Shen et al. (12) designed an end-to-end deep learning architecture, the multi-crop CNN, for low- and high-malignancy lung nodule classification. Kriegsmann et al. (13) used a CNN-based model for the classification of common lung cancer subtypes. The study highlights the potential of CNN image classification models for The MLP IN MLP (MIM) proposed in this study is also architected as a MLP, but it notices the information features between patches and the local information within each patch. In particular, we regarded the local patches (e.g., 16×16) as “visual sentences” and further divided them into smaller patches (e.g., 4×4) as “visual words.” A combination of the global feature information of “visual sentences” and the local feature information of “visual words” was used for modeling. In comparison to CNN and VT, MIM’s network is considered lightweight and less computationally intensive, while still being able to effectively extract both global and local information. 1 Introduction tumor differentiation. Taken together, this demonstrates that deep learning tools are feasible for some aspects of renal pathology classification. Khademi et al. (14) proposed a novel spatio-temporal fusion model using convolutional autoencoder to extract features from CT scans and Swin Transformer to process the time series features of clinical data. The model shows high accuracy and stability in the malignancy prediction of lung cancer. Lung cancer has become one of the major cancer diseases in the world (1). Breast cancer and prostate cancer are more common among women and men, respectively, but the incidence and mortality rates of both cancers are much lower than those of lung cancer. Lung adenocarcinoma is the most common type of lung cancer, accounting for about 50% of lung cancers (2). Microinvasive lung adenocarcinoma is an early type of lung cancer that is detected in a timely manner whose tumor cells can be completely removed through radical surgery (3). Invasive lung adenocarcinoma is relatively more malignant and more complicated to treat than noninvasive ones. Timely and accurate determination of the type of lung cancer can greatly improve the cure rate of patients. Convolutional neural networks (CNNs) (15) and vision transformers (VTs) (16) have emerged as popular deep learning models for various computer vision tasks, including image segmentation and classification (17). While CNNs and VTs have shown promising results, they often require complex network architectures and high computational costs. To address these challenges, a new model called MLP-Mixer (18) has been proposed by the Google team. Unlike CNNs and VTs, MLP-Mixer relies entirely on multilayer perceptron (MLP) and does not use any convolutional operations or self-attentive mechanisms, resulting in a simpler network architecture and lower computational requirements. Therefore, MLP-Mixer offers a promising alternative for efficient and effective image processing tasks. MLP-Mixer only applies MLP to process the global information between patch sequences to achieve classification tasks, which can obtain a performance comparable to those of CNN and VT. The morphology and volume of cell nuclei in histopathological images are different; therefore, these images have a large amount of local information. Moreover, MLP-Mixer destroys the local structure of patches and cannot capture local information; hence, the application of MLP-Mixer model to histopathological images has a shortage. The most reliable scientific basis for physicians to diagnose lung cancer is through pathology (4). OPEN ACCESS Results: The MIM achieves a diagnostic accuracy of 95.31% and has a precision, sensitivity, specificity and F1-score of 95.31%, 93.09%, 93.10%, 96.43% and 93.10% respectively, outperforming the diagnostic results of the common network model. In addition, a number of series of extension experiments demonstrated the scalability and stability of the MIM. Conclusions: In summary, MIM has high classifification performance and substantial potential in lung cancer detection tasks. histopathology image, lung cancer, deep learning, multilayer perceptron, image classification 01 Frontiers in Oncology frontiersin.org 10.3389/fonc.2023.1172234 Liu et al. 2.2 Data preprocessing Dataset pre-processing was performed, and images were subjected to resizing, data enhancement, and data normalization operations. All images were resized to 256×256 pixels. Five types of enhancement techniques (horizontal flip, rotation, scaling, height shift, and width shift) were then applied on the dataset, where the rotation was set to 15° and the height shift and width shift were set to 5% of the image size. These enhancement methods alter the relative position of cancer in histopathological images; therefore, the training set was expanded for training the MIM and no data enhancement operation was taken for the test set. The tumor tissues were extracted and sent to the pathology department; thus, each doctor may use different stains in the process of making tumor sections because of different personal habits. An image 1 Introduction (c) In this stage, the pre-processed data are input into MIM Layer, and the global information features and local information features in it are extracted using a multilayer perceptron. (c) In this stage, the pre-processed data are input into MIM Layer, and the global information features and local information features in it are extracted using a multilayer perceptron. (c) In this stage, the pre-processed data are input into MIM Layer, and the global information features and local information features in it are extracted using a multilayer perceptron. (d) In this stage of classification, the output feature maps are classified using the standard classification head. (e) Finally, our proposed MLP IN MLP is evaluated and the model is evaluated using four metrics: accuracy, precision, specificity, sensitivity and F1-score. (e) Finally, our proposed MLP IN MLP is evaluated and the model is evaluated using four metrics: accuracy, precision, specificity, sensitivity and F1-score. The main contributions of this paper are as follows. (1) A new deep learning based model (i.e., MIM) is proposed, which only uses multi-layer perceptron to do feature extraction for visual sentences and visual words respectively, which can extract local features and global features sufficiently to improve the overall classification performance. (2) A triple classification task was performed to identify the degree of lung adenocarcinoma infiltration, and a digital pathology image dataset for lung adenocarcinoma infiltration detection was prepared in collaboration with pathologists. The experimental results proved that the MIM model can effectively determine the degree of lung cancer infiltration and achieved a good performance of 95.31%. (3) The MIM model has good performance in detecting the degree of lung adenocarcinoma infiltration and shows good generalization performance in other cancer histopathology image classification tasks. 1 Introduction The proposed model was applied to a private lung cancer histopathology dataset. The workflow of MIM is shown in Figure 1: in (a) We use the lung cancer histopathology dataset provided by the First Hospital of Jilin University as the training set, and the details of this dataset are described in Section2. (a) We use the lung cancer histopathology dataset provided by the First Hospital of Jilin University as the training set, and the details of this dataset are described in Section2. D A B E C FIGURE 1 Workflow of the proposed MLP IN MLP model: (A) train date, (B) date process, (C) train process, (D) output classification results, (E) Assessing performance. 1 ow of the proposed MLP IN MLP model: (A) train date, (B) date process, (C) train process, (D) output classification results, (E) Assessing p 02 Frontiers in Oncology frontiersin.org Liu et al. 10.3389/fonc.2023.1172234 different departments, and the diagnosis was collated and labeled by four experienced doctors over a period of 3 months to ensure that each sample had considerable clinical features. Based on this approach, we cropped and cut the histopathological images and gave specific labels for each cut. The dataset includes two parts: training set and test set. Each part contains three categories (Normal, Infiltration, and Micro_infiltration). In the training set there are 7842 data, Infiltration and Micro_Infiltration include 2,614 and 2,614 digital pathology images respectively, and the remaining 2,614 pathology data are Normal. A total of 870 digital pathological images are included the test set. Among which, each category contains 290 lung pathological images. This dataset contains 8712 lung pathology images of size 2048×1500. Among the invasive and microinvasive lung adenocarcinomas, the tumors were predominantly of the adnexal type of growth, whereas microinvasive adenocarcinomas had infiltration foci of ≤0.5 cm in maximum diameter and were of the follicular, papillary, micropapillary, or solid type. Infiltrating adenocarcinomas had infiltration foci with a minimum diameter of >0.5 cm. Normal represented a benign area of lung tissue. Some samples of the specific data are shown in Figure 2. (b) During the data processing, we expanded the dataset by performing data enhancement operation, and then finally did image normalization. (b) During the data processing, we expanded the dataset by performing data enhancement operation, and then finally did image normalization. 2.4 Methods The overall structure of MLP-Mixer consists of three main parts as follows: What makes the MLP-Mixer model effective is that it can fully handle the relationship between several input patches to achieve global information extraction. However, this method also has an obvious drawback, that is, after the input image is split into several patches, it spreads each patch into a 1D vector and thus destroys the local information inside each patch. Therefore, we believe that MLP-Mixer modeling does not take into account the information inside each input patch. 1. Per-patch Fully-connected: Linearly map the input image into a number of p×p×3-sized image patches, flatten each patch and linearly map it into a two-dimensional vector. The input image is thus converted into a two-dimensional (S,D) tensor, where S represents the number of patches and D represents the flattened dimensions of each patch. Specifically, S = HW P2 (where H and W represent the height and width of the input image, respectively, and p represents the size of each patch), and D=3×p2 (where 3 represents the number of channels in the input image). 1. Per-patch Fully-connected: Linearly map the input image into a number of p×p×3-sized image patches, flatten each patch and linearly map it into a two-dimensional vector. The input image is thus converted into a two-dimensional (S,D) tensor, where S represents the number of patches and D represents the flattened dimensions of each patch. Specifically, S = HW P2 (where H and W represent the height and width of the input image, respectively, and p represents the size of each patch), and D=3×p2 (where 3 represents the number of channels in the input image). By analyzing the lung histopathology dataset, we found that the histological features of lung cancer can be broadly classified as adnexal, alveolar, papillary, micropapillary, and solid (21). The cells that are likely to be presented in high magnification view are mostly tubular, columnar oval, and peg-shaped. Pathologists rely on information about these complex shape features to diagnose the type of cancer and the degree of infiltration. 2. Mixer Layer: This layer consists of N identical layers. Figure 3 shows the internal structure of the layers. Each layer consists of two types of MLPs: channel-mixing MLP and token- mixing MLP. Token-mixing MLP acts on the columns of a 2D table and learns the feature information between different spatial locations; it operates independently on each channel. 2.1 Dataset A unique dataset of lung cancer histopathological images provided by the First Hospital of Jilin University was used. The histopathological images were derived from 780 cases. Each case was given a clear diagnosis by a joint consultation of doctors from A B C FIGURE 2 Some representative examples of lung adenocarcinoma data: (A) microinvasive lung adenocarcinoma, (B) invasive lung adenocarcinoma, and (C) images of normal lung tissue. C B FIGURE 2 Some representative examples of lung adenocarcinoma data: (A) microinvasive lung adenocarcinoma, (B) invasive lung adenocarcinoma, and (C) images of normal lung tissue. 03 03 Frontiers in Oncology frontiersin.org Liu et al. 10.3389/fonc.2023.1172234 10.3389/fonc.2023.1172234 degradation over deep gradients and the accuracy degradation of the training set. Layer normalization (20) is a key part of the model for stable training and faster convergence. normalization operation was performed to turn the pathological images into black and white to avoid the color of the stain as a feature during model learning. 3. Finally, MLP-Mixer uses a standard classification head with the global average pooling layer followed by a linear classifier. 3. Finally, MLP-Mixer uses a standard classification head with the global average pooling layer followed by a linear classifier. 2.3 MLP-mixer The MLP-Mixer does not use convolution operations or self- attentive mechanisms. Its architecture is based entirely on repeated application of multilayer perceptrons (MLPs) on spatial locations or feature channels, relying only on basic matrix multiplication routines, changes in data layout (reshaping and transposition), and scalar nonlinearities. Therefore, the model has a simple network and low computational effort. A simple MLP-based model that can compete with the best CNN and VT models available today was constructed. MLP-Mixer implements the convolutional and pooling layers of CNNs and the self-attentive layer of Transformer architecture using the two MLP types and demonstrates a simple MLP-based model that achieves competitiveness with current CNN or VT models, because MLPs require only simple mathematical operations. 2.4 Methods Channel-mixing MLP acts on the rows of a 2D table and learns the feature information between different channels; it runs independently on the same spatial location. The output dimensions of both MLPs always remain the same as the input dimensions. The idea is to explicitly distinguish between per-position (channel-mixing) operations and cross-position (token-mixing) operations. In addition, other standard components are introduced, such as the residual structure (skip-connections) (19), which improves the ability of information interaction and avoids network 2. Mixer Layer: This layer consists of N identical layers. Figure 3 shows the internal structure of the layers. Each layer consists of two types of MLPs: channel-mixing MLP and token- mixing MLP. Token-mixing MLP acts on the columns of a 2D table and learns the feature information between different spatial locations; it operates independently on each channel. Channel-mixing MLP acts on the rows of a 2D table and learns the feature information between different channels; it runs independently on the same spatial location. The output dimensions of both MLPs always remain the same as the input dimensions. The idea is to explicitly distinguish between per-position (channel-mixing) operations and cross-position (token-mixing) operations. In addition, other standard components are introduced, such as the residual structure (skip-connections) (19), which improves the ability of information interaction and avoids network Digital pathological images are more highly complex than natural images with rich details and local information; therefore, the application of MLP-Mixer to medical pathology will be somewhat limited. It will fail to tap the cell and tissue features in different locations if the granularity of patch segmentation is not good enough. This paper proposes an MIM architecture to address the shortcomings of MLP-Mixer. The proposed model considers the global and local information between patches and the internal information of each patch, improving the performance of the model. The specific network structure is shown in Figure 4. We partitioned the input 2D image into n patches uniformly and without overlapping. Let (p, p) be the resolution of each patch and X = [X1, X2, …, Xn] ∈Rn×p×p×3. Each patch was spread into a 1D FIGURE 3 Detailed structure of Mixer Layer. 04 Frontiers in Oncology frontiersin.org Liu et al. Liu et al. 10.3389/fonc.2023.1172234 FIGURE 4 Internal structure of MLP IN MLP. vector with a length of 3 × p². Finally, linear mapping was performed to obtain a 2D tensor table about patches (n, c) i.e. X∈Rn×c. 2.4 Methods MLP- Mixer just designs a mixer layer to handle the relationship between n image patch sequences, unlike MIM, where two standard mixer layers extract the global and local information in the image. In MIM, we consider each patch as a visual sentence representing an image. Each patch will be further divided into m sub-patch visual words, that is, a visual sentence consists of a series of visual words: Xi ! [Xi1, Xi2, …, Xim] ∈Rn×P×P×3, where Xij ∈Rs×s×3 is the j-th visual word of the i-th visual sentence, and (s, s) is the resolution size of the subpatch, j=1, 2, …, m. Each subpatch was also spanned into a 1D vector during the segmentation of the subpatch, and the 2D tensor table (n’, c’) about the subpatch was obtained after linear mapping. For example, the size of the visual sentence is 16×16×3. Here, p=16; therefore, the size of the visual word is 8×8×3. Each visual sentence was divided into four visual words, that is Xij ∈R8×8×12. words. For example, in a patch of human face, a word corresponding to the eye is more related to other words of eyes and interacts less with forehead part. For the sentence level, the sequence of visual words are transformed into the domain of visual sentence by linear projection and added into the visual sentence as follows: Ul i = Xl i + FC(LN(Yl ij)) (3) (3) With the above addition operation, the representation of sentence sequences is augmented by word-level features. We used the standard mixer layer for transforming the visual sentences as follows: Sl i = Ul i  +  (W2s(W1(LN( Ul i))T))T (4) Ql i =  Sl i  +  (W4s(W3(LN( Sl i))T))T (5) Sl i = Ul i  +  (W2s(W1(LN( Ul i))T))T (4) (4) Ql i =  Sl i  +  (W4s(W3(LN( Sl i))T))T (5) (5) In MIM, we have two data flows in which one flow operates across the visual sentences, and the other processes the visual words inside each sentence. For the visual words, we utilize a small mixer layer as follows to explore the relation between visual words: This big mixer layer is used for modeling the relationships among sentence sequences. Frontiers in Oncology 2.5 Hyperparameter settings proposed MIM and other networks. The results are shown in Table 2. The accuracy, precision, sensitivity, specificity and F1- score of MIM on the test set were 95.31%, 93.09%, 93.10%, 96.43% and 93.10%, respectively. The accuracy, precision, sensitivity, specificity and F1-score of MLP-Mixer on the test set were 92.43%, 88.97%, 88.96%, 94.24% and 88.97%, respectively. The five evaluation metrics of the proposed MIM were on average nearly 3% higher than those of MLP-Mixer. In addition, MIM had higher evaluation metrics than ResNet50 and Swin Transformer (25). The effectiveness of modeling using a combination of global and local information in the MIM model was demonstrated for the large amount of local information present in lung adenocarcinoma. proposed MIM and other networks. The results are shown in Table 2. The accuracy, precision, sensitivity, specificity and F1- score of MIM on the test set were 95.31%, 93.09%, 93.10%, 96.43% and 93.10%, respectively. The accuracy, precision, sensitivity, specificity and F1-score of MLP-Mixer on the test set were 92.43%, 88.97%, 88.96%, 94.24% and 88.97%, respectively. The five evaluation metrics of the proposed MIM were on average nearly 3% higher than those of MLP-Mixer. In addition, MIM had higher evaluation metrics than ResNet50 and Swin Transformer (25). The effectiveness of modeling using a combination of global and local information in the MIM model was demonstrated for the large amount of local information present in lung adenocarcinoma. Finding the optimal values of hyperparameters is one of the crucial tasks for building a robust model. Besides fully extracting image information, the hyperparameters values have a great influence on the fast convergence of the model. MIM was used to train the image of the lung cancer pathological tissue dataset for 200 epochs. In each epoch, batch size is set to 256. It uses a method to train the lung adenocarcinoma infiltration level recognition task from scratch. AdamW (23) was used as the optimizer, and its parameters were set as 3×10−5 learning rate. In addition, the dropout ratio (24) was set to 0.1 in the training process. The superior performance of MIM can be further demonstrated by the correlation model confusion matrix shown in Figure 5. In the test confusion matrix, the classifications of normal lung pathological data are very accurate. The classification errors of ResNet and Swin Transformer are shown in Figures 5B and C, respectively. Both methods misclassified Infiltration as Micro_infiltration. 2.7 Performance metrics Accuracy, precision, sensitivity, specificity and F1-score were calculated to evaluate the performance of MIM. Here, accuracy indicates the proportion of the number of samples correctly predicted by the model to the total number of samples. Precision indicates the proportion of samples correctly predicted by the model as a percentage of all samples predicted by the model to be positive. Sensitivity indicates the proportion of the number of samples correctly predicted by the model as a percentage of the total number of positive samples; the specific value indicates the proportion of the number of samples correctly predicted by the model as a percentage of the total number of negative samples. F1-score combines the results of precision and sensitivity. True positive, true negative, false positive, and false negative were used in the definition of these five criteria in Table 1. 3 Result A hybrid dataset consisting of the publicly available pathological dataset of gastric cancer from Northeastern University (26) and our private dataset was used to come up with extended experiments to explore the performance of the model on lung tissue and other histopathological data. The experimental setup was the same as the main experimental setup. The gastric 2.4 Methods In summary, the inputs and outputs of the MIM layer include the sequence of visual word and sequence of visual sentences as shown in Figure 4; hence, MIM can be formulated as: Zl ij =  Xl ij  +  (W2s(W1(LN( Xl ij))T))T (1) Yl ij =  Zl ij  +  (W4s(W3(LN( Zl ij))T))T (2) (1) Y ,  Q = MIM(X ,  U) (6) (6) (2) In our MIM layer, the small mixer layer is used to model the relationship between visual words for local feature extraction, and the big mixer layer captures the intrinsic information from the sequence of visual sentences. We built the MIM network by stacking the MIM layer for N times. Finally, MIM uses a standard classification head with the global average pooling layer, followed by a linear classifier. where l = 1, 2, …, L is the index of the l-th block, L is the total number of stacked blocks, T stands for transpose, s is an element-wise nonlinearity (GELU) (22), W is a fully connected layer process, Z is the output after token-mixing MLP, and Y is the output after channel-mixing MLP. This process builds the relationship among visual words by computing the interactions between any two visual Frontiers in Oncology 05 05 frontiersin.org frontiersin.org 10.3389/fonc.2023.1172234 Liu et al. 2.6 Experimental environment The experiments were carried out on a local workstation with 32 GB RAM and Windows 10 operating system. The GPU of the workstation consists of NVIDIA RTX 3090 and CPU Intel Core i9- 10875H 2.30 GHz. We used the Python 3.7 programming language and PyTorch 1.8.0. 2.5 Hyperparameter settings In Figure 5D, the misclassification of MLP- Mixer is due to the confusion caused between Infiltration and Micro_infiltration. The main reason for these phenomena are that the Infiltration and Micro_infiltration samples contain tumors, but the only difference is whether the infiltrated area is more than 5 mm. Moreover, the model gave wrong judgments for some samples near the threshold. As shown in Figure 5A, MIM effectively alleviated these problems. Using MIM, only 21 Infitration data were incorrectly predicted as Micro_infiltration data, and 13 Micro_Infitration data were incorrectly predicted as Infitration data. The effectiveness of the MIM modeling approach using local and global information was further demonstrated in terms of the degree of infiltration of lung adenocarcinoma, allowing the effective identification of differences in infiltrative and microinfiltrative pathological features. 3.1 Experimental results and analysis In the test phases, accuracy, precision, sensitivity, specificity and F1-score were calculated to evaluate the performance of the TABLE 1 Criteria and corresponding definitions for image global detection evaluation. TABLE 1 Criteria and corresponding definitions for image global detection evaluation. Criterion Definition Criterion Definition Accuracy TP + TN TP + TN + FP + FN Precision TP TP + FP Sensitivity     TP TP + FN Specificity TN   TN + FP F1-score 2  Precision  Sensitivity Precision + Sensitivity TABLE 1 Criteria and corresponding definitions for image global detection evaluation. Criterion Definition Criterion Definition Accuracy TP + TN TP + TN + FP + FN Precision TP TP + FP Sensitivity     TP TP + FN Specificity TN   TN + FP F1-score 2  Precision  Sensitivity Precision + Sensitivity 06 10.3389/fonc.2023.1172234 Liu et al. TABLE 2 Results of MLP IN MLP and other networks on private datasets (In %). TABLE 2 Results of MLP IN MLP and other networks on private datasets (In %). Model Accuracy Precision Sensitivity Specificity F1-score MLP IN MLP 95.31 93.09 93.10 96.43 93.10 MLP-Mixer (18) 92.43 88.97 88.96 94.24 88.97 ResNet50 (19) 93.95 91.46 91.14 95.36 91.30 Swin (25) 94.60 92.48 92.06 95.85 92.27 histopathology database from Northeastern University has three sizes (160×160, 120×120, and 80×80 pixels), and two data types (normal and abnormal). Some examples are shown in Figure 6. In this experiment, 160×160 size data were used and resized to the same size (256×256) as our private dataset during data processing. We use 10080 normal data images and 6670 abnormal data images. Considering the number of training and test sets in our private dataset, we randomly partitioned the training and test sets in a 9:1 ratio for the histopathological data of gastric cancer. The mixed data set is a five-category task. The combined dataset’s data settings are shown in Table 3. Six representative models were selected for the comparative experiments with MIM, including two CNN models [VGG16 (27) and ResNet50] two VT models [ViT and XCiT (28)], and two MLP models [ResMLP (29) and MLP-Mixer]. The experimental settings were the same as the previoussetting. Table 4 shows the results of the models on the test set of the combined dataset. 3.1 Experimental results and analysis ViT obtained the highest accuracy rate of 95.50% among the six models, and MLP- D A B C FIGURE 5 The confusion matrix obtained by the model on a private dataset: (A) MLP IN MLP, (B) Swin Transformer, (C) ResNet50, (D) MLP-Mixer. A B B A C D D C 07 Frontiers in Oncology frontiersin.org Liu et al. Liu et al. 10.3389/fonc.2023.1172234 A B FIGURE 6 Some examples in the gastric cancer dataset: (A) normal stomach tissue data, (B) abnormal stomach tissue data. B FIGURE 6 Some examples in the gastric cancer dataset: (A) normal stomach tissue data, (B) abnormal stomach tissue data. dataset, LC25000 (30), was incorporated to the private dataset, and extended experiments were performed on the hybrid dataset to validate the generalization ability of MIM on other lung cancer subtype pathology datasets. The experimental setup was the same as the main experimental setup. The LC25000 dataset has five different data types about lung and colon histopathology: normal lung histopathology image (Lung_n), lung squamous carcinoma histopathology image (Lung_scc), lung adenocarcinoma histopathology image (Lung_aca), normal colon histopathology image (Colon_n), and colon adenocarcinoma histopathology image (Colon_aca). Some examples are shown in Figure 7. The dataset has 4500 images in the training set and 500 images in the test set for each type, for a total of 25000 images for the five data Mixer had the lowest accuracy rate of 91.45%. Among the models, the proposed MIM model achieved the highest test accuracy of 96.50%, which was nearly 1% higher than the accuracy of ViT and nearly 5% more accurate than MLP-Mixer. In addition, the precision, sensitivity, specificity and F1-score of MIM were also the highest at 93.95%, 93.89%, 98.91% and 93.92%, respectively. The results show the effectiveness of the proposed MIM model. Mixer had the lowest accuracy rate of 91.45%. Among the models, the proposed MIM model achieved the highest test accuracy of 96.50%, which was nearly 1% higher than the accuracy of ViT and nearly 5% more accurate than MLP-Mixer. In addition, the precision, sensitivity, specificity and F1-score of MIM were also the highest at 93.95%, 93.89%, 98.91% and 93.92%, respectively. The results show the effectiveness of the proposed MIM model. 3.2.2 Extended experiment on combined dataset: private data and public data Model Accuracy Precision Sensitivity Specificity F1-score MLP IN MLP 96.50 93.95 93.89 98.91 93.92 ResNet50 (19) Vgg16 (27) XCiT (28) ViT (16) ResMLP (29) MLP-Mixer (18) 94.90 93.68 95.05 95.50 94.87 91.45 92.44 93.70 87.40 92.49 92.43 87.12 92.34 93.53 89.66 92.37 92.10 87.19 98.46 98.12 97.85 98.53 98.39 97.62 92.39 93.62 88.53 92.43 92.27 87.16 A B C D E FIGURE 7 Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. B D A C D C B E A FIGURE 7 Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. not detected; therefore, the accuracy of MIM in this set was 97.42%. For colon adenocarcinoma histopathological images, 996 images were detected by the MIM model, and only four images were not detected; therefore, the accuracy of the model for colorectal images was 99.60%. The LC2500 dataset contains lung cancer subtypes different from invasive lung cancer in addition to the colon histopathological data that are also present. The findings indicate that MIM has good classification performance between different lung cancer subtypes and performs well for colon histopathological data. types. Both datasets contain normal lung histopathology data; hence, we set all normal lung tissue data as one category, forming a seven-category hybrid dataset. Table 5 shows the data settings. The results of the comparison tests between MIM and six classical deep learning models in the lung cancer and colon cancer detection task are shown in Table 6. Obviously, among the six classical models, ConViT (33) performed the best in the test set with accuracy, precision, sensitivity, specificity and F1-score values of 97.83%, 96.70%, 96.43%, 99.59% and 96.52%, respectively. The accuracy, precision, sensitivity, specificity and F1-score values of MIM on the test set were 98.09%, 97.17%, 97.04%, 99.67% and 97.11%, respectively. The specific detection results of MIM are shown in Table 7. For the five types of lung pathological images, 2309 images were correctly detected by the MIM model, and only 61 images were 3.2.2 Extended experiment on combined dataset: private data and public data In addition to exploring the sensitivity between different histopathologies, we also incorporated other lung cancer subtypes to further increase the complexity of the experiment. The public TABLE 3 Data setting for training and test sets. TABLE 3 Data setting for training and test sets. Class/Dataset Train Test Sum Micro_infiltration 2614 290 2904 Infiltration 2614 290 2904 Normal 2614 290 2904 Stomach_Abnormal 6013 657 6670 Stomach_Normal 9072 1008 10080 Sum 22927 2535 25462 08 Liu et al. 10.3389/fonc.2023.1172234 Liu et al. TABLE 4 Comparison results of models on the test set of the combined dataset (In %). TABLE 4 Comparison results of models on the test set of the combined dataset (In %). TABLE 4 Comparison results of models on the test set of the combined dataset (In %). Model Accuracy Precision Sensitivity Specificity F1-score MLP IN MLP 96.50 93.95 93.89 98.91 93.92 ResNet50 (19) Vgg16 (27) XCiT (28) ViT (16) ResMLP (29) MLP-Mixer (18) 94.90 93.68 95.05 95.50 94.87 91.45 92.44 93.70 87.40 92.49 92.43 87.12 92.34 93.53 89.66 92.37 92.10 87.19 98.46 98.12 97.85 98.53 98.39 97.62 92.39 93.62 88.53 92.43 92.27 87.16 A B C D E FIGURE 7 Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. Model Accuracy Precision Sensitivity Specificity F1-score MLP IN MLP 96.50 93.95 93.89 98.91 93.92 ResNet50 (19) Vgg16 (27) XCiT (28) ViT (16) ResMLP (29) MLP-Mixer (18) 94.90 93.68 95.05 95.50 94.87 91.45 92.44 93.70 87.40 92.49 92.43 87.12 92.34 93.53 89.66 92.37 92.10 87.19 98.46 98.12 97.85 98.53 98.39 97.62 92.39 93.62 88.53 92.43 92.27 87.16 A B C D E FIGURE 7 Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. Frontiers in Oncology frontiersin.org 09 4 Discussion First, in our experiments, we found that the performance is better when the number of MIM blocks is set to 8, when the parameter size TABLE 5 Data setting for training and test sets. TABLE 5 Data setting for training and test sets. g g Class/Dataset Train Test Sum Micro_infiltration 2614 290 2904 Infiltration 2614 290 2904 Lung_aca 4500 500 5000 Lung_scc 4500 500 5000 Lung_n 7114 790 7904 Colon_aca 4500 500 5000 Colon_n 4500 500 5000 Sum 30342 3370 33712 09 Frontiers in Oncology Liu et al. 10.3389/fonc.2023.1172234 TABLE 6 A comparison of image classification results on the mixed test set. TABLE 6 A comparison of image classification results on the mixed test set. TABLE 6 A comparison of image classification results on the mixed test set. Model Accuracy Precision Sensitivity Specificity F1-score MLP IN MLP 98.09 97.17 97.04 99.67 97.11 EfficientNet (31) 96.96 95.22 95.45 99.32 95.34 InceptionV2 (32) 96.76 95.29 95.31 99.43 95.30 Swin (24) 97.51 96.09 95.97 99.53 96.03 ConViT (33) 97.83 96.70 96.43 99.59 96.52 gMLP (34) 96.41 96.09 95.16 99.35 95.63 MLP Mixer (17) 95.54 92.71 92.07 99.00 92.39 (In %). TABLE 7 The result of MLP IN MLP in classification task. Data Type Correct Incorrect Accuracy Lung histopathology image 2039 61 97.42 Colon histopathology image 996 4 99.60 Sum 3305 65 98.09 (In %). positively correlated with the number of patches. The fullest possible exploitation of the internal local information of patches should also be considered. Therefore, the resolutions of vision sentences and vision words were set to 16×16 and 4×4, respectively, considering the computational power and training time of the model. of MIM is about 165 MB. We had 7842 training data and used 200 epochs to train MIM, and the total time was about 230 minutes. We lightened the model according to the actual needs of clinical work (35). First, we used a quantization technique to reduce the number of bits required to store each weight from 32 bits to 16 bits. Then, the size of the model parameters was reduced to 83 MB. Consequently, the classification performance was almost unchanged, and the total training time was reduced by about 55 minutes. Third, the optimizer and activation function should be chosen carefully. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 5 Conclusion This work was supported in part by the Science and Technology Department of Jilin Province of China under Grants 20210204193YY, 20220203133SF, and YDZj202201ZYTS137, by the National Key Research and Development Project of China under Grant 2019YFC0409105, and by the Shenyang Science and Technology Plan Public Health R&D Special Project under Grant 21-172-9-18. Support by Graduate Innovation Fund of Jilin University. In the experiments, MIM was tested on a lung cancer pathology dataset with 95.31% accuracy, showing the model’s potential in lung cancer determination tasks. The lung pathological images have a large amount of information on histological shapes, such as pegs and papillae. The local structure of patches was destroyed in the MLP-Mixer model. Compared with the conventional MLP-Mixer, MIM can better preserve and model local information for lung histopathology identification tasks. In the extended experiments, MIM is more efficient and performs better than CNN, MLP and VT models proving its powerful generalization ability. Ethics statement Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability statement The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors. 4 Discussion Although various optimizers (e.g., stochastic gradient descent (36) and Lagrangian optimizer) and activation functions (e.g., CroReLU) (37, 38) are implemented with deep networks to solve different classification problems, we applied AdamW and GELU because of their efficiency in the proposed architecture with our datasets. AdamW solves the problem of parameter over-fitting with Adam optimizer (39) by introducing the L2 regularization terms of parameters in the loss function. It is the fastest optimizer for gradient descent speed and training neural networks that is used in all models. GELU has been widely used in the recent Transformer model. Randomness is introduced to make the model training process more robust and avoid the problem of gradient disappearance well. Loss functions have an important role in a deep network architecture. Hybrid loss functions have been proposed in several works to improve the performance of model. We used the default loss function [i.e., cross-entropy (40)] because of its low computational cost and efficiency with our pathological images. Second, the segmentation granularity of patches varies in terms of the feature richness that can be extracted from the image. By contrast, the feature extraction layer of MIM has two different data streams, namely, visual sentences and visual words, and MIM was designed to capture the global feature information between patches and mine the local feature information within patches. In the pre-experiments, we set different resolutions for the visual sentences and visual words. For the fairness of the experiments, the number of feature extraction layers in the experiments was set to 8. The rest of the experimental settings and the datasets used were the same as those in the main experiments. The specific experimental details and results are shown in Table 8. The highest accuracy rate of 95.31% was achieved with a visual sentence resolution of 16 and a visual word resolution of 4. The finer the granularity of patch segmentation, the more the number of patches. Additionally, the amount of model computation was TABLE 8 Comparison test of different patch sizes. Model Visual Sentences Visual word Accuracy MIM 32 8 93.24 32 4 94.12 16 8 94.78 16 4 95.31 (In %). Model Visual Sentences Visual word Accuracy MIM 32 8 93.24 32 4 94.12 16 8 94.78 16 4 95.31 (In %). 10 Frontiers in Oncology frontiersin.org Liu et al. Liu et al. 10.3389/fonc.2023.1172234 9. Roy TS, Sirohi N, Patle A. Classification of lung image and nodule detection using fuzzy inference system, in: International conference on computing, communication & automation IEEE (2015), 1204–7. Author contributions Finally, although the proposed model showed a prominent performance in the classifying degree of lung cancer infiltration, this study encountered some challenges. Our work was limited to two types of lung cancer data, namely, infiltration and microinfiltration, because of data inadequacy. Moreover, we only considered digital pathological images. Other medical imaging techniques, such as computed tomography and magnetic resonance imaging, can also be taken under consideration in future work. In addition, this work can be extended to patients’ medical history (age, gender, physical condition, etc.) with image datasets for more accurate predictions. The conception or design of the work: ML, LYL, and TZ; the acquisition, analysis, or interpretation of data: YGL, FW, TZ, and YPL; drafting the work or revising: YS, XG, and HW; provide approval for publication of the content: NS, JZ, and KS; agree to be accountable for all aspects of the work: ML and LL. 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De, Boer PT, Kroese DP, Mannor S. A tutorial on the cross-entropy method. Ann operations Res (2005) 134(1):19–67. doi: 10.1007/s10479-005-5724-z 12 12 Frontiers in Oncology frontiersin.org
https://openalex.org/W4390476044
https://dergipark.org.tr/tr/download/article-file/3549925
Turkish
null
Bioactive Contents of Different Parts of Strawberry Tree (Arbutus unedo L.) Plant
DergiPark (Istanbul University)
2,023
cc-by
3,945
ÖZ Çalışma, Piraziz (Giresun) ilçesinde yetiştirilen kocayemiş bitkisinin farklı kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flavonoid ve antioksidan aktivitesini (DPPH ve FRAP testlerine göre) belirlemek amacıyla yürütülmüştür. En yüksek toplam fenolik içeriği yaprak (1.10 g 100 g-1) ve çiçekte (1.08 g 100 g-1) belirlenirken, en düşük meyvede (0.81 g 100 g-1) tespit edilmiştir. Toplam flavonoid içeriği en yüksek yaprakta (3.38 g 100 g-1), en düşük ise meyvede (0.04 g 100 g-1) bulunmuştur. En yüksek antioksidan aktivite her iki yönteme göre de yaprak (sırasıyla 7.72 mmol 100 g-1 ve 2.58 mmol 100 g-1) ve çiçekte (sırasıyla 7.84 mmol 100 g-1 ve 2.58 mmol 100 g-1) tespit edilirken, en düşük meyvede (sırasıyla 1.21 mmol 100 g-1 ve 1.50 mmol 100 g-1) belirlenmiştir. Sonuç olarak, incelenen özellikler bakımından en iyi sonuçlar bitkinin yaprak ve çiçek kısımlarından elde edilmiştir. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri ehmet Fikret BALTA1 , İzzet YAMAN1 , Orhan KARAKAYA2* , Umut ATEŞ2 1Bahçe Bitkileri, Ziraat Fakültesi, Ordu Üniversitesi, Türkiye 2Bahçe Bitkileri, Ziraat Fakültesi, Sakarya Uygulamalı Bilimler Üniversitesi, Türkiye Anahtar Kelimeler: Yaprak, çiçek, meyve, fenolik, antioksidan. * Sorumlu yazar : orhankarakaya7@gmail.com Keywords: Leaves, flower, fruit, phenolics, antioxidant. Research Article Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 Received: 21-Nov-2023 Accepted: 26-Dec-2023 homepage: https://dergipark.org.tr/tr/pub/joinabt https://doi.org/10.58728/joinabt.1393739 Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 Received: 21-Nov-2023 Accepted: 26-Dec-2023 homepage: https://dergipark.org.tr/tr/pub/joinabt https://doi.org/10.58728/joinabt.1393739 ABSTRACT unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyv ile ağaç çileği olarak da bilinmektedir [5]. Ülkemizde bölgelere göre d yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, d yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. un gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7 olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli m değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta o dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda ku A. unedo‘nın meyve verme süresi diğer pek çok türe göre çok daha meyvelerinin kış aylarında veya kış aylarına yakın bir zamanda alternatif bir besin kaynağı sağlamaktadır [7]. Yüksek biyokimy bozukluklar, kalp damar hastalıkları, kanser, damar sertleşmesi, b hipertansiyon gibi pek çok hastalığa karşı da koruyucu etkisinin söktürücü, enfeksiyon önleyici ve yaşlanmayı geciktirici etkilere s çiçeklerinin astım hastalığı ve yapraklarından hazırlanan çayların ise hastalıklarına karşı koruyucu etkisi bulunmaktadır [16, 17]. Bu çalış kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flav belirlemek amacıyla yürütülmüştür. Materyal ve Yöntem Materyal Çalışma, Giresun iline bağlı Piraziz ilçesinde yürütülmüştür. Çalışm yetişen kocayemiş genotipine ait yaprak, çiçek ve meyveler oluşturmu Yöntem Kocayemiş bitkisinden alınan yaprak ve çiçek örnekleri gölge bir örnekleri, meyvenin kendine özgü rengini aldığı zamanda hasat edilm biyokimyasal analizler yapılıncaya kadar -20°C’de muhafaza edilmişt toplam fenolik, toplam flavonoid ve antioksidant aktivite (DP Giriş Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alanına sahip olup, Asya, Avrupa ve Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkânı bulmuştur [1]. Ülkemizde ise Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayılış göstermektedir. Ülkemiz doğal florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne L. en önemli kocayemiş türleridir [2]. Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alanına sahip olup, Asya, Avrupa ve Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkânı bulmuştur [1]. Ülkemizde ise Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayılış göstermektedir. Ülkemiz doğal florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne L. en önemli kocayemiş türleridir [2]. A. unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki habitusuna [3] ve yuvarlak veya yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyve görünüşlerinin çileğe benzemesi ile ağaç çileği olarak da bilinmektedir [5]. ABSTRACT Ülkemizde bölgelere göre değişmekle birlikte; dağ çileği, dağ yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, davulga, davulgı üzümü, piridim, yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. unedo bitkisi gerek görsel özellikleri gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her dem yeşil olan bitki, formu, uzun süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7] ve meyve tutumundan meyve olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli meyveleri [8] ile süs bitkisi olarak değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A. unedo bitkileri çit bitkisi olarak kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta olmak üzere, jöle, marmelat, reçel, dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda kullanılmaktadır [11, 2, 7, 12, 13]. A. unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki habitusuna [3] ve yuvarlak veya yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyve görünüşlerinin çileğe benzemesi ile ağaç çileği olarak da bilinmektedir [5]. Ülkemizde bölgelere göre değişmekle birlikte; dağ çileği, dağ yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, davulga, davulgı üzümü, piridim, yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. unedo bitkisi gerek görsel özellikleri gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her dem yeşil olan bitki, formu, uzun süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7] ve meyve tutumundan meyve olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli meyveleri [8] ile süs bitkisi olarak değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A. unedo bitkileri çit bitkisi olarak kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta olmak üzere, jöle, marmelat, reçel, dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda kullanılmaktadır [11, 2, 7, 12, 13]. A. unedo‘nın meyve verme süresi diğer pek çok türe göre çok daha uzundur [5, 2]. Özellikle olgun meyvelerinin kış aylarında veya kış aylarına yakın bir zamanda hasadının yapılmasıyla insanlara alternatif bir besin kaynağı sağlamaktadır [7]. Yüksek biyokimyasal içeriği sayesinde nörolojik bozukluklar, kalp damar hastalıkları, kanser, damar sertleşmesi, böbrek rahatsızlıkları, romatizma, hipertansiyon gibi pek çok hastalığa karşı da koruyucu etkisinin yanında, kabızlığı giderici, idrar söktürücü, enfeksiyon önleyici ve yaşlanmayı geciktirici etkilere sahiptir [14, 15]. Bunun yanında çiçeklerinin astım hastalığı ve yapraklarından hazırlanan çayların ise romatizma, idrar yolu ve böbrek hastalıklarına karşı koruyucu etkisi bulunmaktadır [16, 17]. Bu çalışmada kocayemiş bitkisinin farklı kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flavonoid ve antioksidan aktivitesini belirlemek amacıyla yürütülmüştür. Materyal Çalışma, Giresun iline bağlı Piraziz ilçesinde yürütülmüştür. Çalışmanın materyalini Piraziz ilçesinde yetişen kocayemiş genotipine ait yaprak, çiçek ve meyveler oluşturmuştur. Yöntem Kocayemiş bitkisinden alınan yaprak ve çiçek örnekleri gölge bir ortamda kurutulmuştur. Meyve örnekleri, meyvenin kendine özgü rengini aldığı zamanda hasat edilmiştir. Alınan tüm bitki örnekleri biyokimyasal analizler yapılıncaya kadar -20°C’de muhafaza edilmiştir. Biyokimyasal özellikler olarak toplam fenolik, toplam flavonoid ve antioksidant aktivite (DPPH ve FRAP testlerine göre) belirlenmiştir. ABSTRACT ABSTRACT The study was carried out to determine the total phenolics, total flavonoids and antioxidant activity (according to DPPH and FRAP tests) of different parts (leaves, flower and fruit) of the strawberry tree plant grown in Piraziz (Giresun) district. The highest total phenolic content was determined in leaves (1.10 g 100 g-1) and flower (1.08 g 100 g-1), while the lowest was detected in fruit (0.81 g 100 g-1). The highest total flavonoid content was found in the leaves (3.38 g 100 g-1) and the lowest in the fruit (0.04 g 100 g-1). The highest antioxidant activity was detected in leaves (7.72 mmol 100 g-1 and 2.58 mmol 100 g-1, respectively) and flower (7.84 mmol 100 g-1 and 2.58 mmol 100 g-1, respectively) according to both methods, while the lowest was determined in fruit (1.21 mmol 100 g-1 and 1.50 mmol 100 g-1, respectively). As a conclusion, the best results in terms of the investigated properties were obtained from the leaves and flower parts of the plant. Keywords: Leaves, flower, fruit, phenolics, antioxidant. * Sorumlu yazar : orhankarakaya7@gmail.com Publisher: Sakarya University of Applied Sciences Publisher: Sakarya University of Applied Sciences https://www.subu.edu.tr/tr Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Giriş Kocayemiş (Arbutus unedo L.) dünya üzeri Afrika’nın ılıman iklim koşullarında doğ Karadeniz, Marmara, Akdeniz ve Ege bölge florası içerinde de yer alan Arbutus unedo L [2]. A. unedo her dem yeşil olup 1.5-9 m ara yuvarlağa yakın turuncu-kırmızı meyvelere ile ağaç çileği olarak da bilinmektedir [5]. Ü yemişi, ayı yemişi, kocakarıyemişi, kara y yağma, enderek ve zefre yemişi gibi farklı is gerekse besleyici özellikleri ile pek çok kull süre bitki üzerinde görülebilen gösterişli olumuna kadar geçen sürede sarıdan kırmız değerlendirilmektedir [9, 10]. Bunun yanın kullanılmaktadır [3]. Çileğe benzeyen meyv dondurma, pastacılıkta, alkollü içecek yapım A. unedo‘nın meyve verme süresi diğer pe meyvelerinin kış aylarında veya kış aylar alternatif bir besin kaynağı sağlamaktadı bozukluklar, kalp damar hastalıkları, kans hipertansiyon gibi pek çok hastalığa karş söktürücü, enfeksiyon önleyici ve yaşlanm çiçeklerinin astım hastalığı ve yapraklarınd hastalıklarına karşı koruyucu etkisi bulunm kısımlarının (yaprak, çiçek ve meyve) top belirlemek amacıyla yürütülmüştür. ABSTRACT Materyal ve Yöntem Materyal Çalışma, Giresun iline bağlı Piraziz ilçesin yetişen kocayemiş genotipine ait yaprak, çiç Yöntem Kocayemiş bitkisinden alınan yaprak ve Giriş Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alanına sahip olup, Asya, A Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkânı bulmuştur [1]. Ülke Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayılış göstermektedir. Ülke florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne L. en önemli kocayemiş [2]. A. unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki habitusuna [3] ve yuva yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyve görünüşlerinin çileğe b ile ağaç çileği olarak da bilinmektedir [5]. Ülkemizde bölgelere göre değişmekle birlikte; dağ yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, davulga, davulgı üzümü yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. unedo bitkisi gerek görsel gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her dem yeşil olan bitki, fo süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7] ve meyve tutumund olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli meyveleri [8] ile süs bitk değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A. unedo bitkileri çit bitk kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta olmak üzere, jöle, marme dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda kullanılmaktadır [11, 2, 7 A. unedo‘nın meyve verme süresi diğer pek çok türe göre çok daha uzundur [5, 2]. Özell meyvelerinin kış aylarında veya kış aylarına yakın bir zamanda hasadının yapılmasıyla alternatif bir besin kaynağı sağlamaktadır [7]. Yüksek biyokimyasal içeriği sayesinde bozukluklar, kalp damar hastalıkları, kanser, damar sertleşmesi, böbrek rahatsızlıkları, ro hipertansiyon gibi pek çok hastalığa karşı da koruyucu etkisinin yanında, kabızlığı gide söktürücü, enfeksiyon önleyici ve yaşlanmayı geciktirici etkilere sahiptir [14, 15]. Bunu çiçeklerinin astım hastalığı ve yapraklarından hazırlanan çayların ise romatizma, idrar yolu hastalıklarına karşı koruyucu etkisi bulunmaktadır [16, 17]. Bu çalışmada kocayemiş bitkis kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flavonoid ve antioksidan a belirlemek amacıyla yürütülmüştür. Materyal ve Yöntem Materyal Çalışma, Giresun iline bağlı Piraziz ilçesinde yürütülmüştür. Çalışmanın materyalini Piraziz i k i i i i k i k l l Giriş Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alan Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkân Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayıl florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne [2]. A. Antioksidan aktivite (mmol 100 g-1) Antioksidan aktivite (mmol 100 g-1) Bitki örneklerin antioksidan aktivitesi DPPH ve FRAP yöntemlerine göre belirlenmiştir. DPPH yöntemine göre antioksidan aktivitesi Blois (1958)’in rapor ettiği yöntem kullanılarak tespit edilmiştir. Hazırlanan stok çözeltiden yaprak ve çiçek örnekleri için 0.02 mL ve meyve örneği için 0.1 mL alınmış ve son hacim metanol ile 3 mL’ye tamamlanmıştır. Daha sonra örneklerin üzerine 1 mL DPPH çözeltisi ilave edilmiştir. Hazırlanan örnekler spektrofotometrede 517 nm dalga boyunda okunmuştur. Elde edilen absorbans değerleri mmol 100 g-1 olarak ifade edilmiştir. FRAP yöntemine göre antioksidan aktivitesi Benzie ve Strain (1996) tarafından bildirilen yönteme göre belirlenmiştir. Yaprak, çiçek ve meyve örnekleri için hazırlanan stok çözeltiden 0.02 mL alınarak, üzerine 1.23 mL fosfat tamponu ve 1.25 mL potasyum ferrik siyanit ilave edilmiştir. Hazırlanan örnekler 50°C’de 30 dk su etüvde inkübe edildikten sonra üzerine 1.25 mL TCA ve 0.25 mL demir klorür eklenmiştir. Örneklerin absorbans değerleri spektrofotometrede 415 nm’de okunmuştur. Elde edilen sonuçlar mmol 100 g-1 olarak ifade edilmiştir. İstatistiksel Analizler Verilerin değerlendirilmesinde JMP 16 (deneme sürümü) istatistik paket programı kullanılmıştır. İncelenen özellikler bakımından bitki kısımları arasındaki farklılık Tukey çoklu karşılaştırma yöntemine göre %5 önem seviyesinde belirlenmiştir. Toplam flavonoid (g 100 g-1) Toplam flavonoid (g 100 g-1) Toplam flavonoid içeriği Chang ve ark. (2002)’nın bildirdiği yönteme göre belirlenmiştir. Yaprak, çiçek ve meyve örnekleri için hazırlanan stok çözeltiden 0.1 mL alınarak, üzerine 4.2 mL metanol, 0.1 mL alüminyum nitrat ve 0.1 mL amonyum asetat ilave edilmiştir. Hazırlanan örneklerin absorbans değerleri spektrofotometrede 415 nm dalga boyunda okunmuştur. Elde edilen sonuçlar g 100 g-1 olarak ifade edilmiştir. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri 4.7 mL’ye tamamlanmış ve üzerine 0.1 mL folin reaktifi ve 0.3 mL sodyum karbonat ilave edilmiştir. Hazırlanan örnekler spektrofotometrede 760 nm’de okunmuştur. Elde edilen absorbans değerleri g 100 g-1 olarak ifade edilmiştir [18]. 4.7 mL’ye tamamlanmış ve üzerine 0.1 mL folin reaktifi ve 0.3 mL sodyum karbonat ilave edilmiştir. Hazırlanan örnekler spektrofotometrede 760 nm’de okunmuştur. Elde edilen absorbans değerleri g 100 g-1 olarak ifade edilmiştir [18]. Toplam fenolik (g 100 g-1) Toplam fenolik içeriği Folin-Ciocalteu yöntemine göre tespit edilmiştir. Hazırlanan stok çözeltiden yaprak ve çiçek örnekleri için 0.1 mL ve meyve örnekleri için ise 0.5 mL alınarak, son hacim saf su ile Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 106 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Bulgular ve Tartışma Meyve ve sebzelerde önemli miktarda bulunan biyoaktif bileşikler insan sağlığını teşvik eder ve kronik hastalıklar başta olmak üzere kanser ve kardiyovasküler hastalıklar gibi pek çok hastalığın riskini azaltmada önemli rol oynar [22]. Antioksidanlar, hastalıklara neden olduğu bilinen düzensiz reaktif oksijen türleri ve serbest radikallerin zararlı etkisini önleyerek vücudun savunma sistemine önemli derecede katkı sağlarlar [23]. Gıdalardan alınan antioksidanların vücutta üretilenlere göre daha yüksek koruma sağladığı bildirilmiştir [24]. Bu durum, fenolik ve antioksidan içeriği yüksek yenilebilir yabani meyve türlerinin tüketiminin önemini arttırmaktadır. Öyle ki kocayemiş de zengin besin içeriğiyle bu anlamda önemli bir yere sahiptir [4, 11, 12]. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği arasındaki farklılık önemli bulunmuştur (p<0.05). Toplam fenolik içeriği bakımından en yüksek değerler yaprak (1.10 g 100 g-1) ve çiçekte (1.08 g 100 g-1) belirlenirken, en düşük değerler meyvede (0.81 g 100 g-1) tespit edilmiştir (Şekil 1). Toplam fenolik içeriği, Lapseki (Çanakkale) yöresinde yetişen Arbutus unedo türüne ait çiçeklerde 20.7 g 100 g-1 ve meyvelerde 1.40 g 100 g-1 [25], Artvin ilinde yetişen Arbutus andrachne türünün çiçeklerinde Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 107 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri 4.40 g 100 g-1 ve meyvelerinde 0.70 g 100 g-1 [26], Cezayir’de yetişen Arbutus unedo türüne ait yapraklarda 17.5 g 100 g-1 ve meyvelerde 1.30 g 100 g-1 [27] olarak bildirilmiştir. Araştırıcıların bulguları değerlendirildiğinde kocayemiş bitkisinde toplam fenolik içeriği bakımından en yüksek değerlerin yaprak ve çiçekte, en düşük ise meyvelerde olduğu görülmektedir. Benzer sonuçlar mevcut çalışmada da kaydedilmiştir. Buna karşılık toplam fenolik içeriği bakımından yaprak ve çiçeklerden elde edilen değerler araştırıcıların bulgularından farklılık gösterirken, meyveden elde edilen değerler benzer bulunmuştur. Görülen farklılıkların genotipten, ekolojik koşullardan ve meyvenin olgunluk durumundan kaynaklı olduğu düşünülmektedir. Şekil 1. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Şekil 1. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Kocayemiş bitkisinin farklı kısımlarının toplam flavonoid içeriği arasındaki farklılık önemli bulunmuştur (p<0.05). En yüksek toplam flavonoid içeriği yaprakta (3.38 g 100 g-1), en düşük ise meyvede (0.04 g 100 g-1) bulunmuştur (Şekil 2). Toplam flavonoid içeriğini Saral ve ark. (2017) Arbutus andrachne türüne ait çiçeklerde 11.42 g 100 g-1 ve meyvelerde 0.62 g 100 g-1 olarak belirlerken, Asmaa ve ark. (2019) Arbutus unedo türünün yapraklarında 6.52 g 100 g-1 ve meyvelerinde 0.04 g 100 g-1 olarak tespit etmişlerdir. Genel olarak değerlendirildiğinde yaprak ve çiçeklerin, meyvelere göre daha yüksek toplam flavonoid içeriğine sahip olduğu görülmektedir. Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 1.1 Rakip Çıkarlar Yazarlar arasında herhangi bir çıkar çatışması yoktur. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Şekil 4. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (FRAP testine göre) Şekil 4. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (FRAP testine göre) Sonuçlar Kocayemiş bitkisinin farklı kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flavonoid ve antioksidan aktivitesinin (DPPH ve FRAP testlerine göre) incelendiği çalışmada, bitki kısımları arasında incelenen özellikler bakımından önemli farklılıklar belirlenmiştir. Toplam flavonoid içeriği hariç, diğer incelenen özellikler bakımından en yüksek değerler yaprak ve çiçekte belirlenirken, en düşük ise meyvede tespit edilmiştir. Toplam flavonoid içeriği ise en yüksek yaprakta, en düşük ise meyvede bulunmuştur. Sonuç olarak, insan sağlığını teşvik eden maddeler bakımından yaprak ve çiçek bitki kısımlarının kayda değer sonuçlar verdiği tespit edilmiştir. Bitkinin bu kısımlarının farklı ürünlere işlenerek değerlendirilme potansiyelinin yüksek olduğu düşünülmektedir. 1.2 Yazarların Katkıları Tüm yazarlar makaleye eşit katkı sunmuştur. Tüm yazarlar makaleye eşit katkı sunmuştur. Bulgular ve Tartışma Mevcut çalışmada da benzer sonuçlar kaydedilmiştir. Ancak yaprak ve çiçeklere ait toplam flavonoid değerleri araştırıcıların bulgularından düşük bulunmuştur. Toplam flavonoid içeriği bakımından görülen farklılıkların başta genetik yapıdan ve ekolojik koşullardan olmak üzere meyvenin olgunluk durumu ile olgunlaşma dönemindeki güneşlenme süresi ve gece ile gündüz arasındaki sıcaklık farklılığından kaynaklı olabilir. Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 108 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Şekil 2. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Şekil 2. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Antioksidan aktivitesi bakımından kocayemiş bitkisinin farklı kısımları arasında önemli bir farklılık belirlenmiştir (p<0.05). En yüksek antioksidan aktivite her iki yönteme göre de (DPPH ve FRAP) yaprak (sırasıyla, 7.72 mmol 100 g-1 ve 2.58 mmol 100 g-1) ve çiçekte (sırasıyla, 7.84 mmol 100 g-1 ve 2.58 mmol 100 g-1) tespit edilirken, en düşük meyvede (sırasıyla, 1.21 mmol 100 g-1 ve 1.50 mmol 100 g-1) belirlenmiştir (Şekil 3 ve Şekil 4). İlgili çalışmalarda DPPH testine göre antioksidan aktivitesi, Arbutus unedo türüne ait meyvelerde çiçeklere göre daha yüksek bildirilirken, FRAP testine göre ise çiçeklerde meyvelere göre daha yüksek bulunmuştur [27]. Farklı bir çalışmada FRAP testine göre Arbutus andrachne türünün çiçeklerinde meyvelerine göre daha yüksek antioksidan aktivite rapor edilmiştir [26]. Benzer sonuçlar Arbutus unedo türünde de bildirilmiştir [25]. Mevcut çalışmada da en yüksek antioksidan aktivitesi sırasıyla yaprak, çiçek ve meyvede belirlenmiştir. Şekil 3. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (DPPH testine göre) Şekil 3. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (DPPH testine göre) Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 109 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Kaynakça [1] Karadeniz, T., Kalkışım, Ö., & Şişman, T. (2003). Trabzon çevresinde yetişen kocayemiş (Arbutus unedo L.) tiplerinin meyve özellikleri ve çelikle çoğaltılması. Ulusal Kivi ve Üzümsü Meyveler Sempozyumu, 476-480. [2] Şeker, M., Yücel, Z., Nurdan, E. (2004). Çanakkale yöresi doğal florasında bulunan kocayemiş (Arbutus unedo L.) populasyonunun morfolojik ve pomolojik özelliklerinin incelenmesi. Journal of Agricultural Sciences, 10(04), 422-427. [3] Yarılgaç, T., & Pekdemir, M. (2019). Promising strawberry tree genotypes from North Anatolia, Turkey. Erwerbs-Obstbau, 61(1), 79-84. [3] Yarılgaç, T., & Pekdemir, M. (2019). Promising strawberry tree genotypes from North Anatolia, Turkey. Erwerbs-Obstbau, 61(1), 79-84. [3] Yarılgaç, T., & Pekdemir, M. (2019). Promising strawberry tree genotypes from North Anatolia, Turkey. Erwerbs-Obstbau, 61(1), 79-84. 110 110 Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri [4] Özcan, M.M., & Hacıseferoğulları, H. (2007). The strawberry (Arbutus unedo L.) fruits: chemical composition, physical properties and mineral contents. Journal of Food Engineering, 78(3), 1022-1028. [5] Anşin, R., & Özkan, Z. C. (1993). Tohumlu Bitkiler, KTÜ Orman Fakültesi, Genel Yayın No: 167, Fakülte Yayın No: 19, 512 s. [6] Koyu, H., Koyu, E. B., Demir, S., & Baykan, Ş. (2019). Arbutus unedo L.(kocayemiş). Türk Farmakope, 4(3), 29-51. [7] Soufleros, E.H., Mygdalia, S.A., & Natskoulis, P. (2005). Production process and characterization of the traditional Greek fruit distillate “Koumaro” by aromatic and mineral composition. Journal of Food Composition and Analysis, 18(7), 699-716. [8] Gilman, E. F., & Watson, D. G. (1993). Arbutus unedo, Strawberry-Tree. Fact Sheet ST-85. A series of the Environmental Horticulture Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. [9] Males, Z., Plazibat, M., Bilusic Vundac, V., & Zuntar, I. (2006). Qualitative and quantitative analysis of flavonoids of the strawberry tree-Arbutus unedo L.(Ericaceae). Acta Pharmaceutica, 56(2), 245-250. [10] Çelikel, G., Demirsoy, L., & Demirsoy, H. (2008). The strawberry tree (Arbutus unedo L.) selection in Turkey. Scientia Horticulturae, 118(2), 115-119. [11] Ayaz, F.A., Kucukislamoglu, M., & Reunanen, M. (2000). Sugar, non-volatile and phenolic acids composition of strawberry tree (Arbutus unedo L. var. ellipsoidea) fruits. Journal of Food Composition and Analysis, 13(2), 171-177. [12] Oliveira, I., Baptista, P., Malheiro, R., Casal, S., Bento, A., & Pereira, J.A. (2011). Influence of strawberry tree (Arbutus unedo L.) fruit ripening stage on chemical composition and antioxidant activity. Food Research International, 44(5), 1401-1407. [13] Aloğlu, H. Ş., Gökgöz, Y., & Bayraktar, M. (2018). Kaynakça Kocayemiş (dağ çileği-Arbutus unedo L.) meyveli dondurma üretimi, fiziksel, kimyasal ve duyusal parametreler açısından irdelenmesi. Gıda, 43(6), 1030-1039. [14] Pallauf, K., Rivas-Gonzalo, J. C., Del Castillo, M. D., Cano, M. P., & de Pascual-Teresa, S. (2008). Characterization of the antioxidant composition of strawberry tree (Arbutus unedo L.) fruits. Journal of Food Composition and Analysis, 21(4), 273-281. [15] Molina, M., Pardo-de-Santayana, M., Aceituno, L., Morales, R., & Tardío, J. (2011). Fruit production of strawberry tree (Arbutus unedo L.) in two Spanish forests. Forestry, 84(4), 419- 429. [16] Martin, R. M., Gunnell, D., Owen, C. G., & Smith, G. D. (2005). Breast‐feeding and childhood cancer: a systematic review with metaanalysis. International Journal of Cancer, 117(6), 1020- 1031. [17] Clavel, J. (2007). Progress in the epidemiological understanding of gene–environment interactions in major diseases: cancer. Comptes rendus biologies, 330(4), 306-317. [18] Beyhan, Ö., Elmastas, M., & Gedikli, F. (2010). Total phenolic compounds and antioxidant capacity of leaf, dry fruit and fresh fruit of feijoa (Acca sellowiana, Myrtaceae). Journal of Medicinal Plants Research, 4(11), 1065-1072. 111 Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 111 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri [19] Chang, C.C., Yang, M.H., Wen, H.M., & Chern, J. C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10(3). [20] Blois, M.S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 181(4617), 1199-1200. [21] Benzie, I.F., & Strain, J.J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical Biochemistry, 239(1), 70-76. [22] Doré, S. (2005). Unique properties of polyphenol stilbenes in the brain: more than direct antioxidant actions; gene/protein regulatory activity. Neurosignals, 14(1-2), 61-70. [23] Ratnam, D.V., Ankola, D.D., Bhardwaj, V., Sahana, D.K., & Kumar, M.R. (2006). Role of antioxidants in prophylaxis and therapy: A pharmaceutical perspective. Journal of Controlled Release, 113(3), 189-207. [24] Thomas, R.H., Bernards, M.A., Drake, E.E., & Guglielmo, C.G. (2010). Changes in the antioxidant activities of seven herb-and spice-based marinating sauces after cooking. Journal of Food Composition and Analysis, 23(3), 244-252. [25] İşbilir, S.S., Orak, H.H., Yagar, H., & Ekinci, N. (2012). Determination of antioxidant activities of strawberry tree (Arbutus unedo L.) flowers and fruits at different ripening stages. Acta Sci Pol, 11, 223-237. [26] Saral, Ö., Erşen Bak, F., & Ölmez, Z. (2017). © 2020 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 Kaynakça Determining total phenolic content and antioxidant activity in fruits and flowers of naturally grown Arbutus andrachne L. in Artvin. Artvin Coruh University Journal of Forestry Faculty, 18(1), 51-54. [27] Asmaa, N., Abdelaziz, G., Boulanouar, B., Carbonell-Barrachina, Á. A., Cano-Lamadrid, M., & Noguera-Artiaga, L. (2019). 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Anti-demineralizing protective effects on enamel identified in experimental and commercial restorative materials with functional fillers
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Anti‑demineralizing protective effects on enamel identified in experimental and commercial restorative materials with functional fillers OPEN Matej Par1,2*, Andrea Gubler2, Thomas Attin2, Zrinka Tarle1 & Tobias T. Tauböck2 The aim of this study was to investigate whether experimental and commercial dental restorative materials with functional fillers can exert a protective anti-demineralizing effect on enamel that is not immediately adjacent to the restoration. Four experimental resin composites with bioactive glass and three commercial restorative materials were investigated. Enamel blocks were incubated in a lactic acid solution (pH = 4.0) at a standardized distance (5 mm) from cured specimens of restorative materials. The lactic acid solution was replenished every 4 days up to a total of 32 days. Surfaces of enamel blocks were periodically evaluated by Knoop microhardness measurements and scanning electron microscopy. The protective effect of restorative materials against acid was identified as enamel microhardness remaining unchanged for a certain number of 4-day acid addition cycles. Additionally, the pH of the immersion medium was measured. While enamel microhardness in the control group was maintained for 1 acid addition cycle (4 days), restorative materials postponed enamel softening for 2–5 cycles (8–20 days). The materials capable of exerting a stronger alkalizing effect provided longer-lasting enamel protection. The protective and alkalizing effects of experimental composites improved with higher amounts of bioactive glass and were better for conventional bioactive glass 45S5 compared to a fluoride-containing bioactive glass. Scanning electron micrographs evidenced the protective effect of restorative materials by showing a delayed appearance of an etching pattern on the enamel surface. A remotely-acting anti-demineralizing protective effect on enamel was identified in experimental composites functionalized with two types of bioactive glass, as well as in three commercial ion-releasing restorative materials. As secondary caries represents one of the main reasons for the failure of composite ­restorations1, bioactive glasses (BGs) have been investigated as interesting dopants for restorative resin composites due to their potential for releasing remineralizing ­ions2,3, neutralizing ­acid4,5, and precipitating ­hydroxyapatite6,7. BGs encompass vari- ous formulations with adjustable relative ratios of constituent elements. The composition of a particular BG determines its network structure, which in turn reflects on its ­properties8. In this way, subtle compositional adjustments can be made to tailor reactivity, solubility, and ion release for a desired therapeutic ­effect9. Among various BG compositions that have been investigated as functional additives for methacrylate-based restorative composites, fluoride-modified BGs appear especially promising due to their ability for releasing fluoride ions when exposed to an aqueous ­medium2. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Endodontics and Restorative Dentistry, School of Dental Medicine, University of Zagreb, Gunduliceva 5, Zagreb, Croatia. 2Department of Conservative and Preventive Dentistry, Center for Dental Medicine, University of Zurich, Plattenstrasse 11, Zurich, Switzerland. *email: mpar@sfzg.hr Scientific Reports | (2021) 11:11806 Anti‑demineralizing protective effects on enamel identified in experimental and commercial restorative materials with functional fillers OPEN These ions can be incorporated into the enamel to render it more resist- ant to ­demineralization10. An additional potential benefit of fluoride-modified BGs is their capability to form a fluorapatite layer on the composite ­surface11, which can be envisioned as a possible sealing agent for marginal ­gaps12. Besides experimental BG formulations containing various therapeutic elements, the conventional Hench’s BG 45S5 formulation has also been extensively investigated as a possible filler for resin ­composites3–6,13. Notwith- standing the unavoidable trade-offs between its high reactivity and stability of mechanical ­properties14 and the 1Department of Endodontics and Restorative Dentistry, School of Dental Medicine, University of Zagreb, Gunduliceva 5, Zagreb, Croatia. 2Department of Conservative and Preventive Dentistry, Center for Dental Medicine, University of Zurich, Plattenstrasse 11, Zurich, Switzerland. *email: mpar@sfzg.hr | https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 www.nature.com/scientificreports/ Table 1. Bioactive glass and reinforcing fillers used in experimental composites. Bioactive glass 45S5 Experimental fluoride- containing bioactive glass Inert barium glass Silica Particle size (d50) 3 µm 3 µm 1 µm 5–50 nm Composition (wt%) 45.0% ­SiO2 24.5% CaO 24.5% ­Na2O 6.0% ­P2O5 33.5% ­SiO2 33.0% CaO 10.5% ­Na2O 11.0% ­P2O5 12.0% ­CaF2 55.0% ­SiO2 25.0% BaO 10.0% ­Al2O3 10.0% ­B2O3  > 99.8% SiO2 Silanization (wt%) None None 3.2 4–6 Manufacturer Schott, Mainz, Germany Schott, Mainz, Germany Schott, Mainz, Germany Evonik, Hanau, Germany Product name / LOT G018-144 / M111473 Experimental batch GM27884 / Sil13696 Aerosil R 7200 / 157,020,635 Table 1. Bioactive glass and reinforcing fillers used in experimental composites. Table 1. Bioactive glass and reinforcing fillers used in experimental composites. Table 2. Composition of experimental composites. Material designation Filler composition (wt%) Total filler ratio (wt%) Bioactive glass 45S5 Experimental fluoride-containing bioactive glass Reinforcing fillers (inert barium glass : silica = 2:1) Control 0 0 70 70 C-10 10 0 60 70 C-20 20 0 50 70 E-10 0 10 60 70 E-20 0 20 50 70 Table 2. Composition of experimental composites. lack of fluoride release, the traditional BG 45S5 composition represents a promising candidate for functional- izing resin ­composites4,6,15,16. BG embedded within resin composites dissolves when exposed to an aqueous medium, releasing its con- stituent elements into the ­solution2. This ion release is accompanied by an alkalizing ­effect11. The release of remineralizing ions and the increase of the solution pH can be employed to protect dental hard tissues against demineralization caused by bacterial acids. Anti‑demineralizing protective effects on enamel identified in experimental and commercial restorative materials with functional fillers OPEN Such a protective effect has been demonstrated in experimental BG- functionalized composites intended for use as orthodontic adhesives; these materials showed the potential for protecting enamel adjacent to bonded brackets against the formation of white ­spots17–21. To investigate whether the protective effect of BG-functionalized composites can extend beyond the dental hard tissues immediately adjacent to the ­material22, the present in vitro study used enamel blocks incubated in a lactic acid solution at a standardized distance from cured specimens of restorative materials. Experimental composites functionalized with 10 or 20 wt% of conventional BG 45S5 and an experimental fluoride-modified BG were compared to three commercial restorative materials capable of exerting an anti-demineralizing protective effect. Microhardness (MH) was chosen as a convenient indicator of structural integrity of the enamel ­surface23 exposed to simulated cycles of acid attack. Additionally, enamel surface morphology and pH changes in the immersion solution were evaluated. The null hypotheses were that the evaluated parameters (enamel MH, enamel surface morphology, and pH of the immersion solution) would: (1) remain unchanged during the simulated acid attack; and (2) not differ among the investigated restorative materials. Figure 1.   Experimental design. Figure 1. Experimental design. Konstanz, Germany; shade: A2, LOT: 1903000819), a giomer (Beautifil II, Shofu, Kyoto, Japan; shade: A2, LOT: 041923), and a resin-based “alkasite” material (Cention, Ivoclar Vivadent, Schaan, Liechtenstein; shade: universal, LOT: XL7102). The alkasite material contains two types of reactive filers: an ionomer glass based on a calcium barium alumino-fluoro-silicate, and a calcium fluoro-silicate ­glass25,26. Enamel blocks. One hundred twenty-eight intact human third molars were collected as by-products of regular dental treatment. Patients had given written informed consent to use the teeth for research purposes, and all teeth were irreversibly anonymized immediately after extraction. Under these terms, the research complied with the use of anonymized biological material and, consequently, authorization from the local ethics committee was not required (Federal Act on Research involving Human Beings (Human Research Act; article 2, paragraph 2)). The teeth were stored at 8 °C in 0.1% thymol solution and used within 6 months of extraction. h y Enamel blocks (1 block per tooth; 3 × 3 × 1 mm) were prepared from buccal surfaces using a low-speed preci- sion cutting machine (IsoMet, Buehler, Lake Bluff, IL, USA). The buccal sides of enamel blocks were ground using P4000 silicon carbide ­paper27 (Buehler; 2 min at 30 rpm, median particle size = 2.5 µm). After preparation, the enamel blocks were stored in a phosphate-buffered saline solution and used within five days of preparation. From the total of 128 enamel blocks, 64 were used for the MH and pH measurements, while 64 were used for the scan- ning electron microscopy (SEM) study, as described in the schematic representation of the study design in Fig. 1. Restorative material specimens. Discoid specimens (diameter = 7 mm, thickness = 2 mm) were pre- pared by casting the restorative materials into custom-made polyoxymethylene molds, covering their surfaces with Mylar foils, and flattening using glass plates. The glass ionomer material was left to set in the mold for 15 min, while the light-curable materials (giomer and experimental composites) and the dual-curable alkasite material were illuminated using a violet-blue LED curing unit (Bluephase PowerCure, Ivoclar Vivadent, Schaan, Liechtenstein; emission wavelength range: 390–500 nm, radiant exitance: 1,340 mW/cm2) for 20 s, resulting in a total radiant exposure of 26.8 J/cm2). To simulate a clinically realistic scenario, the specimens were exposed to the immersion medium within 15 min after preparation. Materials and methods i l i Experimental resin composites. Experimental resin composites were prepared as described in previ- ous ­studies11,24. The resin system contained bisphenol-A-glycidyldimethacrylate (Bis-GMA, Merck, Darmstadt, Germany) and triethylene glycol dimethacrylate (TEGDMA, Merck) in a ratio of 60:40 wt%. Camphorquinone (0.2 wt%; Merck) and ethyl-4-(dimethylamino) benzoate (0.8 wt%; Merck) were added to photoactivate the resin system. The components of the resin system were mixed using a magnetic stirrer for 48 h.hi The composition of fillers used to prepare experimental composites is given in Table 1. BG 45S5, inert barium glass, and silica were obtained from commercial vendors. The experimental fluoride-modified experimental BG with theoretical network connectivity similar to that of BG 45S5 (2.1)8 was prepared on-demand by Schott (Mainz, Germany) via melt-quench route. By using similar preparation and grinding procedures, comparable particle sizes were obtained for both BG types.i Experimental composites with a total filler ratio of 70 wt% were prepared by replacing 0, 10, or 20 wt% of silanized reinforcing fillers (barium glass and silica) with two types of unsilanized BG (Table 2). The experimen- tal composite containing only reinforcing fillers was used as an inert control. The composites were prepared by mixing the resin system and the fillers for 5 min in a dual asymmetric centrifugal mixing system (Speed Mixer TM DAC 150 FVZ, Hauschild & Co. KG, Hamm, Germany) at 2,000 rpm, and deaerating in vacuum for 48 h. mixing the resin system and the fillers for 5 min in a dual asymmetric centrifugal mixing system (Speed Mixer TM DAC 150 FVZ, Hauschild & Co. KG, Hamm, Germany) at 2,000 rpm, and deaerating in vacuum for 48 h. Materials and methods i l i In addition to the experimental composites, three commercial restorative materials with an acid-protective capability were used as references: a reinforced glass ionomer restorative (ChemFil Rock, Dentsply Sirona, In addition to the experimental composites, three commercial restorative materials with an acid-protective capability were used as references: a reinforced glass ionomer restorative (ChemFil Rock, Dentsply Sirona, Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z www.nature.com/scientificreports/ Enamel blocks incubated with specimens of restorative materials (n = 8 per material) Control C-10 C-20 E-10 E-20 Beautifil II ChemFil Rock Cention Added 5 mL of lactic acid (pH = 4.0) and replenished after 4, 8, 12, 16, 20, 24, 28, 32 days After 4, 8, 16, 32 days: • withdrawn two enamel blocks per material group for scanning electron microscopy After 0, 4, 8, 12, 16, 20, 24, 28, 32 days: • measured microhardness of enamel • measured pH of immersion medium 128 enamel blocks (3 x 3 x 1 mm) 64 enamel blocks for microhardness and pH study 64 enamel blocks for scanning electron microscopy study • withdrawn two enamel blocks per material group for scanning electron microscopy www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 3. Knoop microhardness of enamel blocks (mean values with standard deviations in parentheses). Same letters denote statistically similar microhardness values within a material. Time No. of acid addition cycles Material Control C-10 C-20 E-10 E-20 Beautifil II ChemFil Cention Initial 0 243.8 (38.8) a 243.7 (39.2) ab 255.2 (42.9) a 262.4 (20.5) a 233.8 (45.0) a 252.7 (33.9) a 267.6 (22.2) a 261.9 (28.6) ab 4 days 1 228.3 (29.8) a 254.2 (32.6) a 267.6 (38.1) a 259.3 (25.1) a 263.8 (21.5) a 245.9 (27.2) a 260.5 (22.2) ab 276.4 (29.8) a 8 days 2 174.9 (32.3) b 207.2 (27.3) bc 256.6 (33.9) a 231.1 (34.1) a 245.4 (40.3) a 203.1 (33.1) ab 244.8 (23.8) abc 269.9 (20.5) a 12 days 3 145.8 (26.8) bc 178.5 (29.8) bc 239.7 (40.4) a 220.6 (22.1) b 221.0 (35.1) ab 201.6 (42.7) ab 221.4 (32.7) bc 239.3 (27.8) ab 16 days 4 107.1 (33.0) cd 136.1 (23.4) d 230.2 (51.8) a 167.2 (32.5) c 174.9 (37.5) bc 175.3 (33.1) b 203.5 (39.5) bc 212.8 (28.2) bc 20 days 5 81.8 (13.7) de 113.9 (32.6) d 207.7 (49.7) ab 123.5 (32.4) d 136.1 (40.0) c 156.9 (36.8) bc 198.5 (47.6) c 175.0 (50.1) c 24 days 6 40.7 (20.3) ef 47.1 (11.6) e 147.4 (51.6) bc 55.5 (30.7) e 69.4 (27.9) d 99.3 (36.5) d 129.4 (44.0) d 98.2 (50.3) d 28 days 7 29.4 (10.6) f 38.6 (14.8) e 145.1 (48.1) bc 43.0 (23.2) e 59.2 (19.1) d 106.0 (29.1) cd 112.4 (49.6) d 70.5 (30.6) d 32 days 8 25.3 (5.3) f 30.8 (9.2) e 122.3 (41.5) c 36.0 (11.4) e 50.0 (16.0) d 88.9 (35.3) d 113.4 (48.6) d 63.8 (19.5) d Table 3. Knoop microhardness of enamel blocks (mean values with standard deviations in parentheses). Same letters denote statistically similar microhardness values within a material. Immersion in lactic acid. The enamel blocks and restorative material specimens were immersed in closed vials (Eppendorf; Hamburg, Germany) containing 5 mL of lactic acid solution (pH = 4.0). This experimental setup was adopted and modified according to previous ­studies28–30. Each vial contained one enamel block and one restorative material specimen, which were held at a standardized distance of 5 mm. The immersion solution was agitated using a horizontal laboratory shaker at a speed of 30 revolutions per minute. The environmental temperature ranged between 23–24 °C. www.nature.com/scientificreports/ Surface MH of enamel blocks and pH of the immersion solution were measured at the following time points: 4, 8, 12, 16, 20, 24, 28, and 32 days. At each time point, the immersion medium was replenished with 5 mL of fresh lactic acid solution (pH = 4.0). Microhardness measurements. Knoop MH measurements were performed on the buccal sides of enamel blocks using a digital hardness tester (model no. 1600–6106; Buehler). Indentations were made under a load of 100 g and a dwell time of 20 s at random positions. The indentations were evaluated within 2 min after preparation, with a resolution of 0.015 µm31. Per each specimen and time point, five replicate indentations were made and their mean values were considered as a statistical unit. Eight specimens were used per material (n = 8). pH measurements. A calibrated pH electrode (780 pH Meter, Metrohm, Herisau, Switzerland) was immersed in the solution, and pH values were recorded with a resolution of 0.01 pH units. Per each specimen and time point, three replicate measurements were made and their mean values were considered as a statistical unit. Eight specimens were used per material (n = 8). Scanning electron microscopy. Enamel blocks for the SEM study (n = 8 per material) were subjected to the same acid exposure protocol as the specimens for the evaluation of MH and pH. Upon reaching the time points of 4, 8, 16, and 32 days, two enamel blocks per material were withdrawn, rinsed with distilled water, dried, and sputtered with 5 nm of gold. A scanning electron microscope (SEM; GeminiSEM 450, Zeiss, Oberkochen, Germany) was used at 10 kV and 10,000 × magnification to evaluate surface morphology of enamel blocks. Statistical analysis. Normality of distribution and homogeneity of variances were checked using Levene’s and Shapiro–Wilk’s tests, respectively. Within each restorative material, MH and pH values were compared among time points using repeated-measures ANOVA with Bonferroni correction for multiple comparisons. The statistical analysis was performed using SPSS (version 20, IBM, Armonk, NY, USA) at an overall level of significance of α = 0.05. 1. A significant initial pH increase, followed by a plateau at pH = 6–7. Materials: Control, E-10, Beautifil II, and ChemFil. Figure 1.   Experimental design. Per material, 16 specimens were prepared (8 for the MH and pH study; and 8 for the SEM study). https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | Discussionh This study showed that restorative materials with functional fillers exert an anti-demineralizing effect when incubated with enamel blocks in an acid solution. This effect was evidenced by MH of enamel blocks remaining unimpaired over a material-dependent number of acid attack cycles. To demonstrate that the protective effect does not depend on the close proximity of restorative materials to dental hard tissues, enamel blocks and restora- tive material specimens were separated by a distance of 5 mm. The protective effect was material-dependent and identified for the experimental BG-containing composites, as well as for three commercial restorative materials, leading us to reject both null hypotheses.h g j yp The composition of experimental composites with 10 and 20 wt% of BG followed a previous study in which these materials were shown to neutralize acid similarly to the alkasite material and better than a giomer and a glass ­ionomer11. These results motivated the present study, which aimed to investigate whether the acid- neutralizing and ion-releasing activities of experimental BG-composites can protect remote enamel surfaces against demineralization caused by lactic acid. g y Notwithstanding the complex pathophysiology of tooth caries, the initial damage to the enamel surface stems from an imbalance in the dissolution and precipitation ­processes32. While various sophisticated approaches are beneficial for in-depth evaluations of caries ­progression23, simple measurements of enamel surface MH indicat- ing initial structural changes due to acid exposure have proven useful for evaluations of the anti-demineralizing effect of restorative ­materials33–35. Therefore, our study employed periodic replenishing of the lactic acid solution and evaluated the number of acid addition cycles that enamel blocks withstood before showing signs of surface softening. The initial enamel MH in all experimental groups was within the usual MH range for sound ­enamel36. Statistical comparisons of those initial MH values and MH measured after successive acid additions enabled identifying the point of significant MH decrease, which was considered to signify initial enamel softening. Th b l f h d l f l d l b d y g p gi g yt g The capability of the investigated materials for protecting enamel against demineralization is based on two main effects, namely the neutralization of acid and the release of remineralizing ­ions2,11. The latter mechanism shifts the dissolution/precipitation dynamics between the enamel hydroxyapatite and the immersion solution towards precipitation. Results MH values of enamel blocks measured after acid addition cycles are shown in Table 3. Repeated additions of acid solution significantly decreased enamel MH in all groups. The protective effect of restorative materials against acid was identified as enamel MH remaining statistically similar to initial MH values for a certain number of acid additions. The enamel MH in the control group was maintained for up to 1 acid addition and showed a statistically significant decrease thereafter. In all other groups, enamel MH was maintained for more acid addi- tion cycles, remaining statistically similar to initial MH values for up to 2 cycles (E-10 and ChemFil), 3 cycles (C-10, E-20, and Beautifil II), 4 cycles (Cention) and 5 cycles (C-20). The number of acid additions over which enamel MH remained statistically similar to the initial values is summarized in Fig. 2.hh y g The pH changes in the immersion solution are shown in Fig. 3. The initial pH (measured before incubating enamel blocks and restorative materials) was 4.0 in all groups. Statistically significant pH changes were observed after periodic acid additions, leading to the following three patterns in the pH curves: https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | ntificreports/ 0 1 2 3 4 5 Control E-10 ChemFil C-10 E-20 Beaufill II Cenon C-20 s n oitid d a dic a f o r e b m u N Figure 2. Number of acid addition cycles for which enamel surface MH remained statistically similar to initial values. www.nature.com/scientificreports/ Figure 2. Number of acid addition cycles for which enamel surface MH remained statistically similar to initial values. Figure 2. Number of acid addition cycles for which enamel surface MH remained statistically similar to initial values. 2. A transient peak at pH = 8–10 after 4 days, followed by a pH drop and stabilization at pH = 6–7. Materials: C-10, E-20, and Cention.i 2. A transient peak at pH = 8–10 after 4 days, followed by a pH drop and stabilization at pH = 6–7. Materials: C-10, E-20, and Cention. 3 A significant initial pH increase followed by a plateau at pH 9 10 Material C 20 C 0, 0, a d Ce t o . 3. A significant initial pH increase, followed by a plateau at pH = 9–10. Material: C-20. Results The pH changes in patterns (1) and (2) converged to a plateau at pH = 6–7; this was the most common behavior identified in 7 out of 8 tested materials. Pattern (3) was observed for only one material (C-20) capable of maintaining alkaline pH over the whole observation period.t g p p SEM images of enamel surfaces after 4, 8, 16, and 32 days of immersion in the acid solution are shown in Fig. 4 for the experimental materials and Fig. 5 for the commercial materials. After 4 days, enamel surfaces in all groups showed a scratch pattern from grinding, while enamel blocks immersed with Cention additionally showed a precipitate on their surface. Over the subsequent time points (8, 16, and 32 days), the scratch patterns gradually became less visible and were replaced with etching patterns. For C-20, Beautifil II, and Cention, the scratch pattern remained visible up to 16 days, whereas for other materials they were replaced by etching patterns at earlier times (4–8 days). After 32 days, etching patterns were clearly observable for all materials. Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z Discussionh Sufficiently saturating the immersion solution with calcium and phosphate ions can pre- vent enamel demineralization even in moderately acidic ­environments32. A strong alkalizing effect is therefore not necessary for a protective effect if sufficient concentrations of remineralizing ions are released. This consid- eration can explain the finding that E-10, Beautifil II, and ChemFil demonstrated a better protective effect than the control composite by maintaining enamel MH unchanged for a longer time (8–12 days vs. 4 days for the control composite) despite showing pH curves similar to that of the control composite. Discussionh a b b b b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) Control microhardness unchanged a b bc bc bc bc bc bc c 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) C-20 microhardness unchanged a b b b b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) Control microhardness unchanged a b c c c c c c c 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) C-10 microhardness unchanged a b bc c b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) E-10 microhardness unchanged a b b b b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) Beaufil II microhardness unchanged a b c c c c c c c 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) E-20 microhardness unchanged Beaufil II Time (days) a b c d e e e e e 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) Cenon microhardness unchanged Time (days) Figure 3. pH changes (mean values ± standard deviations) of the immersion medium and time periods for which enamel microhardness remained unchanged compared to initial values. Same letters denote statistically similar pH values within a material. capability, namely C-10, C-20, E-20, and Cention. For these materials, the synergistic effect of the alkalization and ion release led to a longer-lasting protective capability. Discussionh A comparatively better protective effect (MH unchanged for 12–20 days) was identified for the materials with a pronounced alkalizing Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z www.nature.com/scientificreports/ a b b b b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) Control microhardness unchanged a b c c c c c c c 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) C-10 microhardness unchanged a b bc bc bc bc bc bc c 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) C-20 microhardness unchanged a b bc c b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) E-10 microhardness unchanged a b c c c c c c c 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) E-20 microhardness unchanged a b b b b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) Beaufil II microhardness unchanged a b b b b b b b b 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) ChemFil microhardness unchanged a b c d e e e e e 0 1 2 3 4 5 6 7 8 9 10 0 4 8 12 16 20 24 28 32 pH Time (days) Cenon microhardness unchanged Figure 3. pH changes (mean values ± standard deviations) of the immersion medium and time periods for which enamel microhardness remained unchanged compared to initial values. Same letters denote statistically similar pH values within a material. Discussionh Th H i b d f th t l b tt ib t d t th di l ti f l37 hi h l d t The pH increase observed for the control group can be attributed to the dissolution of ­enamel37 which led to acid neutralization, as previously reported in studies that incubated enamel blocks in lactic acid ­solution28,30. As the control group showed a plateau at the pH = 6–7 throughout the whole observation period, the alkalizing effect in other groups had to be sufficiently pronounced in order to become distinguishable from the acid neutralization by enamel blocks alone. Such an effect was observed for C-10, E-20, and Cention showing a transient alkalization, and for C-20 which showed a more notable alkalization by maintaining a plateau at pH = 9–10. The observation of the alkalizing effect of E-10, Beautifil II, and ChemFil being similar to that of the control composite does not The pH increase observed for the control group can be attributed to the dissolution of ­enamel37 which led to acid neutralization, as previously reported in studies that incubated enamel blocks in lactic acid ­solution28,30. As the control group showed a plateau at the pH = 6–7 throughout the whole observation period, the alkalizing effect in other groups had to be sufficiently pronounced in order to become distinguishable from the acid neutralization by enamel blocks alone. Such an effect was observed for C-10, E-20, and Cention showing a transient alkalization, and for C-20 which showed a more notable alkalization by maintaining a plateau at pH = 9–10. The observation of the alkalizing effect of E-10, Beautifil II, and ChemFil being similar to that of the control composite does not https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | www.nature.com/scientificreports/ Figure 4. Scanning electron microscopy images of the surfaces of enamel blocks immersed with the control composite and the composites containing conventional (C-10 and C-20) and experimental (E-10 and E-20) bioactive glass. Figure 4. Scanning electron microscopy images of the surfaces of enamel blocks immersed with the control composite and the composites containing conventional (C-10 and C-20) and experimental (E-10 and E-20) bioactive glass. necessarily imply that these materials lack an acid-neutralizing effect. Discussionh These materials indeed demonstrated a moderate acid-neutralizing effect in a previous ­study11; however, by measuring pH at the end of four-day acid addition cycles in the present study, their contributions to the pH increase were indiscernible from the acid- neutralizing effect of the dissolution of enamel blocks.f gf As the acid-neutralizing effect was eventually exhausted for all materials except C-20, their pH curves con- verged to the plateau values of the control composite at pH = 6–7. Although C-20 maintained a high pH of 9–10 throughout the whole observation period, MH measurements showed that its protective effect on enamel was exhausted after 20 days. This finding suggests that an apparently long-lasting alkalizing capability of C-20 was not sufficient to protect enamel against demineralization. Although the pH = 9–10 was consistently measured at the end of each four-day acid addition cycle, repeatedly exposing the enamel to the low-pH solution undersaturated with calcium and phosphate ions at the beginning of each cycle produced cumulative enamel damage, which was not remineralized over the rest of the cycle despite the alkaline pH being attained towards the end of each cycle. This reasoning also implies that the commonly used approach of measuring the alkalizing effect of restorative materials after particular time ­intervals2,4,6,38 does not necessarily indicate protection against demineralization because it does not take into account the demineralization that occurs before reaching a particular time point.f g p p Whereas all investigated commercial restorative materials showed a better protective effect than the control composite, the best performance among them was identified in a recently launched “alkasite” restorative material (Cention). In addition to showing a transient alkalizing capability, this material also showed the longest protective effect among the commercial materials. It is interesting to note that the other two commercial materials protected enamel better than the control composite, even though their pH curves indicated an acid neutralization capabil- ity similar to that of the control. Their protective effect was apparently more reliant on ion release, particularly of fluoride ions which can be incorporated into demineralized enamel over a 4-day acid exposure cycle, thereby rendering enamel more acid-resistant to the acid attack in the next ­cycle39.hf g y The results for the BG-functionalized composites show that (I) the protective and alkalizing effects were dose-dependent, and (II) the protective and alkalizing effects were better for composites functionalized with the conventional BG 45S5 compared to the experimental fluoride-containing BG. Scientific Reports | (2021) 11:11806 | Discussionh Since these differences imply various extents of material degradation due to the dissolution of functional ­fillers11, the ion release profiles vs. the stability of mechanical properties of experimental composites with these two BG types are being addressed in ongoing studies. These studies will also include the commercial material Cention since the evidence of its high reactivity (protective and alkalizing effects better than all other tested materials except C-20) makes it an interesting candidate for studying long-term degradation of mechanical properties. Although Cention lacks true self-adhesive properties, it was initially marketed as capable of being placed without an adhesive system due to the assumed capability for sealing marginal gaps via hydroxyapatite precipi- tation. These recommendations have recently been revised by the manufacturer, indicating that an adhesive system (preferably the proprietary product Cention Primer) should be applied before restoring the cavity using Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z www.nature.com/scientificreports/ Figure 5. Scanning electron microscopy images of the surfaces of enamel blocks immersed with commercial reference materials. Figure 5. Scanning electron microscopy images of the surfaces of enamel blocks immersed with commercia reference materials. Cention. Therefore, clinical application of this new “alkasite” material type can be regarded as being similar to other dual-curing bulk-fill composites with the additional benefit of protective effect on surrounding dental hard tissues through ion release and alkalization.hifh The SEM micrographs support the finding of the protective effect of restorative materials on enamel. The anti-demineralizing effect was identified as the grinding scratch pattern on the enamel surface remaining visible over a material-dependent period of acid ­immersion30. As the protective effect was exhausted by the periodical replenishing of the acid solution, the scratch pattern progressively became shallower and was eventually replaced by etching patterns. An illustrative example of such a gradual transition was observed for C-20, which showed a gradual thinning of the scratch pattern over 4–16 days, followed by an emergence of the etching pattern after 32 days. y Distinctive etching patterns of Type 1 and 2 according to Silverstone et al.40 were observed on SEM micro- graphs. For example, Type 1 etching pattern characterized by enamel dissolution being more pronounced in prism cores was observed after 32 days for the control group, while Type 2 etching pattern in which dissolution occurs preferentially on prism peripheries was observed after 32 days for C-20 and ChemFil. Conclusions Restorative materials functionalized with reactive fillers showed an anti-demineralizing effect on enamel blocks that were 5 mm away from material specimens. The remotely-acting protective effect was identified in experi- mental composites functionalized with two types of bioactive glass, as well as in three commercial restorative materials. Although the materials which showed pronounced alkalizing capability also showed a longer-lasting protective effect, the protection of enamel against demineralization was also attainable without a pronounced alkalization. www.nature.com/scientificreports/ under the conditions of alkaline pH at the time point of 4 ­days25. As the pH dropped over subsequent time points, the precipitate was dissolved, revealing the underlying scratch pattern on the enamel surface. under the conditions of alkaline pH at the time point of 4 ­days25. As the pH dropped over subsequent time points, the precipitate was dissolved, revealing the underlying scratch pattern on the enamel surface. A simple demineralization model using periodically replenished acid solution was employed in this study because it was a convenient and cost-effective means for demonstrating the protective effects of experimental ­composites30. As such a model does not simulate clinically realistic slowly-advancing subsurface lesions, a mul- tispecies biofilm ­model41 is planned to be used in a future study that will evaluate the capability of experimental bioactive glass-containing composites for preventing the progression of carious lesions along restoration margins. Received: 9 March 2021; Accepted: 25 May 2021 Received: 9 March 2021; Accepted: 25 May 2021 Received: 9 March 2021; Accepted: 25 May 2021 References Remineralization of demineralized enamel via calcium phosphate nanocomposite. J. Dent. Res. 91, 979–984 (2012).i 11. Par, M., Attin, T., Tarle, Z. & Tauböck, T. T. A new customized bioactive glass filler to functionalize resin composites: Acid- neutralizing capability, degree of conversion, and apatite precipitation. J. Clin. Med. 9, 1173. https://​doi.​org/​10.​3390/​jcm90​41173 (2020). ( ) 2. Khvostenko, D., Hilton, T. J., Ferracane, J. L., Mitchell, J. C. & Kruzic, J. J. Bioactive glass fillers reduce bacterial penetration into marginal gaps for composite restorations. Dent. Mater. 32, 73–81 (2016). g g p p 13. Par, M., Spanovic, N., Tauböck, T. T., Attin, T. & Tarle, Z. Degree of conversion of experimental resin composites containing bioac- tive glass 45S5: the effect of post-cure heating. Sci. Rep. 9, 17245. https://​doi.​org/​10.​1038/​s41598-​019-​54035-y (2019).ti gf p g p p g y 14. Par, M., Tarle, Z., Hickel, R. & Ilie, N. Mechanical properties of experimental composites containing bioactive glass after artificial aging in water and ethanol. Clin. Oral Investig. 23, 2733–2741 (2019). g g g 5. Par, M. et al. Curing potential of experimental resin composites with systematically varying amount of bioactive glass: Degree o conversion, light transmittance and depth of cure. J. Dent. 75, 113–120 (2018).i 15. Par, M. et al. Curing potential of experimental resin composites with systematically varying a conversion, light transmittance and depth of cure. J. Dent. 75, 113–120 (2018).i g 16. Par, M. et al. Curing potential of experimental resin composites filled with bioactive glass: A comparison between Bis-EMA and UDMA based resin systems. Dent. Mater. 36, 711–723 (2020).fl y 17. Nam, H.-J. et al. Enamel surface remineralization effect by fluorinated graphite and bioactive glass-containing orthodontic bonding resin. Materials 12, 1308 (2019).f ( ) 8. Nam, H.-J. et al. Fluorinated bioactive glass nanoparticles: Enamel demineralization prevention and antibacterial effect of ortho- dontic bonding resin. Materials 12, 1813 (2019). g 19. 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Bioactivity and physico-chemical properties of dental composites functionalized with nano- vs. micro-sized bioactive glass. J. Clin. Med. 9, 772. https://​doi.​org/​10.​3390/​jcm90​30772 (2020).i g g j 5. Yang, S.-Y. et al. Acid neutralizing, mechanical and physical properties of pit and fissure sealants containing melt-derived 45S5 bioactive glass. Dent. Mater. 29, 1228–1235 (2013). g ( ) 6. Tauböck, T. T. et al. Functionalizing a dentin bonding resin to become bioactive. Dent. Mater. 30, 868–875 (2014). 6. Tauböck, T. T. et al. Functionalizing a dentin bondi 6. Tauböck, T. T. et al. Functionalizing a dentin bonding resin to become bioactive. Dent. Mater. 30, 868–875 (2014). 7 Al-eesa N A Johal A Hill R G & Wong F S L Fluoride containing bioactive glass composite for orthodontic adhesives— 7. Al-eesa, N. A., Johal, A., Hill, R. G. & Wong, F. S. L. Fluoride containing bioactive glass composite for orthodontic adhe Apatite formation properties. Dent. Mater. 34, 1127–1133 (2018). p p p 8. Jones, J. R. Review of bioactive glass: From Hench to hybrids. Acta Biomater. 9, 4457–4486 (2013). 9. Jones, J. R., Ehrenfried, L. M., Saravanapavan, P. & Hench, L. L. Controlling ion release from bioactive glass foam scaffolds antibacterial properties. J. Mater. Sci. Mater. Med. 17, 989–996 (2006). p p J , ( ) 10. Weir, M. D., Chow, L. C. & Xu, H. H. K. Discussionh Another notable finding is a layer of mineral precipitate identified after 4 days on enamel blocks immersed with Cention. Although no detailed characterization of this precipitate was performed, it was likely a hydroxyapatite layer that formed Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z www.nature.com/scientificreports/ Data availabilityh y The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References 10, 10560 (2020) i p p 32. Dawes, C. What Is the critical pH and why does a tooth dissolve in acid?. J. Can. Dent. Assoc. 69, 722–724 (2003).tt 32. Dawes, C. What Is the critical pH and why does a tooth dissolve in acid?. J. Can. Dent. Assoc. 69, 722–724 (2003). 33 Esteves Oliveira M et al Rehardening of acid softened enamel and prevention of enamel softening through CO2 laser irradiation 3. Esteves-Oliveira, M. et al. Rehardening of acid-softened enamel and prevention of enamel softening through CO2 laser irradiation J. Dent. 39, 414–421 (2011). 4. Hara, A. T. & Zero, D. T. Analysis of the erosive potential of calcium-containing acidic beverages: Erosive potential of calcium- containing beverages. Eur. J. Oral Sci. 116, 60–65 (2008). g g ( ) 5. Maltz, M., Scherer, S. C., Parolo, C. C. F. & Jardim, J. J. Acid susceptibility of arrestedenamel lesions: In situ study. Caries Res. 40 251–255 (2006).f ( ) 36. Chuenarrom, C., Benjakul, P. & Daosodsai, P. Effect of indentation load and time on knoop and vickers microhardness tests for enamel and dentin. Mater. Res. 12, 473–476 (2009).lf ( ) 37. Attin, T., Becker, K., Wiegand, A., Tauböck, T. T. & Wegehaupt, F. J. Impact of laminar flow velocity of different acids on enamel calcium loss. Clin. Oral Investig. 17, 595–600 (2013).i g 8. Balbinot, G., Leitune, V. C. B., Ogliari, F. A. & Collares, F. M. Niobium silicate particles as bioactive fillers for composite resins Dent. Mater. 36, 1578–1585 (2020).l 9. Francois, P., Fouquet, V., Attal, J.-P. & Dursun, E. Commercially available fluoride-releasing restorative materials: A review and a proposal for classification. Materials 13, 2313 (2020). i 0. Silverstone, L. M., Saxton, C. A., Dogon, I. L. & Fejerskov, O. Variation in the pattern of acid etching of human dental ename examined by scanning electron microscopy. Caries Res. 9, 373–387 (1975).hi y g py 1. Thurnheer, T., Rohrer, E., Belibasakis, G. N., Attin, T. & Schmidlin, P. R. Static biofilm removal around ultrasonic tips in vitro Clin. Oral Investig. 18, 1779–1784 (2014). Competing interests h p g The authors declare no competing interests. Author contributions M.P. study conception, experimental design, funding acquisition, formal analysis and data interpretation, statisti- cal analysis, visualization, wrote manuscript. A.G. performed experiments, data acquisition and interpretation, resources, revised and approved final manuscript. T.A. study conception, experimental design, funding acquisi- tion, supervision, data interpretation, project administration, resources, revised and approved final manuscript. Z.T. study conception, supervision, data interpretation, project administration, revised and approved final manu- script. T.T.T. study conception, experimental design, funding acquisition, supervision, data interpretation, project administration, resources, revised and approved final manuscript. Fundingh g This work was financially supported by the Research Fund of the Swiss Dental Association SSO (grant number 321-20). Matej Par’s research stay at University of Zurich was funded by a grant from the Swiss Federal Depart- ment of Economic Affairs, Education and Research (2019.0221). References et al. Deposition of fluoride on enamel surfaces released from varnishes is limited to vicinity of fluoridation site. Clin Oral Investig. 11, 83–88 (2007). g 23. Lippert, F. & Lynch, R. J. M. Comparison of Knoop and Vickers surface microhardness and transverse microradiography for the study of early caries lesion formation in human and bovine enamel. Arch. Oral Biol. 59, 704–710 (2014). 4. Par, M., Mohn, D., Attin, T., Tarle, Z. & Tauböck, T. T. Polymerization shrinkage behaviour of resin composites functionalized with unsilanized bioactive glass fillers. Sci. Rep. 10, 15237 (2020). gi p 5. Tiskaya, M., Al-eesa, N. A., Wong, F. S. L. & Hill, R. G. Characterization of the bioactivity of two commercial composites. Dent Mater. 35, 1757–1768 (2019).hf 6. Kelić, K., Par, M., Peroš, K., Šutej, I. & Tarle, Z. Fluoride-releasing restorative materials: The effect of a resinous coat on ion release Acta Stomatol. Croat. 54, 371–381 (2020). ( ) 7. Wiegand, A., Credé, A., Tschammler, C., Attin, T. & Tauböck, T. T. Enamel wear by antagonistic restorative materials under erosive conditions. Clin. Oral Investig. 21, 2689–2693 (2017). https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | www.nature.com/scientificreports/ 28. Kaga, M. et al. Inhibition of enamel demineralization by buffering effect of S-PRG filler-containing dental sealant. Eur. J. Oral Sci. 122, 78–83 (2014).fi 29. Kaga, N. et al. Protective effects of GIC and S-PRG filler restoratives on demineralization of bovine enamel in lactic acid solu Materials 13, 2140 (2020).ffi ( ) 0. Kakuda, S., Sidhu, S. K. & Sano, H. Buffering or non-buffering; an action of pit-and-fissure sealants. J. Dent. 43, 1285–1289 (2015) 30. Kakuda, S., Sidhu, S. K. & Sano, H. Buffering or non-buffering; an action of pit-and-fissure sealants. J. Dent. 43, 1285–1289 (2015). 31 P M M i D Atti T T l Z & T bö k T T Th ff t f id hi h i t it li ht i i h i l ffi ar, M., Marovic, D., Attin, T., Tarle, Z. & Tauböck, T. T. The effect of rapid high-intensity light-curing on micromechanical proper- es of bulk fill and conventional resin composites Sci Rep 10 10560 (2020) ffi 31. Par, M., Marovic, D., Attin, T., Tarle, Z. & Tauböck, T. T. The effect of rapid high-intensity light-curing on micromechanical pr ties of bulk-fill and conventional resin composites. Sci. Rep. 10, 10560 (2020). hf p g -fill and conventional resin composites. Sci. Rep. Additional information Correspondence and requests for materials should be addressed to M.P. Correspondence and requests for materials should be addressed to M.P. 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An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
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An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Agbor, Emmanuel A.1, Obongha, Ukpali E.2, Ita, Emmanuel. E.3 1,2,3Department of Urban and Regional Planning Cross River University of Technology, Calabar ABSTRACT: The Ancient city of ‘CALABAR’ often interpreted as “Come And Live And Be At Rest” has experienced insecurity and safety for over a decade due to rapid population growth and urbanization. The urban settings have also influenced criminal activities and disorder especially the physical form of the metropolis and its diverse economic activities. The increase crime rate is also attributed to the city’s social activity pattern and the structure of its transportation network which distributes traffic to all the neighborhoods within and around Calabar Metropolis. The aim of this study is to appraise the insecurity situation in the ancient city of Calabar and the consequences on the residents in the metropolis. The specific objectives are; to identify the major types of criminal activities in Calabar metropolis; to determine the cause of such criminal activities in the study area; and to examine the physical form and structure of the area under study. The Pearson’s Product Moment statistical techniques was employed to test the relationship between population and crime. At 4.345 degree of freedom and 0.05 level of significance, the P-value was less than the chosen alpha of .05, the null Hypothesis was therefore rejected confirming a significant relationship between population and crime in the study area. The study further revealed increase criminal activities in the inner-city neighborhoods and the Gated and rich neighborhoods in the study area due to the deteriorating quality of urban life and poor urban planning and design. KEYWORDS: Crime, Felonies, Misdemeanors, Population, Safety, Security. International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586 Page No: 1901-1915 International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586 Page No: 1901-1915 International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586 Page No: 1901-1915 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586 Page No: 1901-1915 INTRODUCTION The world is experiencing the second phase of urbanization phenomenon since the industrial revolution in the mid-19th century. The current rate of urbanization and urban growth is unprecedented. The United Nations survey in 1800, revealed that only 2.00 percent of the world population was urbanized. By 1850, not only more than 5.0 percent of the global population was urbanized and no society had more than half of its population living in cities except London which had more than one million people. The percentage of world’s population living in urban centres increased between 1800 and 1985 from 14.00 percent to 43.00 percent as indicated below on table 1. Currently in year 2020 the world urban population has increased to 2.9 billion as reported by the United Nations and Population Reference Bureau (1982). Currently, most African nations are experiencing the fastest rate of urbanization in the world without a corresponding increase in the essential basic infrastructure which is creating serious economic, social, political and environmental related problems. Between 1970-1995 the average African country’s urban population grew by 5.2 percent per annum while its Gross Domestic Product (GDP) declined by 0.66 percent per annum Hicks (1998). Urban population growth has been on the increase in Nigeria since the pre-colonial era. Okeke (1998) asserts that Nigeria urban centres are amongst the fastest growing cities in the world. For instance, in 1921 Nigeria had a total of 29 urban centres. By 1931, the number drop to 27 as a result of the political boundary delineations between the Federal Republic of Nigeria and the British Cameroun protectorate. The number of urban centres reached 56 in 1953 and between 1963 and 1991 the number of urban centres rose to 182 and 359 respectively. There was also an increase in the percentage urban population. A total of 7.18% of Nigeria’s population lived in urban centres while 92.82% were rural inhabitants. The percentage rose to 19.30% and 36.30% respectively between 1963 and 1991 as indicated on table 1 below. The United Nation (2000) projected that by the end of 2050 half of the world’s population will live in urban centres. INTRODUCTION IJSSHR, Volume 04 Issue 07 July 2021 Page 1901 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Table 1: Number and growth of urban cities in Nigeria (Population 20,000 person) Year Population (in thousand) Total population (in thousand) No of Urban Cities % of Urban Pop % from 1921 Rural Urban Rural Urban 1921 17.375 1,345 18,720 29 7.18 1931 18,625 1,431 20,056 27 7.14 0.42 2.79 1953 27.166 3,237 30,403 56 10.65 1.42 2.76 1963 44.925 10,745 55,670 182 19.30 2.29 5.01 1991 57,185 31,807 88.992 359 36.3 1.70 4.52 Source: NPC, 1991 Population census An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross R rity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The increasing Nigeria urban centres and population is occurring without generating the needed resources and employment opportunities to accommodate the surge in population. The concentration of economic activities in urban centres creates disparity in the economic growth between urban and rural areas. The less privileged and poor migrants who move from rural areas to urban centres in search of employment, security and safety, find themselves living in slum settlements within and around urban centres. As more people moved into an urban center voluntarily or forced by administrative compulsion, many social maladies become noticeable in the city. This group of people who suffer extreme deprivation and exclusion later indulge into a wide range of criminal activities. Crime is an action or omission which constitute an offence and is punishable by law. In modern criminal law crime does not have any simple universally accepted definition. A crime or offence is an act lawfully not only to individuals but also to a community, society or the states (Wikipedia Encyclopedia, 2008). Criminologists have grouped crime into two major categories – felonies and misdemeanors. According to Paul Tappen, crime is an international act or omission in violation of criminal law committed without defence or justification and sanctional by state as a felony or misdemeanor. This study seeks to appraise the internal security and safety situation in the ancient city of Calabar arising from rapid population and urbanization, the physical structure and planning of the metropolis. II. There has been immense increase in population and urban expansion over the years in Calabar Metropolis. The official national population census conducted during the colonial administration revealed that Calabar had a total of 46,705 people in 1953 and 100,628 people in 1963 with percentage growth rate of 1.09. The next and most resent official census reported a total of 329,000 in 1991 and 375,196 in 2006 respectively. Urban population growth becomes a significant problem when its consequences impact negatively on the environmental resources and threats to human lives. Among the most serious problems confronting Calabar Metropolis and its inhabitants include lack of employment opportunities, increasing poverty, housing shortage and expansion of squatter settlements, growing insecurity and rising crime rates, uncoordinated urban development and increasing vulnerability to disaster which are the basic issues of mankind in the societies. The ancient city of Calabar is experiencing high rate of insecurity resulting from the presence of less privilege (poor) who suffer extreme deprivation and exclusion. These poor migrants who migrated from rural areas to Calabar in search of security, safety and gainful employment are found living in slum settlements without basic facilities and services. Calabar Metropolis has witnessed a wide range of criminal activities over a decade such as kidnapping, armed robbery, rape, murder, burglary, auto theft, arson etc. These criminal activities occur in different geographical locations and settings (neighbourhoods) such as Bay side, Anantigha, Watt Market, Ekpo Abasi, Akim Qua, Big Qua, Ikot Ansa etc. Other locations that cluster criminal activities are entertainment clubs, super markets, stores, major motor parks and residential neighbourhoods, example the State, Federal and Private Housing Estates inhabited by relatively well-to-do, young and adults, abandoned and uncompleted public and private property etc. III. AIM OF THE STUDY IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1902 III. AIM OF THE STUDY The aim of this study is to appraise the security and safety situation in Calabar Metropolis with a view to finding solution to the problem. Objectives 1. To identify the major types of criminal activities in Calabar Metropolis 2. To determine the causes of such criminal activities in the study area 3. To examine the physical form and structure of the study area The aim of this study is to appraise the security and safety situation in Calabar Metropolis with a view to finding solution to the problem. Objectives 1. To identify the major types of criminal activities in Calabar Metropolis 2. To determine the causes of such criminal activities in the study area 3. To examine the physical form and structure of the study area IJSSHR, Volume 04 Issue 07 July 2021 Page 1902 Page 1902 IV. STUDY AREA The ancient city of Calabar has since 1967 remain the centre of activities and the administrative headquarters of the Cross River State Government of Nigeria. Calabar is located between latitude 4056’ and 5004’ North of the Equator and longitude 8018’ and 8024’ East of the Greenwich Meridian. The City is on the peninsular between the Calabar River on the nearest buildable land to the sea. Calabar remains one of the oldest city and port in the West African Sub-region and a slave depot along the Bight of Biafra (Bonny). It was the first national Administrative capital of Nigeria from 1882-1906 during the early British rule in Nigeria. Calabar is a dynamic urban centre with a rural-urban migrant population. The population of Calabar urban had been estimated to be 82,100 in 1975;159,599 in 1978; 140,200 in 1985, 217,800 in 1995; 320,862 in 1991 and 375,196 in 2006. The indigenes are the Efiks, the Quas and Efuts who are engaged in commercial, industrial, educational and small scale farming. Climatically, Calabar falls within the rain forest belt and the vegetation is mainly tropical. Fig 1: Map of Nigeria Showing Cross River State Source: Cross River GIA Fig 1: Map of Nigeria Showing Cross River State Source: Cross River GIA FIG. 2: Map of Cross River State Showing Calabar Metropolis FIG. 2: Map of Cross River State Showing Calabar Metropolis FIG. 2: Map of Cross River State Showing Calabar Metropolis FIG. 2: Map of Cross River State Showing Calabar Metropolis IJSSHR, Volume 04 Issue 07 July 2021 Page 1903 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Figure 3: Map of Calabar showing the Study Area Source: Cross River GIA he Consequences on Residents in Calabar Metropol Figure 3: Map of Calabar showing the Study Area Source: Cross River GIA he Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Figure 3: Map of Calabar showing the Study Area Source: Cross River GIA An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria urity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Figure 3: Map of Calabar showing the Study Area Source: Cross River GIA An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian Police recorded about 26 criminal offences between 1994 and 2003 as shown on table 3 below An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian Police recorded about 26 criminal offences between 1994 and 2003 as shown on table 3 below. A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian Police recorded about 26 criminal offences between 1994 and 2003 as shown on table 3 below. ble 3: Crime Statistics in Nigeria (1994-2003) OFFENCES RECORDED INCIDENCE Armed Robbery 25,352 Assault 377.568 Child Stealing 1,320 False Pretence 110,913 Forgery of Currency Note 6,759 Murder 17,448 Attempted Murder 2,434 Theft, Larceny and others 505,075 Burglary 64,863 Demand/Menace 1,824 Man Slaughter 259 Suicide 2,305 Attempted Suicide 754 General Hamm and Wounding 156,328 Coining Offence 211 Gambling 3,330 Rape 22,151 Slave Dealing 196 Breach of Public Peace 72.009 Perjury 903 House Breaking 72,574 Store Breaking 39,724 Bribery and Corruption 1,690 Escape from Lawful Custody 4,699 Kidnapping 3,589 Unnatural Offences 4,366 Total 1,488,644 Source: Clean Foundation, 2003 Global trends have shown a tremendous increase in crime rates over the years. Reports have shown an increase in crime from 2,000 to 2,300 for every 1000 people between 1980-2000. This showed that 60 percent of urban residents in developing countries have been victims of crime, over a five-year period with victimization rate of 70 percent in parts of Latin America and the Caribbean and Africa. According to the UN-Habitat, (2007) cities in Africa have the highest reported cases of burglary, with victimization rates of over 8 percent of the population. In the year 2000, the police in South Africa recorded 460 robberies for every 100,000 people. V. LITERATURE REVIEW The whole world including Sub-Saharan Africa (SSA) are experiencing high rare of population growth and urban expansion. The United Nation (2008) reported that only 20 million people were living in urban areas in Sub-Saharan Africa. Todaro (2000) observed that the SSA region recorded the highest urban growth rate in the world with an average of 5% per annum. Hove, Ngwerume and Muchemwa (2013) reported a total urban population of 250 million. Accordingly, the United Nation Population Fund*UNPF 2000) concluded that at this rate of growth, urban population will double between 2000 and 2030 resulting to serve social, economic, physical and environmental consequences. The poor migrant population in urban centres will create informal settlements with extreme human insecurity resulting to high rate of criminal offences ranging from major crimes referred to as felonies or predatory crimes and minor crimes otherwise known as misdemeanors. According to Obeng-Odoom (2011) these poor migrants constitute part of the urban poor who suffer extreme deprivation and exclusion. Rakisits (2008) also observed the growing number of street children like the Amajeris or Skolombos in the Northern and Southern States in Nigeria and destitute in our urban areas who survive through begging, stealing, casual work or crime as major consequences of deprivation. ‘Crime’ does not have a universally accepted definition. It has different meaning by different schools of thought. Paul Tappen, a criminologist, defined crime as an international act or omission in violation of criminal law submitted without defence or justification and sanctioned by state as a felony or misdemeanor. Felonies are major or serious crimes that attract death sentence or incarceration in a prison for several years by government. These include kidnapping, murder, rape, arm robbery, burglary etc. Misdemeanor or minor crimes are unserious crimes such as shoplifting, petty theft, pick pocking, simple assault etc which attract conferment in a local jail for at least a year. Table 2: Major Crime Incidence in the Urban Areas and Rates per 100,000 Populations. S/NO CRIME INCIDENCE CRIME INDEX I Murder 19,645 7.4 Ii Forcible Rape 957,679 36.1 Iii Robbery 537,050 202.1 Iv Aggravated Assault 1,029,814 388 V Burglary 2,501,524 943 Vi Larceny 7,894,620 2,925.9 Vii Auto Theft 1,395,192 525.9 Viii Arson 88,887 44.3 Total 13,562,501 5,078.9 Source: FBI (UCR), 1996 : Major Crime Incidence in the Urban Areas and Rates per 100,000 Populations. IJSSHR, Volume 04 Issue 07 July 2021 Page 1904 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria the most important environmental variables. Security has been defined by the Oxford Learner’s Dictionary as “the state of being free from danger or threat” and is one of the basic human needs. Human security protects communities and their inhabitants from insecurity and violence. Safety on the other hand refers to the environmental factors whose absence may lead to occurrence of crime and gives rise to threat and risk (Kelly and Dian 2009). It is interesting to note that the UN-Habitant (2007) have placed urban safety and security within the wider perspective of human security in our societies. According to Ogboi (2013) the level of safety differ among urban neigbourhoods especially the inner city and the suburban context. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria In Johannesburg for instance, 30 percent of residents were reported to have been victims of robbery. Studies in urban and regional planning have continuously placed safety and security concerns within spatial context by examining design and policy intervention that can create defensible space (Newman, 1972; Frank and Engelke, 2001; Handy, Boarnet, Ewing and Killingswarth 2002). Environmental criminologists have placed much emphasize on the importance of geographical locations and architectural designs as factors that influence crime and its prevalence. It has been urged that certain urban design patterns such as neighbourhood design, street layout, avenues, alleys, boulevards, private homes and business establishments attract criminals and remain “Hotspots” and sites of high crime and deviate rates. Stark (1987) addresses such neighbourhood or communities as deviant neighbourhood which are characterized by dense population, with poor and overcrowded conditions that increase the temptation and opportunity to involve in crime and deviant behavior. The built environment (neighbourhood) also present environmental constraints and opportunities which may be characterized by physical disorder (litter) and social disorder (crime). According to Ross et al (2001); Klinenberg (2002) living in a neighbourhood with such high physical and social disorder generates stress and fear. On the contrary, clean and safe neighbourhood attract outdoor activities. Recent studies have tried to acertain the promotion of a safety and healthy community through the manipulation of urban design, land use and transportation. (Greenwald and Boarnet, 2002); Safety and security have been seen as IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1905 VII. RESEARCH FINDINGS AND DISCUSSIONS VII. RESEARCH FINDINGS AND DISCUSSIONS PRESENTATION OF RESULT Table 4: Trend of population for Calabar metropolis based on official population census S/n Year Population Percentage (%) 1 1953 46,705 5.45 2 1963 100,628 11.82 3 1991 329,000 38.64 4 2006 375,196 44.06 TOTAL 851,529 100.00 Table 4: Trend of population for Calabar metropolis based on official population census d of population for Calabar metropolis based on official population census The results in Table 3 shows the trends in population for Calabar metropolis based on official population census revealed a total population of 851,529. While year 2006 had the highest population of 375,196 (44.06%), followed by 1991 with a population of 329,000 (38.64%), while the least was 1953 which has a population of46,705 (5.45%).. This indicates that there is a geometric increase in the population of the respondents in Calabar metropolis. The result is also presented in a bar chart as shown in Figure 4. The results in Table 3 shows the trends in population for Calabar metropolis based on official population census revealed a total population of 851,529. While year 2006 had the highest population of 375,196 (44.06%), followed by 1991 with a population of 329,000 (38.64%), while the least was 1953 which has a population of46,705 (5.45%).. This indicates that there is a geometric increase in the population of the respondents in Calabar metropolis. The result is also presented in a bar chart as shown in Figure 4. al population of 851,529. While year 2006 had the highest population of 375,196 (44.06%), followed by 1991 with a popula 29,000 (38.64%), while the least was 1953 which has a population of46,705 (5.45%).. This indicates that there is a geom ease in the population of the respondents in Calabar metropolis. VI. The research study made use of the primary and secondary data. The official national population census data was obtained from the National Population Commission while the last official national census of 2006 was projected to 2020 at 3.0% growth rate. Data on crime was obtained from the Cross River State Criminal Intelligence Bureau (SCIB) Headquarters Calabar. Oral personal interview and direct observation were adopted to indentify crime hotspots and most especially the physical form of the city and its social and economic activity pattern, the structure of the city’s street network and neighborhood design. The Pearson’s Product Moment (PPM) statistical instrument was employed to explore the relationship between population and crime variables as shown on tables 4 and 5 below. VII. RESEARCH FINDINGS AND DISCUSSIONS The result is also presented in a bar chart as shown in Figu Figure 4: Bar chart of trends in population for Calabar metropolis based on official population census 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1953 1963 1991 2006 TOTAL 1 2 3 4 POPULATION Percentage (%) Figure 4: Bar chart of trends in population for Calabar metropolis based on official population census 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1953 1963 1991 2006 TOTAL 1 2 3 4 POPULATION Percentage (%) Figure 4: Bar chart of trends in population for Calabar metropolis based on official population cen IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1906 Page 1906 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Table 5: Estimated population figure for Calabar Metropolis S/n Year Estimated population Number of crime committed 1 2006 375,196 326 2 2007 367, 458 211 3 2008 374,363 291 4 2009 386,989 172 5 2010 375,882 152 6 2011 365,605 156 7 2012 379, 553 204 8 2013 386,277 319 9 2014 398,045 325 10 2015 422,286 364 11 2016 448,003 305 12 2017 475,286 335 13 2018 505,231 337 14 2019 534,938 250 15 2020 567,515 500 Total 3, 737, 581 4247 ecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria population figure for Calabar Metropolis An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cros It is evident from the result in table 5 of the estimated population figure for Calabar Metropolis that the total estimated population for the fifteen years (2006-2020) is 3, 737, 581 and the corresponding number of crime committed within these years amounted to 4,247. Year 2020 had the highest crime rate of 500; this was followed by year 2015 that has a crime rate of 364 cases. While the least was year 2010 with an estimated crime rate of 152 accordingly. This implies that there is an arithmetic increase in the rate of crime committed between 2006-2020. The result is further presented in the bar chart in figure 5 for the fifteen years (2006-2020) is 3, 737, 581 and the corresponding number of crime committed within these years amounted to 4,247. It is evident from the result in table 5 of the estimated population figure for Calabar Metropolis that the total estimated population for the fifteen years (2006-2020) is 3, 737, 581 and the corresponding number of crime committed within these years amounted to 4,247. Year 2020 had the highest crime rate of 500; this was followed by year 2015 that has a crime rate of 364 cases. While the least was year 2010 with an estimated crime rate of 152 accordingly. This implies that there is an arithmetic increase in the rate of crime committed between 2006-2020. The result is further presented in the bar chart in figure 5 VII. RESEARCH FINDINGS AND DISCUSSIONS Year 2020 had the highest crime rate of 500; this was followed by year 2015 that has a crime rate of 364 cases. While the least was year 2010 with an estimated crime rate of 152 accordingly. This implies that there is an arithmetic increase in the rate of crime committed between 2006-2020. VII. RESEARCH FINDINGS AND DISCUSSIONS The result is further presented in the bar chart in figure 5 Figure 5: Estimated population figure for Calabar Metropolis 0 100000 200000 300000 400000 500000 600000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 S/n Year Estimated population Number of crime committed Figure 5: Estimated population figure for Calabar Metropolis 0 100000 200000 300000 400000 500000 600000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 S/n Year Estimated population Number of crime committed Figure 5: Estimated population figure for Calabar Metropolis IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1907 Table 6: Major crime statistics in Calabar metropolis (2006-2020) S/n Year Kidnapping Cultism Rape Burglar y Murder Arson Armed robbery Theft Auto theft Vandalism 1 2006 7 - 9 10 18 5 37 240 - - 2 2007 5 - 7 6 7 3 15 168 - - 3 2008 4 - 16 9 15 2 21 224 - - 4 2009 8 - 11 4 15 5 25 103 1 - 5 2010 4 - 8 7 15 - 18 99 1 - Table 6: Major crime statistics in Calabar metropolis (2006-2020) Page 1907 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria 6 2011 6 - 5 4 22 2 31 86 - - 7 2012 7 - 16 5 31 3 36 106 - - 8 2013 9 - 12 10 24 - 63 201 - - 9 2014 7 - 15 8 26 2 24 243 - - 10 2015 4 - 11 6 12 2 14 315 - - 11 2016 9 - 15 16 5 3 45 199 - - 12 2017 15 - 12 9 19 5 52 220 2 1 13 2018 6 6 11 5 22 1 38 238 5 1 14 2019 7 - 4 6 24 4 30 172 3 - 15 2020 8 4 35 10 26 10 41 342 4 16 Tot al 106 10 187 115 281 47 490 2956 16 18 It can be discerned from the statistical analysis in Table 6 of the major crime statistics in Calabar metropolis (2006-2020), theft has the highest with a total of 2956, this was followed by armed robbery 490 while those with murder cases came third with a total of 291 while the least are those who constitute the categories of cultism with a total of 10. VII. RESEARCH FINDINGS AND DISCUSSIONS The results are presented in the bar chart in Figure 6 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cros rity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria It can be discerned from the statistical analysis in Table 6 of the major crime statistics in Calabar metropolis (2006-2020), theft has the highest with a total of 2956, this was followed by armed robbery 490 while those with murder cases came third with a total of 291 while the least are those who constitute the categories of cultism with a total of 10. The results are presented in the bar chart in Figure 6 Figure 6: Bar chart of major crime statistics in Calabar metropolis (2006-2020) 0 500 1000 1500 2000 2500 3000 Major Crime Statistics in Calabar Metropolis (2006-2020) 2006-2020 Figure 6: Bar chart of major crime statistics in Calabar metropolis (2006-2020) 0 500 1000 1500 2000 2500 3000 Major Crime Statistics in Calabar Metropolis (2006-2020) 2006-2020 Figure 6: Bar chart of major crime statistics in Calabar metropolis (2006-2020) VIII. TEST OF HYPOTHESIS Ho: There is no significant relationship between population and crime in Calabar metropolis. The variables involved in this hypothesis are population and crime. To test this hypothesis, Pearson’s Product Moment Correlation Statistics was employed. The choice of this statistical tool was because the scored were measured and the researcher intends to explore relationship between the variables under study. The result is presented in Table 7. IJSSHR Volume 04 Issue 07 July 2021 www ijsshr in Page 1908 Table 7: Relationship between population and crime rate in Calabar metropolis (N=4247) Variables ∑x ∑y ∑x2 ∑y2 ∑xy df LS r-cal p-value Population (x) 63356 1017090 513905720 4245 0.05 .78 .000 Crime rate (y) 66664 2554366 Table 7: Relationship between population and crime rate in Calabar metropolis (N=4247) Variables ∑x ∑y ∑x2 ∑y2 ∑xy df LS r-cal p-value Population (x) 63356 1017090 513905720 4245 0.05 .78 .000 Crime rate (y) 66664 2554366 IJSSHR, Volume 04 Issue 07 July 2021 IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1908 Page 1908 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The result obtained in Table 7 revealed that with 4247 respondents as the estimated population figure for Calabar Metropolis. While X scores has a sum of 63356 sum of all scores squared of 1017090 while crime rate (y) scores has a sum of 66664 and sum of all scores squared of 2554366. The sum of product of x and y scores is 513905720 accordingly. At 4,345 degrees of freedom and 0.05 level of significance, the p-value was found to be .000 which is less than the chosen alpha of .05. Thus, the null hypothesis is rejected. This implies that there is a significant relationship between population rate and crime rate in Calabar metropolis. Aptly put, the greater the population the higher the rate of crime in Calabar metropolis. Calabar Metropolis comprises of Calabar Municipality and Calabar South. The study revealed that the inner city neighborhoods are the crime Hotspots and further identified other crime endemic areas in the metropolis as shown on fig. 7. SSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page Parliamentary Extension Ikot Effanga Jonathan By Pass Akai Effa CALABAR MUNICIPALITY Satellite Town Obutong AsariIso IkotIshie Ikot Ansa Ekorinim Lemna INNER CITY NEIGHBORHOODS:  Henshaw Town  Duke Town  Watt Market  Etim Edem Park CALABAR SOUTH Efut Ekondo Efut Abua Edibe-Edibe Mbukpa Ekpo Abasi Anatigha Esuk Nsidung Afokang Itiat Ekpe (Bayside) FIG 7. CRIME HOTSPOTS IN THE CALABAR METROPOLIS INNER CITY NEIGHBORHOODS:  Akim Qua Town  Edeba Town  Big Qua Town  Diamond Town  Essien Town Source: Field Survey (2020) Inner City Neighborhoods - - - Local Govt. Boundary  Surrounding Settlements FIG 7. CRIME HOTSPOTS IN THE CALABAR METROPOLIS Lemna Itiat Ekpe (Bayside) Ekpo Abasi Inner City Neighborhoods - - - Local Govt. Boundary  Surrounding Settlements Source: Field Survey (2020) IJSSHR, Volume 04 Issue 07 July 2021 Page 1909 Page 1909 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The geographic locations, architectural and urban design patterns, such as neighborhood design, street layouts, avenue, alley ways, boulevards, private homes and business establishments like the popular Watt market, Eka Ika Qua and Mbukpa market among others in the Calabar metropolis are the primary factors that influence crime and its prevalence. Other locations are Jonathan Bye-pass, Parliamentary extension, Akai Effa, Ikot Ishie, Ikot Ansa, etc in Calabar Municipality and Anatigha, Ekpo Abasi, Mbukpa, Edibe-Edibe etc in Calabar South. Table 6 above shows constant increase in crime except for auto theft, vandalism and cultism from 2012-2014 which is the last quarter of the previous administration and from 2015-2020 towards the end of the present administration. Criminal activities dropped significantly during CONVID-19 pandemic which held criminal elements hostage (indoors) for fear of the deathly virus which is on-going in both developed and developing nations of the world. The year 2020 also witnessed serious civilian protest against the Nigerian Police Force tagged ENDSARS along the streets of Nigerian urban centres and Calabar in particular. This protest led to insecurity and safety of life and property in the Calabar metropolis. The period recorded an increase in theft, arm robbery, rape, murder, vandalism, burglary, arson, kidnapping, auto-theft and cultism respectively as shown on table 6 above. Within the period under study several public and private offices, business and commercial buildings were set ablaze, theft, arm robbery, rape, murder and vandalism were at their peak. Below are places destroyed and looted within Calabar Metropolis during the ENDSARS protest - the Calabar International Conference Centre, the Newspaper Cooperation (Chronicle), the Cross River State Ministry of Works at Ekorinim which comprises the Vehicle Inspection Office (VIO), Government Warehouse, Training School, Mechanical Workshops, Cross River Independent Electoral Commission (INEC), West African Examination Council Office, Governor’s Petrol Station, Garment Factory, Roll Back Malaria Office, Senators Houses, Access Bank. First Bank, National Television Authority (NTA) Value Mart Supermarket, University of Calabar Microfinance Bank etc. IJSSHR, Volume 04 Issue 07 July 2021 Page 1910 Page 1910 www.ijsshr.in Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The study revealed that residents in the crime endemic neighbourhoods (Inner city) of the metropolis are imporrished ms of resources availability with fewer opportunities to bridge the gap between the rich and the poor hence the tendency ulge in criminal activities. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Among these crime endemic neighborhoods are Duke Town, Henshaw Town, Esuk, Watt Market, Etim Edem in Calab th and Akim Qua, Big Qua Town, Diamond Town, Essien Town, Edeba, Ika-Ika Qua Market in Calabar Municipality. T eriorating conditions and quality of life in these neighborhoods makes them criminogenic. The study further revealed that vio An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The study revealed that residents in the crime endemic neighbourhoods (Inner city) of the metropolis are imporrished in terms of resources availability with fewer opportunities to bridge the gap between the rich and the poor hence the tendency to indulge in criminal activities. The study revealed that residents in the crime endemic neighbourhoods (Inner city) of the metropolis are imporrished in terms of resources availability with fewer opportunities to bridge the gap between the rich and the poor hence the tendency to indulge in criminal activities. Among these crime endemic neighborhoods are Duke Town, Henshaw Town, Esuk, Watt Market, Etim Edem in Calabar South and Akim Qua, Big Qua Town, Diamond Town, Essien Town, Edeba, Ika-Ika Qua Market in Calabar Municipality. The deteriorating conditions and quality of life in these neighborhoods makes them criminogenic. The study further revealed that violet crime and delinquency rates are more prevelant among the lower socio-economic groups living in these neighborhoods and other crime locations. IJSSHR, Volume 04 Issue 07 July 2021 Page 1911 www.ijsshr.in XI. RECOMMENDATIONS Crime can be reduced through the encouragement of neighborhood watch and frequent policing of target areas of crime hotspots in Crime can be reduced through the encouragement of neighborhood watch and frequent policing of target areas of crime hotspots in Calabar Metropolis. Government should have democratized urban design and planning to accommodate all social groups) that is Integration between urban and suburban areas to reduce high crime rate. There should be stiffer penalties, faster court trails and sentences for crime victims. The public should be informed through public media to always take precautionary measures to safeguard their cars and home doors. The use of hard drugs by the youths should be closely monitored by the Nigerian Police Force. Brujak (1999) observed that the higher the proportion of young people especially the male counterpart in a city, the greater the incidence of violent crime. He further contended that the youth populations are more predisposed to crime than the elderly people. This shows that there is a relationship between age structure of population of a city and incidence of crime. In its study, the FBI (1996) crime index shows that about 55.6 percent of offenders are between 18-25 years of age. Orphanage homes in the Calabar Metropolis should be a transformed as foundation for the welfare of youths and should be equipped to deal with the problem of child abandonment and street children popularly known as Skolombo in Calabar Metropolis, Amajiris and Damiskas in Northern Nigerian cities, Agberos and Alayes in Western parts of Nigeria and touts, street urchins and juveniles. The higher income class should be informed through public media to avoid extravagant and flamboyant lifestyles. Government and the organize private sector should introduce youth empowerment programme or employment opportunities for the poor youths in our societies to reduce inequality and denial justice which fosters and engenders criminal activities. There should be synergy among ethnic, religious and political groups to co-exist in harmony with one and another. Government curfew laws should be enforced to reduce crime in hot spots of the Calabar metropolis as shown on fig 7. Urban and regional planners should develop safety base designs for urban spaces such as residential and commercial neighborhoods, parks and recreational grounds, street networks and urban renewed projects. CAUSES OF CRIME IN CALABAR METROPOLIS Oral interview of selected groups of residents in the inner city neighborhoods and other crime locations and hotspots in th metropolis revealed the following factors as being responsible for crime in the study area. Extravagant and flamboyant lifestyles; Driving and walking alone on dark streets; Building of high wall fences in rich residential neighborhoods; Poverty and deprivation; Leaving with house doors open; Leaving car keys in the ignition; Exposing valuable properties in the car; Indecent dressing code by women; Disintegration of urban and suburban areas through urban planning and architectural design; Poor policing of crime hotspots and lack of patrol vehicles; Unemployment; Rapid population and urbanization; Drug usage; The structure of political power and corruption; Religion and intergenerational transmission of violence; Family conditions; Lack of access to legitimate opportunities for minority groups; Deteriorating condition of urban neighborhoods and quality of life; Neighborhood streets with poor linear visibility and dead-ends; Incessant use of artifacts which include grafitis on city buildings and walls such as “Beware of dogs”, cars parked at owner’s risk etc are criminogenic; Poor funding of security personnel; Lack of security gargets such as CCTV cameras; Poor urban planning, design and management. X. CONSEQUENCES OF CRIME ON RESIDENTS OF CALABAR METROPOLIS  There has been serious problem of insecurity and safety due to high rate of criminal activities in the Calabar Metropolis.  Many business have folded up and businessmen have loss their savings to armed robbers and fraudsters in recent times.  Rape victims for instance have suffered humiliation, embarrassment shame and serious depression. The psychological and physical trauma will live with the residents for a long time.  Many business have folded up and businessmen have loss their savings to armed robbers and fraudsters in recent times.  Rape victims for instance have suffered humiliation, embarrassment shame and serious depression. The psychological and physical trauma will live with the residents for a long time.  Millions of Naira, personal properties and many lives have been lost especially during the ENSARS protest in Calabar. Insurance claims and premium are now high and many victims of vandalization may not be able to cope. Most victims and property owners suffered physical assault and majority are permently disabled due to high medical cost.  Millions of Naira, personal properties and many lives have been lost especially during the ENSARS protest in Calabar. Insurance claims and premium are now high and many victims of vandalization may not be able to cope. Most victims and property owners suffered physical assault and majority are permently disabled due to high medical cost.  Under the present economic down turn government may find it difficult to maintain the cost of security systems and many other crime prevention measures and apparatus. There is general fear in the system especially the frequent cases of kidnapping and assassination of high profile politicians and businessmen within the metropolis and its environ.  Under the present economic down turn government may find it difficult to maintain the cost of security systems and many other crime prevention measures and apparatus. There is general fear in the system especially the frequent cases of kidnapping and assassination of high profile politicians and businessmen within the metropolis and its environ. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Oral interview with the Nigerian Police Force in Calabar revealed Youth involvement in crime from the age of 12-21 and above which according to them is a reflection of the degree of social disorganization and deteriorating quality of urban life. According to the police force these crime endemic locations have the highest population of youth gangs who have masterminded the kidnapping of high profile government officials and business mogul for ransom to get rich quick. The study further revealed high incidence of crime in the Gated and rich neighborhoods such as the Satellite Town, Ekorinim, State Housing Estate, Federal Housing Estate, House of Assembly village and other private housing estates within the metropolis. These rich neighborhoods are characterized with high wall fences which negates the Eye-on-the-street theory by Ross (2000) and Eyler and Vest (2002), Narrow and winding lonely streets with Dead-ends, and grafitis on fences and buildings such as “Beware of dogs”, car parked at owners risk” etc which attract criminality as shown on the plates below. HR, Volume 04 Issue 07 July 2021 www.ijsshr.in Pa IJSSHR, Volume 04 Issue 07 July 2021 Page 1912 Page 1912 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria XII. CONCLUSION Urbanization is a product of rapid population growth of an area. It is an important component for the economic and social development of an area with its attendant consequences such as unemployment, poverty, crime, insecurity and safety. Calabar Metropolis has witnessed a wide range of criminal activities, at different geographical locations by both physical and social disorder. Other factors that influenced crime and its prevalence in Calabar Metropolis include architectural and urban design pattern such as neighborhood design, the structure of the city’s street layouts, private homes, offices business establishments etc. It must be noted that security and safety are the major objectives of physical planning to reduce crime rate in our urban centres. Urban planners should develop appropriate designs and opportunities to minimize the rate of criminal activities in the urban centres. Government in partnership with the private sector should employ appropriate security apparatus, skill security personnel, modern technology and other measures to promote the security and safety of residents in the study area. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria activities in the cities. Other crime prevention technology such as electric fencing, security breach alarm systems, auto-breach sensor and so on, should be utilized to reduce auto-theft, license plate recognition technology, and the use of Geographic positioning systems (GPS) should also be used to monitor criminal activities in our urban areas. ACKNOWLEDGEMENTS We acknowledge the Authors whose materials and publications guided us in this research as indicated in the Bibliography section. We also want to thank our wives and children who allowed us to be away from them at the course of this research and the cab man who took us round the city of Calabar in order to get important information and photographs needed for this study. Above all, we give God the Glory for wisdom, understanding and knowledge bestowed on us to complete this work. XI. RECOMMENDATIONS The “Eye on the street” defined by Jacobs (1961) as a natural surveillance that is a deterrent of criminal activities should be adopted in street network design. It is seen as the resident ability to view public streets. The design orientation of buildings with windows directly facing the street increases natural surveillance for residents of rich neighborhoods such as Ekorinim, Satellite Town, Public and private residential estates in Calabar Metropolis. High wall fences and heavy landscaping should be discouraged in rich neighborhoods as they block view and create hideouts for criminal activities like rape and handbag snatching. Street lighting along major and minor streets should be encouraged as well as public open spaces and parking lots to scare criminals and hoodlums. Undeveloped plots of land should be cleared and maintained while uncompleted buildings and open parks should be policed more frequently by law enforcement officers. The Cross River State Government should key into the crime prevention technology in major urban areas in the state through the use of video surveillance (Close Circuit Television Camera (CCTV) to monitor criminal IJSSHR, Volume 04 Issue 07 July 2021 Page 1913 www.ijsshr.in REFERENCES 1) Brantingham, P. (2005) “Daily Urban Crime and Disorder: Is there a simple solution? Canada. Institute for Urban Research Studies. Simon Frazer University. 2) Bryjak, G. J. (1999) “Multiple Reasons for Lower Crime Rates” San Diego Union Tribune. March 28. 2) Bryjak, G. J. (1999) “Multiple Reasons for Lower Crime Rates” San Diego Union Tribune. March 28. 3) Federal Bureau of Investigation (1996) “Uniform Crime Report” Washington D.C. Government Offi 4) Federal Bureau of Investigation (1996) Crime in the United States” Washington D.C. Government office. 4) Federal Bureau of Investigation (1996) Crime in the United States” Washington D.C. Government office. 5) Frank L. D. and Engelke P. O. 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REFERENCES (1998) “Enhancing the Productivity of Urban Africa” Being paper Published in the Proceedings of an International Conference Research Community for the Habitat Agenda: Linking Research and Policy for the Sustainability of Human Settlement held in Geneva, July 6-8. 1998. 9) Hicks, J. (1998) “Enhancing the Productivity of Urban Africa” Being paper Published in the Proceedings of an International Conference Research Community for the Habitat Agenda: Linking Research and Policy for the Sustainability of Human Settlement held in Geneva, July 6-8. 1998. y 10) Hove, M., Ngwerume E. T. and Muchemwa C. (2013) “The Urban crisis in Sub-Saharan Africa: A threat to Human Security and Sustainable Development”. Stability: International Journal of Security and Development 2(1). 11) Jacobs, J. (1961) “The Death and Life of Great American cities, Vintage Books, New York. 12) Kelly E. & Dian D. C. (2009) Securing the Built Environment: An Analysis of Crime Prevention through Environmental Design Ball University, Munie Indiana. ) Kelingberg, E. (2002) Heat Wave: A social autopsy of disorder in Chicago. The University of Chicago P 14) Newman, O. (1972) Dejensible space: Crime Prevention through Urban Design. New York. Macmillan. 15) Nnodu V. C, Okoye, Onwuka (2008) Urban Environmental problems in Nigeria. Rex Charles and Patrick Limited, Booksmith House, Harmony Place, Nimo, Anambra State, Nigeria. ) NPC (1998), 1991 Population Census of the Federal Republic of Nigeria. Nigeria Population Commissio 17) Obeng-odoom, F. (1981) :Informal sector in Ghana under Siege” Journal of Developing Societies, Vol. 27 no. 3-4 DOI: http:/dxi.doi/org/10.1177/0169796x1102700406. 18) Ogboi, K. C. (2013) “Enhancing Urban Security and safety in Nigeria through Parks, S. E. Honsemann R. A. and Brownson R. C. (2002) Differential Correlates of Physical Activity in Urban and Rural Adults of various socioeconomic Background in the U.S. Journal of Epidemolog and Community Health. Vol. 57, Pp. 29-35. IJSSHR, Volume 04 Issue 07 July 2021 Page 1914 Page 1914 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nige 19) Okeke, D. C. (2002) Environmental and Urban Renewal Strategies: Theoretical and Analytical Frameworks. Institute for Development Studies, University of Nigeria, Enugu Campus, Nigeria. 20) Rakisits, C. (2008) “Child soldiers in East of Democratic of Congo”. Refugee Survey Quarterly, 27(4): 108-122. DOI:htt:dxi.doi.org/10.1093 req/hdno54. 21) Rose, C. E. (2000)”Walking Exercising, and smoking: Does neighborhood matter? Social Science and Medicine. Vol. 51, No. 2, Pp.265-274. 22) Stark, R. (1987) “Deviant Places: A theory of the Ecology of Crime” Criminology V 23) Todaro, M. (2000) Economic Development, Essex: Pearson Education Limited. 23) Todaro, M. (2000) Economic Development, Essex: Pearson Education Limited. 24) United Nations, (1996) An Urbanizing World: Global Report on Human Settlements 1996. A United Nation Publication. New York. 25) UN-Habitat (2008) State of the World’s Cities Report, Nairobi, Kenya: UN-Habitat United Nations Population Fund (2007), State of World Population 2007: Unleashing the potential of urban growth, accessed online at www.un7pa, on 3 Jan. 2013. United Nations Department of Economic and Social Affairs (DESA) 2011) Population Distribution, Urbanization, Internal Migration and Development: An International Perspective. New York City, NY. IJSSHR, Volume 04 Issue 07 July 2021 Page 1915 www.ijsshr.in
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Mössbauer Spectroscopy in Heterogeneous Catalysis
Advances in catalysis
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Mo¨ ssbauer Spectroscopy in Heterogeneous Catalysis JEAN-MARC M. MILLET Institut de Recherches sur la Catalyse et l’Environnement de Lyon, IRCELYON, UMR 5256 CNRS- Universite´ Claude-Bernard, Lyon 1, 2 avenue A. Einstein, F-69626 Villeurbanne Cedex, France; E-mail: jean-marc.millet@ircelyon.univ-lyon1.fr Mo¨ ssbauer spectroscopy dates back to 1958 and for characterization of a solid catalyst was first used in 1971; the technique achieved a high point in the 1980s and has been applied only modestly for catalyst characterization since then. The declining use of the technique may be related to the experimental difficulties and the complexity of the spectral analysis. However, the adaptability of Mo¨ ssbauer spectroscopy to the investigation of catalysts under working conditions and the progress made in the field are evidently leading to a reawakening of interest in its application in catalysis. The value of Mo¨ ssbauer spectroscopy in catalyst characterization is illustrated by results of investigation of Fischer–Tropsch catalysts by 57Co Mo¨ ssbauer emission spectroscopy performed at temperatures up to 723 K and pressures up to 40 bar corresponding to industrial working conditions. Furthermore, synchrotron Mo¨ ssbauer techniques have reached a level of maturity that today allow their use for applications from which catalysts characterization would benefit. This review includes a brief summary of the technique and an overview of ap- plications in catalysis. The majority of investigations have dealt with the isotopes 57Fe, 119Sn, and 57Co (in emission mode). In contrast, investigations focusing on other isotopes, such as 197Au, 121Sb, 125Te, 193Ir, or 99Ru, have been limited in number but very fruitful. Opportunities for future applications to working catalysts are presented and discussed. New techniques related to the use of synchrotron radiation as energy source are emphasized. Such techniques have been applied to isotopes other than 57Fe, such as 169Tm, 119Sn, 83Kr, 181Ta, 151Eu, 161Dy, and 149Sm. The in- vestigation of isotopes such as 197Au, 197Pt, 61Ni, 186W, 99Ru, and 99Rh, is also possible and should strongly benefit catalyst characterization. An effort has been made here to present the techniques, results, and developments in a didactic manner. I. Introduction The nature and breadth of the physical techniques used to investigate solid catalysts continue to increase rapidly in complexity. This statement pertains specifically to Mo¨ ssbauer spectroscopy, which was applied to the characterization of solid catalysts as early as 1971 (1). A retrospective analysis of the use of Mo¨ ssbauer spectroscopy in catalysis showed that it has consistently accounted for 3–10% of the communica- tions presented at the International Congresses on Catalysis (ICC) (7%t at the ICC in Paris in 2004). Such continuity over the years reflects the high value of this technique in catalyst characterization. The theoretical basis of Mo¨ ssbauer spectroscopy as well as its applications to catalyst characterization was reviewed in Advances in Catalysis in 1989 (2). This thorough article summarizes the physical basis of the technique and significant contributions to the characterization of solid catalysts. Since 1989, Mo¨ ssbauer spectroscopy has not underwent major developments, and its applications to catal- ysis have been largely limited to catalysts that were not in reactive atmospheres, notwithstanding the impressive advances that have been made with other techniques in characterizing catalysts under working conditions. Because this volume is focused on characterization of working catalysts, this chapter is short, including a brief summary of the technique and the underlying physical principles, with a focus on its potential and limitations for investigation of catalysts as they function. Only the essential aspects necessary to understand the technique are presented here. The summary of recent applications emphasizes in- vestigations with less commonly used isotopes and on investigations of catalysts at temperatures and pressures relevant to catalytic reactions. The practical aspects of the use of Mo¨ ssbauer spectroscopy are not treated in detail, and only a few examples are discussed. Readers searching for more complete reviews of research done using Mo¨ ssbauer spectroscopy should consult the reviews published in recent years (3–6). This article concludes with an evaluation of potential innovations that may arise in the near future and that have not yet been applied for investigations of heterogeneous catalysis. Mo¨ ssbauer Spectroscopy in Heterogeneous Catalysis Abbreviations: ADC, analog-to-digital converter; CEMS, conversion electron Mo¨ ssbauer spectroscopy; DCEMS, depth selective conversion electron Mo¨ ssbauer spectroscopy; DFT, density functional theory; EMS, emission Mo¨ ssbauer spectroscopy; ESRF, European synchrotron radiation facility; FeZSM-5, iron-containing Zeolite Society Mobil constructed from five-membered-ring building units; GC, gas chromatography; HTP, high throughput; IMS, imaging Mo¨ ssbauer spectroscopy; MCM-41, mobil composite material/mesoporous composite materials of type 41; MERDJ, Mo¨ssbauer Effect Reference and Data Journal; NBD, nuclear Bragg diffraction of synchrotron radiation; NFS, nuclear forward scattering of synchrotron radiation; NIS, nuclear inelastic scattering of synchrotron radiation; NQES, nuclear quasi-elastic scattering of synchrotron radiation; NR, nuclear reflectometry of synchrotron radiation; NRS, nuclear resonance scattering; NSAS, small-angle scattering of synchrotron radiation; SCA, single channel analyzer; SOD, second-order Doppler shift; XANES, X-ray absorption near edge spectroscopy; ZSM-5, Zeolite Society Mobil constructed from five-membered-ring building units. 1 A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES Mo¨ ssbauer spectroscopy provides measurements of the resonant absorption of g-rays by nuclear transitions from a ground state to an excited state. Like other nuclear techniques, it is based on a phenomenon that is specific to a given isotope and for which no interference from other isotopes is possible. In conventional Mo¨ ssbauer spectroscopy, X-rays with energies corresponding to nuclear transitions (5–150keV) can be produced only by use of radioactive sources containing a parent isotope of the absorbing nucleus in an appropriate excited state from which it decays into the ground state with emission of a g-quantum. For spec- troscopic applications, the g-radiation must be variable. The chemical perturbations 2 at the nuclear levels are very small relative to the transition energy (i.e., 108–1011). Variations of the energy of the parent radiation of this order of magnitude can be achieved by the Doppler effect. For this purpose the radiation source has to be moved linearly at a low velocity v (generally few mm/s) that will allow variation of the incident energy E0: E ¼ E0 1  v c   . ð1Þ ð1Þ Experimentally, the most convenient way to achieve such movement is to fix the source on a constant-acceleration oscillator allowing imposition of velocities in positive and negative direction depending on whether the source moves toward or away from the absorber: E ¼ E0 (17v/c) (Fig. 1). For convenience, Mo¨ ssbauer spectrum is taken to correspond to the plot of the intensity of the transmitted or diffused X-rays as a function of the Doppler velocity v instead of the corresponding energy. The observation of resonance is governed by the probability of emission (in the source) and absorption (in the absorber) of the gamma photons. This probability (Eq. (2)) is named the Lamb–Mo¨ ssbauer or recoil-free factor. The probability of the interaction of a photon with a nucleus that is at the basis of the absorption is proportional to the square of the matrix element of the interaction Hamiltonian which excites the initial state i to the final state f: f ¼ exp  4p2E2 l x2   ðhcÞ2 ! , ð2Þ ð2Þ where x is the vibrational amplitude of the absorbing or emitting atoms in the solid lattice, Eg the energy of the photon, and h is Planck’s constant. A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES The factor f can be expressed in the Debye model as follows: f ¼ exp  3ER 2kTD 1 þ 4 T TD  2 Z T=TD 0 y dy ey  1 " # ( ) , ð3Þ ð3Þ sample detector radioactive source FIG. 1. Basic apparatus for measuring a Mo¨ ssbauer spectrum in transmission mode. FIG. 1. Basic apparatus for measuring a Mo¨ ssbauer spectrum in transmission mode. 3 where ER is the recoil energy ER ¼ E2 Y=2Mc2  ; T the temperature, TD the Debye temperature of the solid, and y ¼ hv=kT: When T5TD and T5TD, the expression may be approximated by where ER is the recoil energy ER ¼ E2 Y=2Mc2  ; T the temperature, TD the Debye temperature of the solid, and y ¼ hv=kT: When T5TD and T5TD, the expression may be approximated by f TTD ¼ exp  3ER 2kTD 1 þ 2p2 3 T TD  2 " # ( ) and f TTD ¼ exp  6ER kTD T TD   . ð4Þ ð4Þ ð4Þ These equations are useful because they allow visualization of practical limits of the technique in terms of suitable isotopes, characteristics of the solids, and exper- imental conditions. Indeed, to have sufficiently large values of f, M should not be too small (Z40), Eg not too large (r150 keV), TD not too small (which corresponds to relatively strong chemical bonds), and T not too high. y g ) g As a result of these constraints, 79 isotopes have been found for which the Mo¨ ssbauer effect can be observed. For 38 of them, chemical perturbations of the nuclear levels result in resonance shifts or splittings of the order or greater than the observed width Gexp of the resonance line (this parameter is defined in the next paragraph (Eq. (9)) and can thus be used for Mo¨ ssbauer spectroscopy. This list of 38 isotopes may be further restricted because of experimental difficulties encoun- tered in their use. These difficulties are related either to their large recoil energies, which require a very low recording temperature for the spectra (4.2 K), or the short half-lives of the parent nucleides used as sources. A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES For higher values of t, the proportionality no longer holds, and corrections have to be made to use the intensity values, for example, to establish the relative amounts of different phases. Equation (5) gives the maximum value of the resonance absorption cross-section, which varies as follows with the energy: s ¼ s0 1 þ 4ðE  E0Þ2 G2 0 " #1 , ð7Þ ð7Þ where E is the incident energy and G0 the width of the excited nuclear state. This width is given by Heisenberg’s uncertainty relationship: G0 ¼ h 2pt , ð8Þ ð8Þ where t is the mean (or natural) exponential lifetime of the excited state (1.44  the half-lifetime). Because the source radiation and the absorber cross-section have line width values of G0, the minimum width actually observable in a transmission experiment is 2G0. This corresponds approximately to 0.2 mm/s of velocity Doppler shift for 57Fe (and 0.65 mm/s for 119Sn). The broadening of the line width beyond this theoretical value is related to inhomogeneities arising from imperfections in the source or the absorber or from a saturation effect. The observed experimental line width is evaluated, and an approximate value can be calculated from the following equation: ð9Þ Gexp ¼ G0 2 þ 0:27tA ð Þ, ð9Þ where tA, called sample or absorber thickness, equals fAnAas0t, with fA being the Lamb–Mo¨ ssbauer factor of the absorber, nA the number of resonant isotope species per cubic centimeter, and a the fractional abundance of the Mo¨ ssbauer isotope. Generally, tA has to be optimized experimentally, and values close to or smaller than 1 are used. Because the cross-section has a Lorentzian shape 1/(1+x2) form around E0 (Eq. (7)), the resonance peak has the same shape. A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES In the former case, cryogenic cooling of both the source and the sample during the experiment may be needed; in the latter case, the manufacturing of the source requires rapid access to a nuclear reactor or accelerator. In practice, by far the most commonly used Mo¨ ssbauer isotopes in heterogeneous catalysis (>90% of all publications) are 57Fe and 57Co (in the emission mode). 119Sn represents more than half of the remaining investigations, and most of the others have been done with a group of nuclei including 197Au,193Ir, 121Sb, 125Te, and 151Eu. In view of the importance of Ni, Ta, and Ru in catalysis, Mo¨ ssbauer investi- gations of 61Ni, 181Ta, or 99Ru would have been of great interest. Unfortunately, the aforementioned difficulties encountered with these isotopes lead to the realization that although Mo¨ ssbauer spectroscopy is in principle possible, almost no investi- gations have been reported using these isotopes. The magnitude of the observed absorption depends on the absorption cross- section for g-rays to excite a transition between the nuclear ground and excited states at resonance. This cross-section per nucleus s0 is given by the following equation: s0 ¼ 1 2p h2c2 E2 0 2Ie þ 1 2Ig þ 1 1 ð1 þ aÞ , ð5Þ ð5Þ where c is the velocity of light, E0 the transition energy, Ie and Ig the excited and ground state spins, and a the internal conversion coefficient (a describes the relative probability of radiative (g-ray) and non-radiative (electron conversion) processes of the transition (2)). 4 The magnitude of the observed resonance depends not only on s0 but also on the effective thickness of the absorber, which may be expressed as follows: The magnitude of the observed resonance depends not only on s0 but also on the effective thickness of the absorber, which may be expressed as follows: t ¼ nf s0, ð6Þ ð6Þ t ¼ nf s0, where n is the number of resonant isotope species per square centimeter. For small values of t (r0.5) there is proportionality between the observed resonance intensity and t. For higher values of t, the proportionality no longer holds, and corrections have to be made to use the intensity values, for example, to establish the relative amounts of different phases. where n is the number of resonant isotope species per square centimeter. For small values of t (r0.5) there is proportionality between the observed resonance intensity and t. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS The nuclear ground and excited levels involved in the Mo¨ ssbauer transition are shifted or split because of the electrostatic interactions between the nuclear charge and the surrounding electric charge (Fig. 2). The first interaction, sometimes called the electric monopole interaction, shifts only the nuclear levels and is related to the perturbation resulting from the electrons inside the nuclear volume. This shift is 5 5 absorber energy -3/2 +3/2 -1/2 +1/2 ±1/2 ±3/2 +1/2 -1/2 source I=3/2 I=1/2 m δ δ ∆ δ Transmission Transmission Transmission velocity velocity velocity FIG. 2. Schematic representation of the hyperfine interactions in the case of a D transition 1/2-3/2 like that of 57Fe. Transmission Transmission FIG. 2. Schematic representation of the hyperfine interactions in the case of a D transition 1/2-3/2 like that of 57Fe. different for the ground and excited states because the nuclear volumes are different in the two states. Furthermore, these shifts are different for the source and the absorber because emitting and absorbing nuclei have different electron densities inside the nuclear volumes because of different chemical states and electronic en- vironments, and the absorption will be observed at a velocity characteristic of the absorber relative to the source. This measured shift, called isomer shift (IS), is expressed as follows: IS ¼ 2 3 e2pZSðZÞD r2   rAð0Þ  rSð0Þ , ð10Þ ð10Þ where D r2   is the variation of the mean-square radius of the nucleus between the excited and ground states of the Mo¨ ssbauer transition, S0ðZÞ is a correction for the relativistic effects, and the term in brackets, rAð0Þ  rSð0Þ; the difference between the electron density at the nucleus in the absorber A and the source S. Isomer shifts measured and defined as in Eq. (10) depend on the source. For a comparison of results reported by different authors, these must be stated with respect to that of a standard compound. In the case of 119Sn, the usual standards 6 are BaSnO3 and SnO2, and in the case of 57Fe, they are a-Fe metal or sodium nitroprusside. The latter compound is, however, used less and less. For Mo¨ ssbauer nucleides other than these two, there are generally no specific prescriptions regard- ing standards. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS In some cases, the natural line width is very broad relative to those of iron or tin, or the change in nuclear radius between the ground and the excited state is very small so that the measurement of isomer shifts is difficult or virtually impossible. The use of an isomer shift standard thus became insignificant. Although IS is independent of T, variations with temperature are observed experimentally because ISOBS is in fact a linear combination of IS and a second shift which is sample-dependent, being called a second-order Doppler shift (SOD): ISOBS ¼ IS þ ISSOD. ð11Þ ð11Þ This shift arises from the relative motions associated with atomic vibrations, via a second-order relativistic effect; it is equal to the following: This shift arises from the relative motions associated with atomic vibrations, via a second-order relativistic effect; it is equal to the following: ISSOD ¼  V 2   2c2 Ey, ð12Þ ð12Þ where V 2   is the mean-square velocity of the probe nucleus in the sample, which can be derived from the Debye model of the vibrations: ISSOD ¼  1 2c 9kTD 8M 1 þ 8 T TD  4 Z T TD 0 y3dy ey  1 " # . ð13Þ ð13Þ It is evident that this term depends on the temperature and also on the source and the absorber (through M and TD). A second perturbation of the nuclear levels is caused by the magnetic interaction between the nuclear magnetic moment and the surrounding electron spin density, creating an electric field gradient at the nucleus. This electric field gradient is a tensor that is described by the spatial orientations of its three principal axes UXX, UYY, and UZZ. This interaction partially removes the degeneracies of nuclear states in which the nuclei have non-zero quadrupole moments Q (spin I>1) into 2I+1 nuclear states characterized by the value of the magnetic quantum number mI, and it shifts them. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS In the general case when the electrical field gradient has no axial symmetry, the energy shift of the levels can be expressed as follows: EQ ¼ eUZZQ 3m2 I  IðI þ 1Þ 4Ið2I  1Þ 1 þ Z2 3  1=2 , ð14Þ ð14Þ where Q is the quadrupole moment of the nucleus in the excited state of the Mo¨ ssbauer transition, mI the magnetic quantum number, UZZ the main component of the electric field gradient tensor UZZ j j4 UYY j j  UXX j j ð Þ; and Z the asymmetry parameter ðZ ¼ ðUXX  UYYÞ=UZZÞ; which is zero in the case of axial symmetry. In the case of 57Fe, the ground state is an I ¼ 1/2 state without a quadrupole moment and is thus not affected by the electric field gradient. The first excited state is an I ¼ 3/2 state with a quadrupole moment and has a degeneracy of 4 that is 7 partially lifted. It is symmetrically split into two substrates. This splitting, called quadrupolar splitting (QS or D), is given by the following: partially lifted. It is symmetrically split into two substrates. This splitting, called quadrupolar splitting (QS or D), is given by the following: D ¼ 1 2 eUZZQ 1 þ Z2 3  1=2 . ð15Þ ð15Þ As a result, the Mo¨ ssbauer spectrum will consist of two lines, called a doublet, with a 2G0-line width and separated by D, the isomer shift being measured at the center of the doublet (Fig. 2). The relative intensities of the two absorption lines of the doublet depend on the orientation of Uzz relative to the incident X-ray direction (Table I). For a fine powder absorber, in which all directions are uniformly distributed, the two lines of the corresponding doublet have equal intensities. A third perturbation of the nuclear energy levels occurs in the presence of a magnetic field and yields different energies for the different orientations of the nuclear magnetic moment mI relative to the field direction. Each nuclear level of non-zero spin is shifted into 2I+1 sub-levels reached by shifting it by – gmNmIH, where H is the magnitude of the magnetic field, g the nuclear factor of the con- sidered sub-level, and mN the nuclear Bohr magneton. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS For example, in the case of iron, the ground level will undergo a symmetric splitting (I ¼ 1/2, mI ¼ +1/2, mI ¼ 1/2) and the first excited state is split into four sub-levels (I ¼ 3/2, mI ¼ +3/2, mI ¼ +1/2, mI ¼ 1/2, mI ¼ 3/2). Among the eight possible transitions between the two sub-levels of the ground state and the four sub-levels of the excited state only six are allowed by the quantum mechanical selection rule, and the resonance spectra consist of six lines with a 2G0-line width called a sextet. The isomer shift is measured at the center of the sextet (Fig. 2). H is proportional to the difference between the positions of two lines of the spectrum; the outermost ones are generally considered in the data analysis for better precision. TABLE I Line Position and Relative Intensities in 57Fe Spectra Obtained in the Presence of an Electric Field Gradient (Doublet) and both an Electric Field Gradient and a Magnetic Field (Sextet) Line Spin state Line position (mm/s) Relative intensities Ig Ie Doublet 1 71/2 71/2 dD/2 2+3sin2ygVZZ 2 71/2 73/2 d+D/2 3(1+cos2ygVZZ) Sextet 1 +1/2 3/2 d(Z+3)(z/2)+e 3(1+cos2ygH) 2 +1/2 1/2 d(Z+1)(z/2)e 2(2sin2ygH) 3 +1/2 +1/2 d(Z1)(z/2)e 1(1+cos2ygH) 4 1/2 1/2 d(Z1)(z/2)e 1(1+cos2ygH) 5 1/2 +1/2 d(Z+1)(z/2)e 2(2sin2ygH) 6 1/2 +3/2 d+(Z+3)(z/2)+e 3(1+cos2ygH) ygVZZ and ygH are respectively the angles between the gamma ray direction, Vzz and H. Ig and Ie are the spin states of the ground and excited states; D the quadrupolar splitting (Eq. (15)), and e the quadrupolar shift parameter (Eq. (16)). Z the ratio between the g factor of the ground (gi) and excited state (ge), and z ¼ gemNH. TABLE I Line Position and Relative Intensities in 57Fe Spectra Obtained in the Presence of an Electric Field (Doublet) and both an Electric Field Gradient and a Magnetic Field (Sextet) TABLE I ygVZZ and ygH are respectively the angles between the gamma ray direction, Vzz and H. Ig and Ie are the spin states of the ground and excited states; D the quadrupolar splitting (Eq. (15)), and e the quadrupolar shift parameter (Eq. (16)). Z the ratio between the g factor of the ground (gi) and excited state (ge), and z ¼ gemNH. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS 8 In the presence of a small simultaneous electric quadrupolar interaction, the split levels are shifted by an amount e, which is called the quadrupole shift parameter and is given by the following:  ¼ e2UzzQ 8   ½2  ð3  Z cos 2fÞsin2y, ð16Þ ð16Þ where f and y are the azimuthal and polar angles of the magnetic field direction in the principal axes coordinate system of the electric field gradient. The relative in- tensities of the six lines of the sextet depend on the orientation of the magnetic field relative to the incident g-ray beam (Table I). The magnetic field can have several origins and may be written as the sum of up to five contributions that can be added vectorially to give the following expression: ð17Þ H ¼ HS þ HL þ HD þ HM þ HE, ð17Þ where HS, HL, and HD are, respectively, the fields attributed to the s-electron spin polarization term, to the on-site orbital electron current, and to the dipole inter- action related to the local electron spin moments on the probe site. HM is the field resulting from the magnetization of the particle within which the nucleus is located. It depends on the shape and magnetic domain structure of the particles. HE is an external magnetic field that can be applied to the sample. C. SPECTRAL ANALYSIS AND INTERPRETATIONS In the preceding section the hyperfine parameters (d, D, H, and f ) that can be extracted by spectral analysis were presented. These parameters have complex re- lationships with physico-chemical properties that are presented in a rather simpli- fied manner in the following section, showing how local electronic, magnetic, structural, and chemical environments can be characterized. Equation (10) shows that the isomer shift IS is a direct measure of the total electronic density at the probe nucleus. This density derives almost exclusively from s-type orbitals, which have non-zero electron densities at the nucleus. Band electrons, which have non-zero occurrence probabilities at the nucleus and s-type conduction electrons in metals may also contribute, but to a lesser extent. Figure 3 shows the linear correlation that is observed between the experimental values of 121Sb Mo¨ ssbauer isomer shift and the calculated values of the valence electron density at the nucleus rvð0Þ. The total electron density at the nucleus rAð0Þ (Eq. 10) is the sum of the valence electron density rvð0Þ and the core electron density rcð0Þ, which is assumed to be constant. This density is not only determined by the s-electrons themselves but also by the screening by other outer electrons (p-, d-, or f-electrons) and consequently by the ionicity or covalency and length of the chem- ical bonds. IS is thus a probe of the formal oxidation state of the isotope under investigation and of the crystal field around it (high- and low-spin Fe2+ may be differentiated). The variation of IS with temperature can be used to determine the Debye temperature of a compound (see Eq. (13)). 9 9 9 FIG. 3. Linear correlation between the experimental values of the 121Sb Mo¨ ssbauer isomer shift d (relative to BaSnO3) and the calculated values of the valence electron density rvð0Þ at the nucleus for a series of compounds. rsSð0Þ ¼ rvð0Þ þ rcð0Þ; where rcð0Þ is the core electron density, which is assumed constant. Figure according to Lippens (7). FIG. 3. Linear correlation between the experimental values of the 121Sb Mo¨ ssbauer isomer shift d (relative to BaSnO3) and the calculated values of the valence electron density rvð0Þ at the nucleus for a series of compounds. rsSð0Þ ¼ rvð0Þ þ rcð0Þ; where rcð0Þ is the core electron density, which is assumed constant. Figure according to Lippens (7). C. SPECTRAL ANALYSIS AND INTERPRETATIONS The quadrupolar splittings depend mainly on the electric field gradient at the nucleus, which is caused by the spatial distribution of the electrons around it, via electric Coulomb forces. Both electrons (valence contribution Uval) and neighboring anions and cations (lattice contribution Ulat) in the vicinity of the nucleus contrib- ute to this electric field gradient U, which can be expressed as follows: U ¼ Uvalð1  RÞ þ Ulatð1  gÞ, ð18Þ ð18Þ where R and g are screening and anti-screening Sternheimer parameters. Values of R range between 0 and 1, and values of g between 10 and 100. Because of these two contributions, the quadrupolar splitting is strongly related to site symmetry and local structure. For example, in the case of iron, if the site being characterized is a Fe3+(4sx3d 5) ion, inner atomic shells are spherically symmetric, and qval is the 10 major component of the electric field gradient). In contrast, Fe2+(4sx3d 6) ions have asymmetric distributions of their valence electrons, and qval has a dominant con- tribution to the electric field gradient. The quadrupolar splitting characterizes the degree of local asymmetry at the probe site. Such local asymmetry is caused by various forms of structural, chemical, and magnetic disorder, such as lattice defects, chemical substitutions of neighboring anions, local moment disorder in magneto- volume and magneto-elastic materials that can be characterized and quantified in solid catalysts. For example, the Mo¨ ssbauer spectrum of a BaFe3Al9O19 catalyst for combustion of methane is given in Fig. 4 (8). This spectrum has been fitted with four sextets that correspond to the four octahedral crystallographic sites of the structure that may be occupied by the ferric ions, thus leading to a different atomic environment for each of them. From the relative intensities of the four sextets used for the fit and con- sidering equal f factors for the corresponding species, it is possible to determine the respective occupation of these sites by iron. Alternatively, only a complete Rietveld analysis of the powder X-ray diffraction pattern of the catalyst would have enabled such a determination. The internal magnetic field H is a reliable signature of a given species and allows a good identification of magnetic phases containing these species. C. SPECTRAL ANALYSIS AND INTERPRETATIONS Assuming that particles for which trotL and tr>tL contribute exclusively to one of the two com- ponents (paramagnetic and magnetic), the temperature at which tr ¼ tL and at which the hyperfine split takes place can be determined; V is calculated from Eq. (19) at the temperature T at which the spectral areas of the two components are equal). The dependence of the two spectral areas on temperature in the range in which both components are observed yields the particle size distribution. One can often describe a solid catalyst as a dispersed set of active species on the surface of a support, present as more or less isolated atoms or small particles. These species, because of their small size and their bonding to the support, present specific features different from those of bulk material. Many studies of supported catalysts have shown that the f factor for small particles is influenced by the vibration of the particles relative to the atomic environment. It may therefore be difficult to unravel contributions of surface vibrations to the f factors of small particles. On the other hand, when active species are weakly adsorbed at the surface of a support, they can also vibrate or even diffuse on the surface, leading to a decrease of their f factors and to a broadening of the lines in the Mo¨ ssbauer spectra that can result in the disappearance of the signal. Furthermore, if they only vibrate, the mean-square vibration amplitude perpendicular to the surface is not the same as that parallel to the surface, leading to an anisotropy of the f factor. This effect (referred to as Karyagin–Goldanskii effect) implies a dependence of f upon y (the angle between Uzz and the direction of the incident g-radiation). In the case of a doublet, an asymmetry of the intensity of the two lines is observed; this may complicate spectral analysis. Another difficulty may arise from the deviation from a Lorenzian line shape of the absorption signal. Such deviation corresponds to either homogeneous or in- homogeneous broadening. Inhomogeneous broadening relative to the natural line width is caused by variable environments of the probe nuclei that create variations in the electronic or structural characteristics. C. SPECTRAL ANALYSIS AND INTERPRETATIONS In catalyst char- acterization, one of the major interests in this last parameter relates to its variation with temperature, which can be used to evaluate particles sizes and particle size distributions when the particles are small enough. Indeed, when particles of a magnetic phase are small, a collective reorientation of the magnetic moment is observed in these particles. In a large particle, once a particular orientation has been reached below the magnetic ordering temperature, it does not change, and an in- ternal magnetic field is observed leading to a sextet in the Mo¨ ssbauer spectrum. When particles are small, the thermal excitation energy can be sufficiently high to invert all of the spins simultaneously, thereby reversing the magnetization of the lattice. If the relaxation time (tr) of these inversions is smaller than the nuclear Larmor precession time (tL), the magnitude of the magnetic field at the nucleus averages to zero, and no magnetic splitting is observed. The solid behaves as a paramagnetic solid (superparamagnetic behavior), which results in the observation of a doublet instead of a sextet in the Mo¨ ssbauer spectrum. x107 Velocity (mm/s) Counts 1.36 1.34 1.32 1.30 -10 -5 0 5 10 FIG. 4. Mo¨ ssbauer spectrum of a BaFe3Al9O19 catalyst recorded at 298 K in air. Solid lines are derived from least-square fits. Figure according to Naoufal et al. (8). FIG. 4. Mo¨ ssbauer spectrum of a BaFe3Al9O19 catalyst recorded at 298 K in air. Solid lines are derived from least-square fits. Figure according to Naoufal et al. (8). 11 11 The relaxation time can be expressed in a simplified manner as follows: tr ¼ t0eKV=kT, ð19Þ ð19Þ where K is the anisotropy constant and V the volume of the particle. It is clear that tr increases with decreasing temperature and can become larger than tL. In such cases, the spectrum changes, and a sextet is again observed. A simplified method was proposed by Ku¨ ndig et al. (9) allowing evaluation of the particle size and the size distribution of a solid by analysis of its spectrum as a function of temperature. By variation of the temperature it is possible to follow the variation of the relative spectral areas of the sextet and doublet. C. SPECTRAL ANALYSIS AND INTERPRETATIONS This broadening can be considered to be a consequence of the superposition of many sub-spectra and can relatively easily be taken into account by fitting the experimental spectra considering a distribution of sub-spectra with Lorenzian line shapes. Homogeneous broadening originates from time variation in the electronic environment. Such effects, referred to as re- laxation effects, imply the modification of the line width of the elemental spectrum. These broadenings may coexist and significantly complicate the analysis and in- terpretation of Mo¨ ssbauer spectra. 12 D.1. Emission Mo¨ssbauer Spectroscopy (EMS) In Mo¨ ssbauer absorption spectroscopy, as described in the preceding section, an emitting moving source with a single-line resonance is used to scan the energy levels of an absorber that is the material under investigation. In contrast, in Mo¨ ssbauer emission spectroscopy, the source corresponds to the material under investigation and a reference single-line absorber has to be used. The studied emitting material can be moved but most of the times it is the reference absorber that is moved (Fig. 5). This technique has been used successfully to investigate CoMo/Al2O3 catalysts, which are well known for their use in hydrotreating processes. The tech- nique has contributed to identify the active species of these catalysts (10,11). The catalysts to be investigated with this method should be synthesized with radioactive 57Co; this constitutes the major experimental challenge of the technique. Another challenge may be encountered in the interpretation of the MES spectra. This interpretation may be hampered by the so-called chemical aftereffects. The transition from 57Co to 57Fe occurs in the catalyst via electron capture, whereby the electronic structure of the atom is changed. This change is followed by a reshuffling of the electrons in the material that may take time to occur, especially if the material is an insulator. If this required time is longer than the lifetime of the excited state, the probed electronic structure and, thus, the energy levels may have changed. Therefore, EMS is generally applied as a fingerprint technique, with spectra being compared to each other and to those of reference compounds. radioactive sample reference absorber radioactive sample detector detector FIG. 5. Basic equipment for measuring an emission Mo¨ ssbauer spectrum. The sample is prepared with a radioactive isotope and used as the source. FIG. 5. Basic equipment for measuring an emission Mo¨ ssbauer spectrum. The sample is prepared with a radioactive isotope and used as the source. 13 13 The single-line absorber is generally potassium (or sodium) nitroprusside, K4Fe(CN)6,3H2O, enriched with 57Fe. The single-line absorber is generally potassium (or sodium) nitroprusside, K4Fe(CN)6,3H2O, enriched with 57Fe. Besides hydrotreating catalysts (12–20), supported cobalt Fischer–Tropsch catalysts (21–25) and multi-phasic molybdate-containing catalysts for propene oxidation (26) have also been characterized by this technique. The technique can also be applied for investigations involving 107Cd, 133Cs, 195Pt, 99Rh, and 83Rb. D.2. Conversion Electron Mo¨ssbauer Spectroscopy (CEMS) A. IDENTIFICATION OF CATALYST COMPONENTS Mo¨ ssbauer spectroscopy has been quite successful in identifying catalyst com- ponents. Mo¨ ssbauer spectroscopy provides quantitative site populations, easily discriminating between various metal oxidation states and anion coordinations, and it can lead to phase compositions or distributions between phases of the isotope under investigation. It also gives quantitative population distributions of local dis- tortion environments and local chemical environments, via extracted quadrupolar splitting distributions. 57 The majority of investigations in this category has dealt with the isotopes 57Fe, 119Sn, and 57Co (in emission mode). In contrast, investigations focusing on other isotopes, such as 197Au, 121Sb, 125Te, 193Ir, or 99Ru, have been limited in number but very fruitful. D.2. Conversion Electron Mo¨ssbauer Spectroscopy (CEMS) It is possible to analyze the fluorescence radiation emitted from the decay of the excited state in the solid instead of the absorption of the gamma X-rays. This can be done by detecting and analyzing the conversion electrons arising from the internal conversion process in which the de-excitation leads to the ejection of an inner-shell electron instead of being used in the production of a g-radiation (Fig. 6). This internal conversion process is much more frequent than X-ray emission. The initial kinetic energy of an ejected conversion electron is equal to the energy of the tran- sition minus its original atomic binding energy. Because emitted electrons lose energy as they interact with the sample, their path length is limited to 0.1–0.2 mm, and therefore CEMS is a surface-sensitive technique. It is possible to analyze the energy distribution of the ejected electrons and to obtain spectra with various depth resolutions (depth-selective CEMS is called DCEMS). Ultrahigh-vacuum and high- resolution spectrometers allow analysis of surface layers with thicknesses as low as 1 nm with almost atomic-scale depth resolution. This technique is used only infrequently in catalysis research. For finely dispersed catalysts, the size of the particles is smaller than the depth analyzed, and spectra are radioactive source e- channeltron high vaccuum chamber FIG. 6. Basic equipment for measuring a conversion electron Mo¨ ssbauer spectrum. FIG. 6. Basic equipment for measuring a conversion electron Mo¨ ssbauer spectrum. 14 consequently similar to those recorded in the conventional transmission mode. Furthermore, the high-resolution analysis of a fine-powder sample is sometimes difficult to achieve, and the use of ultrahigh vacuum may lead to surface modi- fication of the sample. Therefore, CEMS has found limited applications in the field of catalysis. y This technique has been reviewed recently, and applications to surface charac- terization were described in Reference (27). It is noteworthy that a low-temperature apparatus has been described recently that allows g-ray and conversion electron Mo¨ ssbauer spectroscopy measurements at the same time, in the temperature range from 10 K to room temperature (28). III. Applications of Mo¨ ssbauer Spectroscopy in Heterogeneous Catalysis In this section, we summarize results characterizing catalytic materials by Mo¨ ssbauer spectroscopy. An analysis of the literature leads to the following clas- sifications of the objectives of this work: 1. Identification of catalyst components in terms of active phase or active sites and search for correlations between these components and one or more of the cat- alytic properties. 1. Identification of catalyst components in terms of active phase or active sites and search for correlations between these components and one or more of the cat- alytic properties. 2. Investigations of catalysts under working conditions and analysis of activation or deactivation processes that may take place with time on stream. 2. Investigations of catalysts under working conditions and analysis of activation or deactivation processes that may take place with time on stream. 3. Study of chemisorption of reactants or molecular probes. Progresses and recent results obtained in each category are presented below. Our intent is to give a broad view of the results generated recently, and this review is not exhaustive. A.1. Iron-Containing Catalysts 57Fe Mo¨ ssbauer spectroscopy has been used extensively to characterize catalysts, constituting the large majority of the Mo¨ ssbauer investigations of catalysts. The 15 publications dealing with the Mo¨ ssbauer spectroscopy of iron-containing catalysts cover a broad range, and only a partial coverage of the work is included here. These investigations focus mainly on well-known reactions for which iron-containing catalysts are among the best. These reactions are the Fischer–Tropsch synthesis (29–50), ethylbenzene dehydrogenation (51–58), ammonia synthesis (59–71), the water gas shift (72–75), and oxidation reactions (76–90). In addition to these cat- alysts for these reactions, several others have been the focus of Mo¨ ssbauer inves- tigations, including those concerned with catalytic materials that have attracted recent attention, such as iron supported on ZrO2 (91–104) or MgO, either directly prepared from MgO (105–107) or from substituted hydrotalcite (108–111). Zeolites and mesoporous materials (112–125), especially ZSM-5 (120–125) and MCM-41 (112–128) were also investigated. Furthermore, bimetallic alloys have also been characterized by Mo¨ ssbauer spectroscopy (129–149) to determine the active phases and active sites. A.2. Tin-Containing Catalysts The characterization of tin-containing catalysts by Mo¨ ssbauer spectroscopy has been focused mainly on supported bimetallic alloys (150–162). These catalysts, which consist of Pt, Pd, Rh, or Ru as primary metals with Sn used as a promoter, are generally supported on SiO2 or Al2O3. These catalysts are used in reforming and hydrogenation processes in the petroleum industry (151,152) and have recently drawn attention for other applications, such as low-temperature CO oxidation, alkane and alcohol dehydrogenation, metathesis reactions (153–160), and even ni- trate removal for drinking water processing (161). The Mo¨ ssbauer transition in the isotope 119Sn is of the same type as that oc- curring in 57Fe (1/2-3/2). Depending upon the presence or absence of an electric field gradient at the nucleus, single lines or doublets are observed. Mo¨ ssbauer spectroscopy appears to be efficient for identification and quantifications of various compounds that are formed, depending upon the nature of the support, the metal relative contents and the preparation conditions; these include Pt3Sn, PtSn, PtSn2, or SnO and SnO2. Preparation of these catalysts by the contacting of silica- supported rhodium with tetra-n-butyl-tin, led to relatively stable solids with organometallic fragments bonded to the rhodium surface. These materials may exhibit very high selectivities and activities for hydrogenation of a,b unsaturated aldehydes or nitrobenzene (159). Mo¨ ssbauer spectroscopy has allowed identifica- tion of all the species that were encountered when tetra-n-butyl-tin interacted with the surfaces of silica or silica-supported rhodium (Fig. 7). At room temperature, tetra-n-butyl-tin was physically adsorbed on the surfaces (characterized by a single line d ¼ 1.42 mm/s). After reaction under H2 at 373 K, the formation of grafted organometallic fragments on the rhodium surface (characterized by a doublet with d ¼ 1.39 and D ¼ 2.26 mm/s) as well as a well-defined bimetallic RhSn compound (characterized by a doublet d ¼ 1.42 and D ¼ 0.85 mm/s) were identified, whereas with pure silica, surface-bound Si–O–Sn(n-C4H9)3 moieties were observed (characterized by a doublet d ¼ 1.39 and D ¼ 2.69 mm/s). After treatment at 523 K, the rhodium-grafted organometallic species were completely decomposed, 16 x106 x106 6.165 1.70 (a) (b) (c) (d) 1.68 1.66 1.64 6.160 6.155 6.150 6.145 velocity (mm/s) velocity (mm/s) velocity (mm/s) velocity (mm/s) x106 x106 5.72 5.73 9.94 9.95 9.93 9.92 5.71 5.70 5.69 5.68 FIG. 7. A.2. Tin-Containing Catalysts Experimental Mo¨ ssbauer spectra of samples recorded at 78 K: (a) Sn(n-C4H9)4 physisorbed on SiO2, (b) SiOSn-(n-C4H9)3, (c) Rhs[Sn(n-C4H9)2]/SiO2, and (d) Rhs[Sn(n-C4H9)2]/SiO2 treated at 523 K. Solid lines are derived from least-square fits. Figure according to Millet et al. (158). x106 1.70 (b) (d) 1.68 1.66 1.64 velocity (mm/s) x106 6.165 (a) 6.160 6.155 6.150 6.145 velocity (mm/s) (b) (a) (d) d) y ( ) velocity (mm/s) x106 9.94 9.95 9.93 9.92 (c) y ( ) velocity (mm/s) x106 5.72 5.73 5.71 5.70 5.69 5.68 velocity (mm/s) velocity (mm/s) FIG. 7. Experimental Mo¨ ssbauer spectra of samples recorded at 78 K: (a) Sn(n-C4H9)4 physisorbed on SiO2, (b) SiOSn-(n-C4H9)3, (c) Rhs[Sn(n-C4H9)2]/SiO2, and (d) Rhs[Sn(n-C4H9)2]/SiO2 treated at 523 K. Solid lines are derived from least-square fits. Figure according to Millet et al. (158). and formation of a well-defined bimetallic RhSn compound was observed. Because the isomer shift of Pt–Sn alloys of varying composition varies linearly with the Sn mole fraction (160), it was possible to determine the composition of the alloy-like structures and consequently the relative amount of the grafted species. This quan- tification was confirmed by use of X-ray photoelectron spectroscopy. Mesoporous tin-containing analogues of MCM-41 and tin oxide-modified me- soporous SBA-15 have also been characterized by Mo¨ ssbauer spectroscopy (162,163). In the first case, the results suggest that tin was incorporated in the structure of the silicate and, in the second, they indicated that two types of sup- ported tin species were formed, depending on the tin content. One would corre- spond to atomically isolated species stabilized in the wall of the pore and susceptible to reduction to Sn2+ under reductive treatment conditions and the other to large oxide clusters distributed in the external pore structure. A.3. Gold-Containing Catalysts Highly dispersed supported gold has been shown to have high catalytic activity for various oxidation reactions 197Au spectroscopy has emerged as one of the 17 methods of choice for investigation of catalysts consisting of gold particles on oxide supports, such as TiO2, Fe2O3, SiO2, Al2O3, MgO, and MnOx (164–170). The 197Au resonance is characterized by Ig and Ie, respectively, equal to 3/2 and 1/2. Con- sequently, the presence of an electric field gradient at the nucleus does not affect the excited state, although the ground state is symmetrically split into two sub-states. This splitting leads to the presence of doublets in the spectra. Early investigators proposed the presence of both metallic and ionic gold species (AuIII) in the active catalysts (165), but more recent investigations suggest the pres- ence of only metallic species (166,167). Mo¨ ssbauer spectra obtained for gold cat- alysts supported on TiO2 and SiO2 are shown in Fig. 8. The solid lines in the figure are derived from least-squares fits and show both the total fits and the sub-spectra. The spectra are fitted using a single line (IS ¼ 1.22 mm/s), which is attributed to bulk gold, and a doublet that has been attributed to surface metallic species (1.5 mm/s oIS o0.75 mm/s and 1.25 mm/soQSo2.5 mm/s). Indeed, since the earlier Mo¨ ssbauer investigations of gold compounds (168,169), it was observed that isomer shifts (IS) and quadrupolar splittings (QS) vary over a wide range. This variation could have rendered difficult the attribution of oxidation states to the detected species, but it was shown that a linear correlation between IS and QS values exists for AuI and AuIII compounds (170). This relationship has been 100.0 Au/TiO2 5.2 nm Au/TiO2 spent propene epox Au/TiO2/SiO2 3.2 nm Au/SiO2 6 - 30 nm 99.5 99.0 100.0 100.0 99.5 99.0 98.5 99.5 99.0 100.0 99.5 -10 -8 -6 -4 -2 0 Velocity (mm/s) Transmission (%) 2 4 6 8 10 FIG. 8. Mo¨ ssbauer spectra recorded at T ¼ 4.2 K of gold catalysts on various supports used for propene epoxidation. The average size of the particles is given in the legend. Figure according to Goossens et al. (165). FIG. 8. Mo¨ ssbauer spectra recorded at T ¼ 4.2 K of gold catalysts on various supports used for propene epoxidation. The average size of the particles is given in the legend. Figure according to Goossens et al. (165). A.3. Gold-Containing Catalysts 18 explained by the fact that for a given oxidation state the effective charge on the gold atom does not vary as a result of p-back donation, and only transfers between the d- and s-shells are observed, which affect both IS and QS values. This is possible because the lattice contribution to QS is at least one order of magnitude smaller than the contribution of the valence electrons (Eq. (18)). To determine whether the observed doublet could be attributed to ionic species, the IS/QS combinations have thus been considered, and it has been observed that they did not fit within ranges corresponding to AuI and AuIII. Moreover, the isomer shifts always remained close to that of the bulk species. It was thus proposed that they correspond to metallic surface species, the presence of a QS being only related to the large electric field gradient generated at the surface of the gold particles (166). The Au/TiO2 spectrum (upper plot of Fig. 8) still showed a small contribution that might be attributed to AuV that has not been explained but which disappeared after catalysis and was never observed on any other support. A recent study on Au/Fe2O3 confirmed the exclusive presence of metallic gold Au0 on the catalysts but only after heat treat- ment at high temperature (4001 C) (170). At lower temperature, at which the catalysts were more active, both AuIII (probably related to the presence of AuOOH,H2O) and Au0 were observed. Although discrepancies remain in the lit- erature on the existence and role of the gold species, Au0 and/or AuIII were most of the time identified by Mo¨ ssbauer spectroscopy in the catalysts and may both play a role; conversely AuI was never observed in the catalysts suggesting that its involvement is very unlikely. A.4. Antimony-Containing Catalysts 121Sb Mo¨ ssbauer spectroscopy has been used to investigate the tin-antimony oxide catalysts used for selective oxidation of hydrocarbons (171). Recent inves- tigations have been conducted to characterize FeSbO catalysts for ammoxidation of propene (172) and VSbO (173) and MoVSbNbO (174) catalysts for ammoxidation of propane. p p The Mo¨ ssbauer transition for the 121Sb isotope is of the type 5/2-7/2. Broad, asymmetric lines indicating unresolved quadrupolar split spectra are observed for the oxide compounds (Fig. 9). However, the relative variation of the nuclear radius D r2    is rather large, and therefore the isomer shift for this system becomes a sensitive parameter not only for changes in oxidation state but also for changes in the local environment. Isomer shifts allow the identification and characterization of various ionic species. In the cases of VSbO and FeSbO, the investigations evidenced the presence of b-Sb2O4 formed during the synthesis or by segregation under catalytic condition. In the case of MoVSbNbO, it allowed a determination of the relative SbIII/ SbV contents of the two main phases of the complex catalysts. The results obtained in the latter case were consistent with X-ray absorption near-edge spectra (174). A.5. Catalysts Containing other Elements Other characterization of catalysts have dealt with isotopes such as 125Te, 193Ir, 99Ru and 51Eu. The tellurium-containing catalysts were MoVTeNbO ammoxidation catalysts (175) and Te-doped Cr2O3 hydration–dehydration catalysts (176) and the 19 19 Iz = 7/2 Iz = 5/2 ±7/2 m Electrical quadrupole interaction ±5/2 ±3/2 ±1/2 ±5/2 ±3/2 ±1/2 FIG. 9. Schematic representation of the electrical quadrupolar splitting in the case of a transition 5/ 2-7/2 like that of 121Sb and corresponding the experimental spectra of FeSbO catalysts (173). Electrical quadrupole interaction FIG. 9. Schematic representation of the electrical quadrupolar splitting in the case of a transition 5/ 2-7/2 like that of 121Sb and corresponding the experimental spectra of FeSbO catalysts (173). iridium-containing catalysts, iridium/iron bimetallic methanol synthesis catalysts (177); 99Ru and 51Eu spectroscopies were applied for the characterization of carbon- supported ruthenium–tin catalysts (used for the reduction of a,b-unsaturated aldehydes) (178) and the study of the hydrogen reduction of supported europium catalysts (179). These investigations are scarce, and all the published data have been cited here to illustrate the breadth of the applications of Mo¨ ssbauer spectroscopy. B. INVESTIGATIONS OF CATALYSTS IN REACTIVE ATMOSPHERES AND UNDER WORKING CONDITIONS Characterization of catalysts in reactive atmospheres and during catalysis has led to insights that have not emerged from other characterizations. The introduction of EMS (57Co) to investigate Co–Mo/Al2O3 hydrodesulfurization catalysts in reactive atmospheres was a key technological advance in the 1980s; it provided the first detailed insight into the nature of the promoter atoms in the active sulfided state of the catalysts and showed that the catalytic activity was related to the presence of the Co–Mo–S edge structures (10,11). Since that time, Mo¨ ssbauer spectroscopy has been used widely to characterize catalysts in reactive atmospheres, leading to continuous progress in the under- standing of structure/catalytic property relationships (180–194). 20 20 Methodologies for investigation of solid catalysts under real working condi- tions with simultaneous measurements of catalytic activity data have advanced markedly in recent years, and Mo¨ ssbauer spectroscopy has played an important role. Only isotopes that can be investigated at room temperature or higher temperatures (57Fe,57Co, 119Sn, and 151Eu) have been used in this context; they are powerful because they allow the characterization of the local atomic environ- ment of the working catalytic species, giving rise to unique fingerprints of the catalytic sites. It is important in this section to differentiate between the catalyst characteriza- tions with Mo¨ ssbauer spectroscopy under real working conditions and those per- formed under controlled environments after quenching of the reaction. Only recently (in the preceding 12 years) has this distinction been made with some clarity; the unclear term ‘‘in situ’’ has been used often in the literature for both types of investigations; the reader should be aware of this issue and carefully review the experimental conditions when the term ‘‘in-situ’’ is used. B.1. Investigations of Functioning Catalysts Investigations of functioning catalysts with Mo¨ ssbauer spectroscopy have been performed for a wide range of samples and applications. The reactions include hydrodesulfuration (15), the Fischer–Tropsch reaction (20,180), selective oxidation or oxidative dehydrogenation (181–186), and acetonitrile synthesis (187). We have seen in the first part of this article that the time required for obtaining a Mo¨ ssbauer spectrum is rather long. The application of the technique to follow time- dependent changes has been limited, and in most cases it is catalysts operating under steady-state conditions that have been characterized. A great advantage of Mo¨ ssbauer spectroscopy is that the measurements can be done under extreme working conditions, at both, high pressures and high temperatures. This essential point, which sets Mo¨ ssbauer spectroscopy apart from numerous other methods for characterization of catalysts, is illustrated by the example of an industrial cobalt- containing Fischer–Tropsch catalyst investigated by 57Co Mo¨ ssbauer emission spectroscopy (24). A cell has been constructed that allows investigation of catalysts at temperatures up to 723 K and pressures up to 20 bar with injection of steam at 10 bar, permitting the production of wax and continuous catalyst test runs lasting up to 2 months (Fig. 10). The stainless steel cell is composed of two compartments. The inner one which corresponds to the high temperature and high pressure reactor and the outer one which is there to safely confine the radioactive 57Co in case the inner one fails. The thickness of the beryllium windows of the two compartments have been optimized to support high pressure without absorbing too much g-rays. The catalyst is placed onto a sintered metal filter with pore size diameter of 5 mm that allows the reactive gas to flow from top to bottom through the catalyst powder. This filter also allows draining of the produced wax downwards in a specially designed reservoir. It is stressed that only few physical chemical techniques can be used under such drastic conditions. To investigate the deactivation of these catalysts, a 57Co-containing catalyst was prepared by adding ca. 1000 ppm of 57Co to a cobalt and platinum supported on 21 FIG. 10. Photograph and schematic representation of high-pressure Mo¨ ssbauer emission spectroscopy cell (24). FIG. 10. Photograph and schematic representation of high-pressure Mo¨ ssbauer emission spectroscopy cell (24). alumina catalyst. B.1. Investigations of Functioning Catalysts This catalyst, the initial composition of which was 15Co/0.025Pt/ 100Al2O3, was then reduced in hydrogen at 653 K for 16 h (20). The Mo¨ ssbauer emission spectrum showed about 70% reduction of cobalt. The catalyst was sub- sequently treated at 423 K in the cell in a hydrogen–water gas mixture with H2/ H2O ¼ 1 (molar) at 1, 5, and 10 bar, corresponding to a water partial pressures of 0.5, 2.5, and 5 bar, respectively. The Mo¨ ssbauer emission spectra showed that the amount of metallic cobalt increased from 70% at 1 bar to 86% at 10 bar total pressure. These results show that the catalysts resisted oxidation under the con- ditions used and became more and more reduced when the total pressure (and thus the water partial pressure) increased. The investigation also showed that water was not responsible for the deactivation of the cobalt-containing Fischer–Tropsch cat- alysts. It was inferred that the deactivation should better be described as a surface poisoning or a carbon deposition, as had been proposed (188,189). alumina catalyst. This catalyst, the initial composition of which was 15Co/0.025Pt/ 100Al2O3, was then reduced in hydrogen at 653 K for 16 h (20). The Mo¨ ssbauer emission spectrum showed about 70% reduction of cobalt. The catalyst was sub- sequently treated at 423 K in the cell in a hydrogen–water gas mixture with H2/ H2O ¼ 1 (molar) at 1, 5, and 10 bar, corresponding to a water partial pressures of 0.5, 2.5, and 5 bar, respectively. The Mo¨ ssbauer emission spectra showed that the amount of metallic cobalt increased from 70% at 1 bar to 86% at 10 bar total pressure. These results show that the catalysts resisted oxidation under the con- ditions used and became more and more reduced when the total pressure (and thus the water partial pressure) increased. The investigation also showed that water was not responsible for the deactivation of the cobalt-containing Fischer–Tropsch cat- alysts. It was inferred that the deactivation should better be described as a surface poisoning or a carbon deposition, as had been proposed (188,189). With the same type of cell, it has recently been possible to investigate the sulfidation of calcined Co–Mo/Al2O3 hydrodesulfurization catalysts under 22 realistic hydrotreatment conditions, with an activation of the catalysts at 673 K and 40 bar (20). B.1. Investigations of Functioning Catalysts In another example, a complex multi-component BiMoCoFeO catalyst used in the partial oxidation of propene to acrolein was characterized by Mo¨ ssbauer spectros- copy. This example has been chosen because it provides a good demonstration of the high efficiency of Mo¨ ssbauer spectroscopy for the characterization of working catalysts (181,182). y ( ) In this investigation, Co1xFexMoO4 as pure solid solutions and in mixtures with Bi2(MoO4)3 were characterized at 688 K under the conditions of propene oxidation to acrolein (C3H6:O2:N2 ¼ 1:1:5.6 (molar ratios) at atmospheric pressure). The mechanical mixture of the mixed iron and cobalt molybdate with the bismuth molybdate has been shown to be a good model for the industrial catalysts, since both selectivity to acrolein and propene conversion larger than 95% could be simultaneously achieved on such catalyst (190). The data showed that under catalytic reaction conditions, no reaction was taking place between the two solid phases, which remained stable except when iron loadings were high and Fe2(MoO4)3 was formed. A comparison of the data obtained with the pure solid solution and the mixtures of phases showed that cobalt stabilized Fe2+ sites in the solid solution while Bi2(MoO4)3 stabilized Fe3+ sites in the solid solution. The presence of both species was proposed to be a key to the mild oxidation of propene by creating a high electrical conductivity, which favors the electron exchange in the Mars–van Krevelen mechanism. Furthermore, because iron sites have different environments in a and b polymorphic forms of Co1xFexMoO4, it was possible to determine the relative ratio of the two forms under the conditions of the catalytic test. It was confirmed that the b-type solid solution was more efficient than the a-type. Because the former was preferentially formed at high iron contents, it has been suggested that an optimal content of iron was present, which was sufficiently high to stabilize the b-phase but not too high, preventing the formation of ferric molybdate. C. INVESTIGATION OF CHEMISORPTION OF REACTANTS AND MOLECULAR PROBES Heterogeneous catalysis results from interactions between molecules in a gas or liquid phase and active sites at the surface of a solid. Mo¨ ssbauer spectroscopy, which is an elemental probe technique, may be used to investigate adsorption sites containing appropriate isotopes and thereby gain information about adsorption and reaction processes. Several examples of chemisorption investigations with Mo¨ ssbauer spectroscopy appeared in the literature of the 1980s (2). In contrast, only very few investigations were reported in recent years. This lack may be explained on the one hand by the development of other techniques such as infrared spectroscopy, which are better adapted, more reliable, and easier to perform than Mo¨ ssbauer spectroscopy, and on the other by the difficulties of interpretation of Mo¨ ssbauer spectra. These difficulties have several origins. For example, f factors of surface species, especially in their component perpendicular to the surface, will change extensively as a result of the adsorption or desorption of molecules. In the case of carbon- supported Pt–Fe catalysts, the doublet corresponding to iron atoms at the surface of the alloy particles was detected only when hydrogen was adsorbed (195). In the case of small particles dispersed on a support, the discrimination of resonant species at the surface of the particles from bulk species is possible only if the former are sufficiently numerous compared to the latter. This requirement implies a particle size that does not exceed about 20 nm. Furthermore, these surface species are not always easily differentiated from bulk species. The major parameter that allows such differentiation is the quadrupolar splitting which may strongly vary because of the larger electric field gradient related to the structural discontinuity at the surface. In the case of magnetic compounds, the hyperfine field at the surface has to be different from that in the bulk. Though agreement seems to have been reached on this point, it remains questionable in most cases whether the change may be de- tectable. Furthermore, it is often difficult to separate surface effects from size effects (such as superparamagnetism or collective magnetic excitations). Chemisorbed molecules have been shown to affect the total magnetic anisotropy constant of small particles and thus change their superparamagnetic relaxation times. Partial recon- struction of the surface may also occur and complicate the data analysis even more. Among the few investigations reported on chemisorption of molecules, two are cited. B.2. Investigations of Quenched Catalysts It is not always possible to record Mo¨ ssbauer spectra under catalytic reaction conditions, and characterization with this technique sometimes requires that spectra be measured at room temperature or at cryogenic temperatures. The latter limi- tation pertains to investigations involving isotopes that, by their very nature, re- quire low temperature and to iron when phases are superparamagnetic at room temperature or higher temperatures. Most of the work reported in this category concerns 57Fe; however, some investigations have been reported that deal with the characterization of PtSn particles (with the isotope 119Sn) used in the selective hydrogenation of crotonaldehyde (which occurs at 353 K), the oxidation of CO (which occurs at room temperature) or the dechlorination of 1,2-dichloroethane (which occurs at 473 K) (191–193). In these investigations the spectra were recorded at 77 K under the reactive atmosphere after rapid quenching. It is noteworthy that Mo¨ ssbauer spectroscopy applies not only to solid–gas reactions but also to solid–liquid reactions. It has been used, for example, to 23 23 characterize (at 77 K) active site heterogeneity in pyrolyzed carbon-supported iron porphyrin catalysts for the electrochemical reduction of oxygen (194). characterize (at 77 K) active site heterogeneity in pyrolyzed carbon-supported iron porphyrin catalysts for the electrochemical reduction of oxygen (194). C. INVESTIGATION OF CHEMISORPTION OF REACTANTS AND MOLECULAR PROBES Both concern N2 chemisorption on FeZSM-5 (196) and on small aFe particles supported on carbon (197). In the first case, the affinity of nitrogen for certain ferrous centers after adsorption at 623 K was clearly evidenced. In the second case nitrogen was shown to react with surface iron atoms only at temperatures up to 700 K, resulting in a number of different types of environments for the latter depending upon the number of nitrogen atoms in their proximity. The other such investigations that have been reported deal with the adsorption of gases such as H2, CO, and SO2 (198,199). 24 There are reports of the use of iron species as probes (chemisorbed species) to characterize supports and their adsorption properties. Burger et al. (200) used Mo¨ ssbauer spectroscopy to characterize submicroscopic droplets of Sn(IV) and Fe(III) complexes carried in an alkane/naphthalene mixture. The analysis of the Mo¨ ssbauer parameters gave a qualitative picture regarding the solution structure inside the pores and the adsorption and wetting properties of the solid. Pol’shina et al. (201) investigated the introduction of heteroatoms such as O or N into active carbons by measuring the adsorption of ferric ions. Analysis by Mo¨ ssbauer spectroscopy allowed them to distinguish and quantify ferric ions that form surface complexes with O-containing groups (carboxylate complexes). A.1. Mo¨ssbauer Spectroscopy of Catalysts under Working Conditions The preceding several years have witnessed an evolution in Mo¨ ssbauer investi- gations in catalysis toward investigations of catalysts under working conditions, consistent with a general trend in spectroscopic characterization of catalysts. The trend in spectroscopy of catalysts is toward extreme conditions of temperature and pressure. Mo¨ ssbauer spectroscopy is well suited to such conditions. The development is still at an early stage, and so far there has been no report illustrating the coupling of Mo¨ ssbauer spectroscopy of a working catalyst with another technique, except for on-line gas chromatography for product analysis. There is a great interest in simultaneous catalyst characterization with comple- mentary spectroscopic methods applied in ways that ensure that the data are all internally consistent. Mo¨ ssbauer spectroscopy is expected to play a role in such combined investigations of catalysts under working conditions (202). A.2. Mo¨ssbauer Spectroscopy of Catalysts below 4.2K With the development of nanoscaled catalysts, several recent studies have pointed out the great interest of recording Mo¨ ssbauer spectra below 4.2 K up to 0.055 K (100,203,204); it allowed the identification at the surface of various support such as mesoporous silica or ZrO2, the formation of nanometric iron oxide clusters; this identification was not possible in classical low temperature studies conducted above 4.2 K, which concluded to the presence of larger particles. The analysis by Mo¨ ssbauer spectroscopy performed at lower temperature enabled to show that these larger particles were agglomerates of nanometric iron clusters and allowed to reach another level of resolution of ferric particles structures (204). Technological Advances and Theoretical Evaluation of Mo¨ssbauer Parameters In the preceding decade there have been no drastic changes in the design of Mo¨ ssbauer spectrometers. However, two new innovations have recently been 25 25 proposed that both reduce the acquisition time and improve the precision of the measurements (205,206). One of the problems of the conventional method of Mo¨ ssbauer spectroscopy is the occurrence of pulse overlapping at high-count rates. The single channel analyzer (SCA) that is generally used to register pulses of the signal almost always records interfering noise pulses. Such pulse overlapping disturbs the amplitude spectrum, reduces the signal-to-noise ratio, and limits the maximum count rate of the data acquisition system, thereby increasing the duration of an experiment. To solve this problem, new instrumentation has been proposed that includes a fast analog-to-digital converter (ADC) together with a modern digital signal processor, which significantly increases the count rate as a consequence of the elimination of pulse overlapping (205). The proposed instrumentation allows calculation of the exact amplitude of each pulse, which may be combined with the current velocity discrimination, therefore leading to the recording of a two-dimensional Mo¨ ssbauer spectrum (Fig. 11). The application of such two-dimensional data acquisition sys- tems allows one to choose the optimal energy window in the amplitude spectrum after the experiment and to measure g-quanta absorption and X-ray emission spectra simultaneously in the same transmission experiment. The new instruments also re- duce data acquisition time, because amplitude and Mo¨ ssbauer spectra are collected simultaneously. Even more interesting is a new spectrometer that has been built with a novel moving system that synchronizes the movement of the radioactive source with that of the detector (206). This system allows the accumulation of Mo¨ ssbauer spectra either in constant acceleration or constant velocity modes, therefore giving better signal-to-noise ratios and also reducing the time necessary for accumulation of a spectrum. Narrower line widths were also obtained with this equipment design. 80000 70000 60000 50000 40000 Counts Energy, Kev Velocity, mm/s 30000 20000 10000 0 3 7 11 15 19 -9 -7-5 -3 -11 3 5 7 9 FIG. 11. Two-dimensional Mo¨ ssbauer spectrum of an iron foil, measured with a proportional counter. Figure according to Maltsev et al. (205). 80000 70000 60000 50000 40000 Counts Energy, Kev Velocity, mm/s 30000 20000 10000 0 3 7 11 15 19 -9 -7-5 -3 -11 3 5 7 9 FIG. 11. A.4. Mo¨ssbauer Spectroscopy and High-Throughput Catalyst Characterizatio The implementation of combinatorial chemistry and automated methods for rapid synthesis, testing, and characterization of catalysts, has opened a wide range of new opportunities in catalysis. However, so far, Mo¨ ssbauer spectroscopy has not been introduced into this methodology. Two hurdles must be overcome for Mo¨ ssbauer spectroscopy to become important in high-throughput catalyst char- acterization: the system for recording spectra must be scaled down, and the data acquisition and exploitation systems must be adapted. The first condition could easily be fulfilled if progress made in spatial resolution were exploited. For example, an instrument has been developed that allows the measurement of Mo¨ ssbauer spectra of metallurgical slags with a spatial resolution of approximately 500 mm (209,210). By use of a 57Co source with high-specific activity, the g-ray beam can be collimated from the conventional diameter of ap- proximately 1 cm to a diameter of 500 mm. The sample is mounted in an epoxy resin, ground to a thin disk, and then mounted on an x–y stage directly behind a lead shield with a 500 mm hole. This technology should be easily applied to a sample set such as those generated with synthesis robots. With regard to the second condition, advances in Mo¨ ssbauer data analysis have progressed so far that it is currently possible to handle the fitting processes of a Mo¨ ssbauer spectrum in a completely automated manner by using genetic algo- rithms and fuzzy logic; both software and hardware have been developed (211,212). Nevertheless, it is not trivial in many cases to relate the extracted hyperfine pa- rameters to the underlying structure and properties of the material without a sig- nificant amount of experience, and many pitfalls have been documented (213). The development of such integrated approaches to spectral analysis will benefit greatly from the large data bases and reference banks developed with the Mo¨ ssbauer Effect Data Indexes covering 1958–1976 and the Mo¨ssbauer Effect Reference and Data Journal database (MERDJ: Vols. 1–19) covering 1976 to the present (http://www.unca.edu/medc/Journal.html). Technological Advances and Theoretical Evaluation of Mo¨ssbauer Parameters Two-dimensional Mo¨ ssbauer spectrum of an iron foil, measured with a proportional counter. Figure according to Maltsev et al. (205). 26 Furthermore, the theoretical evaluation of the Mo¨ ssbauer hyperfine parameters (isomer shift (IS), quadrupolar splitting (QS), and magnetic hyperfine field (H)) is now possible with first-principles electronic structure calculations based on density functional theory (DFT) (207,208). The quality of the data depends on the size of the atomic cluster chosen to represent the solid and the charge densities around the nucleus under investigation. A detailed understanding of the relationships between the electrical field gradient or magnetic field and their various causes can come only from comparisons with sufficiently complete electronic structure calculations. Such calculations for realistic local and longer-range environments are rare and are very much needed, especially for the characterization of surface species. A.5. Mo¨ssbauer Spectroscopy for Characterization of Membranes and Monoliths Another advantage of Mo¨ ssbauer spectroscopy that should benefit future re- search is that it can be used in a non-destructive way. This advantage should allow the investigation of membranes and monoliths without them being damaged, even 27 27 sample detector radioactive source FIG. 12. Basic equipment for measuring a Mo¨ ssbauer spectrum in diffusion mode. radioactive source FIG. 12. Basic equipment for measuring a Mo¨ ssbauer spectrum in diffusion mode. under catalytic reaction conditions. This application can be achieved by experi- ments in the scattering or diffusion mode (Fig. 12). Furthermore, local micro heterogeneities in a monolithic or membrane sample may be characterized by use of an imaging Mo¨ ssbauer spectroscopic device system (IMS), which can record 256 simultaneous Mo¨ ssbauer spectra along a sample. The experimental system, which has been applied for the characterization of metallic ribbons, is based on a linear position-sensitive proportional counter that facilitates measurements with a spatial resolution on a length of about 50 mm (214). Other improvements in spatial resolution in the future should allow more accurate investigation of conventional powdered samples such as catalysts. This will likely be made possible as a result of developments in synchrotron investigations of nuclear forward scattering (presented in the next section) (215). The development of a Mo¨ ssbauer electron microscope that would focus conversion electrons using conventional electron optics has also been mentioned (216). B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation Synchrotrons produce photons with energies in the range of nuclear Mo¨ ssbauer transitions and can, in principle, be used to excite these transitions. However, syn- chrotron radiation can be monochromatized to only about 1 meV with new mon- ochromators. Because the accessible nuclear levels are extremely narrow (between 109 and 108 eV), it is only about 103 of the incident photons that can excite the nuclear levels (excitation cross-section could be as much as 103 G0). This is far weaker than radiation that is non-resonantly scattered by the electronic processes in the solid arising from the scattering of the entire 1 meV width of the incident radiation. This limitation has been overcome by use of the property of synchrotron radi- ation that can be emitted from electron clouds (bunches) circulating in the storage rings. Bunches are a few centimeters in length, corresponding to 100 ps, and the time between two successive pulses is 2.8 ns to 2.8 ms (at the European synchrotron radiation facility). When a material absorbs X-rays, resonant and non-resonant scattering occurs. However, because of the lifetime of the nuclear excited state, the resonant scattering is delayed with respect to the non-resonant scattering (the nat- ural lifetime of the first nuclear excited state for 57Fe is 141 ns). By suppressing data acquisition during the short non-resonant pulses, it is possible to measure only the nuclear forward scattered intensity. A typical NFS experiment may be described as follows (218): the pulsed syn- chrotron radiation impinges on the sample as bunches, as they are produced in the storage ring (Fig. 13a). The typical pulse duration is 100 ps, and the period of pulse repetition is of the order of 200 ns, corresponding to the electron bunch distance. A fast detector records the transmitted and forward scattered radiation, and the time evolution of this intensity is used to discriminate the resonant from the non-resonant intensity (Fig. 13b). The black spikes corresponding to the exciting pulses, which define the ‘‘time zero’’ of the nuclear excitation are suppressed by a gating acquisition system in the course of the experiment. The signal between these spikes represents the nuclear forward scattered intensity that is recorded and which is delayed with respect to the exciting pulse as a consequence of the lifetime of the nuclear excited states. B. FUTURE DEVELOPMENTS AND APPLICATIONS The main future developments of Mo¨ ssbauer spectroscopy will undoubtedly be based on the use of synchrotron radiation as a source for Mo¨ ssbauer experiments, taking advantage of its high flux, brilliance, pulse structure, and energy tunability. In the field of catalysis, new experimental opportunities include the use of parentless Mo¨ ssbauer isotopes and time resolution. Catalyst characterizations should be pos- sible over a wide range of time scales, including investigations of activation and deactivation under working conditions on time scales of tens to hundreds of sec- onds, investigations of transport processes such as diffusion on time scales from micro- to picoseconds, and investigations of processes such as relaxation and elec- tron transfers on the intrinsic lifetime scale of the Mo¨ ssbauer nuclei. All these techniques are referred to as nuclear resonance scattering (NRS) of synchrotron radiation; they include a wide spectrum of experimental techniques, such as nuclear forward scattering (NFS), nuclear inelastic scattering (NIS), nuclear 28 28 Bragg diffraction (NBD), nuclear small-angle scattering (NSAS), nuclear reflect- ometry (NR), nuclear quasi-elastic scattering (NQES), and quasi-elastic and inelastic scattering with nuclear resonance energy analysis (217). In this review, we focus only on NFS and NIS, which are the two main techniques, and we attempt to describe the advantages of using them for the characterization of solid catalysts. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation Figure 14 shows three 57Fe case studies of the time behavior of the photons re- emitted in the forward direction and a comparison with the typical spectra obtained in Mo¨ ssbauer spectroscopy. Figure 14a corresponds to the case for which there is no hyperfine interaction. The nuclear levels are not split, and only one transition between ground and excited state is possible. In that case, the Mo¨ ssbauer spectrum shows a single-absorption line and contains only g-quanta of equal energy. In the presence of an electric field gradient (Fig. 14b), the splitting of the excited state is 29 29 (a) (b) 200 ns sample detector 200 ns FIG. 13. Basic equipment for an NFS experiment (a) and time structure of the detected radiation (b). (a) detector sample (b) FIG. 13. Basic equipment for an NFS experiment (a) and time structure of the detected radiation (b). 13. Basic equipment for an NFS experiment (a) and time structure of the detected radiation (b). observed, and the Mo¨ ssbauer spectrum has two absorption lines (a doublet). Accordingly, the NFS spectrum shows characteristic modulations caused by the interference of the two forward re-emitted radiations containing g-quanta with two different energies (quantum beats). The equal probability of the two transitions is directly evidenced by the symmetry of the doublet and is evaluated on the basis of the contrast between the maximum and the minimum of the beats. This contrast vanishes with increasing inequality of the two probabilities. When a magnetic hyperfine field is present (Fig. 14c), both ground and excited states split into sub-levels, which leads to the observation of six absorption lines corresponding to the six transitions allowed by selection rules (sextet) in the Mo¨ ssbauer spectrum. In the NFS spectrum, these transitions give rise to interfer- ences and the superposition of quantum beats of different frequencies. The latter superposition leads to complex patterns that are difficult to interpret. In that case, the intrinsic polarization of the incident synchrotron radiation (the radiation is linearly polarized in the plane of the synchrotron ring) may advantageously be used to reduce the number of free parameters (i.e., to reduce the number of hyperfine radiation components and consequently reduce the number of quantum beats). The polarization of the synchrotron radiation mentioned above is an important pro- perty of the source, which is different from the source used for conventional Mo¨ ssbauer experiments, because radioactive sources emit non-polarized radiation. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation The mechanism producing synchrotron radiation guarantees almost complete linear 30 velocity (mm/s) time (ns) FIG. 14. Examples of experimental NFS time patterns with corresponding schematic conventional Mo¨ ssbauer spectra: (a) stainless steel (221), (b) iron porphyrin complex [Fe(CH3COO)(TPpivP] (spec- trum taken at 30 K in a 4 T field) (222), and (c) a-Fe (221). FIG. 14. Examples of experimental NFS time patterns with corresponding schematic conventional Mo¨ ssbauer spectra: (a) stainless steel (221), (b) iron porphyrin complex [Fe(CH3COO)(TPpivP] (spec- trum taken at 30 K in a 4 T field) (222), and (c) a-Fe (221). polarization. Furthermore, optical elements can convert synchrotron radiation into circularly polarized radiation with high efficiency. When experimental NFS spectra of samples with finite thickness are recorded, the time decay also shows modulations attributed to coherent multiple scattering oc- curring in solids (dynamical beats). These superimposed modulations have different (non-periodic) time modulation and are taken into account when the spectra are fitted. At this point, if we consider the Mo¨ ssbauer parameters calculated in conventional Mo¨ ssbauer spectroscopy, the isomer shift (IS) is not accessible from NFS spectra, because the latter only shows interference between radiation fields corresponding to different radiations. Isomer shifts will have to be measured relative to a second reference absorber that must be placed behind or in front of the sample along the g-ray beam. When the nuclei are subjected to an electrical quadrupole interaction, the NFS pattern shows quantum beats with a single frequency corresponding to the energy difference between the sub-levels of the exited state (DEQ), which is equal to the 31 quadrupolar splitting (D). The time dependence of the intensity of the delayed radiation can be expressed approximately as follows: quadrupolar splitting (D). The time dependence of the intensity of the delayed radiation can be expressed approximately as follows: IðtÞ exp  t t   cos2 DEQt 2_   , ð20Þ ð20Þ where t is the natural lifetime of the nuclear transition (t ¼ 141 ns for 57Fe). This expression contains two time-dependent terms. The first, exp ðt=tÞ; corresponds to the nuclear decay and the second, cos2ðDEQt=2_Þ; to the quantum beat pattern; DEQ, which is equal to h2p/F (F is the period of the quantum beats), can be estimated using the following approximate relationship: DEQ½mm=sF½ns 87. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation ð21Þ ð21Þ For magnetic compounds, the determination of the internal magnetic field re- quires the decomposition of the complex spectra of multi-quantum beats. Computer codes now allow one to attempt to perform such decomposition and to interpret the spectra. The determination of the complex quantum beat structure requires spectra recorded over long periods for high accuracy. In spite of that, analysis of complex spectra remains very often difficult and NFS does not compete against conventional Mo¨ ssbauer spectroscopy. However, it offers a major advantage over the latter technique by being a time-resolved technique, with accumulation times in the range of minutes or less. This advantage is gained without loss of the advantages of the Mo¨ ssbauer technique, such as site selectivity, quantitative analysis, and applica- bility to catalysts under extreme working conditions (high pressure and high tem- perature). The NFS technique benefits from the outstanding properties of synchrotron radiation, such as the high collimation of the photon beam that pre- vents Mo¨ ssbauer line asymmetry resulting from the finite dimensions of the source. Furthermore, it can easily be combined with several complementary spectroscopic techniques. As in conventional Mo¨ ssbauer spectroscopy, 57Fe is the most widely used isotope in nuclear resonant scattering of synchrotron radiation. Experiments with other isotopes, such as 169Tm, 119Sn, 83Kr, 181Ta, 151Eu, 161Dy, and 149Sm have been reviewed recently (219). Other isotopes with very short half-life time sources should also be usable. The applicability of such isotopes represents another strong advan- tage that can be exploited in the future for catalyst characterization. The inves- tigation of isotopes such as 197Au (Eg ¼ 77.34 keV; source, 197Pt, half-life, 18 h); 61Ni (Eg ¼ 67.40 keV; source, 61Co, half-life, 99 min); 186W (Eg ¼ 122.5 keV; source, 186Re, half-life, 90 h); 99Ru (Eg ¼ 89.36 keV; source, 99Rh, half-life, 16 days); or 125Te (Eg ¼ 35.46 keV; source, 125I, half-life, 60 days) may be expected. g As was stated in Section II.A, the energy resolution of the radioactive sources used in conventional Mo¨ ssbauer spectroscopy is typically 109 eV. This resolution is determined by the natural line width and the maximum energy range obtained by Doppler-shifting techniques. In the case of synchrotron radiation, the energy res- olution, which is related to the time period following the excitation of the isotope, is superior to that in conventional Mo¨ ssbauer spectroscopy. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation This period can be as short as 2.8 ms, which leads to an energy resolution of about 1010 eV. However, the 32 32 recordable period is determined by the bunch separation, which depends on the operationing mode of the synchrotron. The energy resolution, which depends on this period, will be superior to that of the radioactive source only if the bunch separation is larger than the lifetime of the excited nuclear state. The reported examples of experimental characterization of solids mainly concern biological inorganic solids (220) or geological samples (221). In the latter case, the measure- ments were made at pressures up to 1.5 Mbar. B.2. Nuclear Inelastic Scattering (NIS) of Synchrotron Radiation The one phonon term is given by where f is the Lamb–Mo¨ ssbauer factor, d(E ) a Dirac function describing the elas- tic part of absorption (zero phonon term) and Snp(E ) a series of terms from which the npth term represents the inelastic absorption accompanied by creation - (annihilation) of np phonons. The one phonon term is given by S1ðEÞ ¼ ERgð E j jÞ Eð1  ebEÞ ð23Þ ð23Þ and the subsequent terms by the following when the harmonic approximation is valid: and the subsequent terms by the following when the harmonic approximation is valid: S1ðEÞ ¼ 1 np Z þ1 1 S1ðE0ÞSnp1ðE  E0Þ dE0. ð24Þ ð24Þ In these expressions, b ¼ ðkTÞ1; ER ¼ _2k2=2Mis the recoil energy of a free nu- cleus; k the wave vector of the g-ray quantum and M the mass of the nucleus. The function g(E) is the normalized density of phonon states: gðEÞ ¼ V 0 1 ð2pÞ3 X j Z d~qd E  _ojð~qÞ ; ð25Þ ð25Þ where V 0 is the volume of the unit cell, and the index j enumerates the branches of the dispersion relation ½_ojð~qÞ with ~q being the phonon momentum. The integral is taken within the first Brillouin zone. This technique, besides allowing determination of the Lamb–Mo¨ ssbauer factor, provides direct access to the density of phonon states for the probe isotope in a solid. It thus provides information about lattice dynamics that is excluded by the limitations of Mo¨ ssbauer spectroscopy. This technique could be valuable in inves- tigations of adsorption with the adsorbing element as the probe and showing the modifications brought about by the adsorbate on the dynamic properties of the probe. The frequency distribution of vibrations can be different in various directions, although Mo¨ ssbauer spectroscopy did not show it (225). Few examples are available to illustrate the use of the technique. Furthermore, most of these examples have been chosen to show basic applications of the technique. Temperature and pressure de- pendences of nuclear inelastic absorption in iron metal (a-Fe) have been investigated (226), as has the anisotropy dependence in a 57FeBO3 single crystal (225). NIS has also been used to investigate complexes in solution (57Fe[2,20-(bipyridyl)3]Cl2 in water and in glycerol) (227). A recent publication reports the characterization of perovskite-related oxides ((Sr,Ca)(Fe,Co)O3d and (Ba,Ca)(FeCo)O3d) that may find applications in fuel cells or oxygen-permeable membranes (228). B.2. Nuclear Inelastic Scattering (NIS) of Synchrotron Radiation This technique comprises measurement of the energy distribution of phonons excited by the inelastic absorption of X-rays at the resonance. The Mo¨ ssbauer effect comprises the absorption and re-emission of X-rays that occur in an elastic manner. This excitation can also be achieved if the radiation energy is not exactly equal to the resonance energy E0, but instead is equal to E07DE when a phonon of energy DE is annihilated providing the lacking energy or a phonon of energy DE is created, taking up the excess energy. Annihilation or emission of several phonons can be involved in the process. When resonance absorption takes place with detuned syn- chrotron radiation, the delayed re-emitted g-quanta or conversion electrons are emitted into a solid angle of 4p and can be counted by a detector placed at a position out of the incoming beam (Fig. 15). The normalized probability of nuclear inelastic absorption W(E) can be decom- posed in terms of a multi-phonon expansion (222,223): WðEÞ ¼ f dðEÞ þ X 1 np¼1 SnpðEÞ ! , ð22Þ ð22Þ Resonant sample Ionization chamber Detector #1 Detector #2 High-heat-load monochromator High-resolution monochromator Storage ring Undulator e− Detector #1 Detector #2 FIG. 15. Basic equipment for measuring a nuclear inelastic scattering spectrum. Detector 1 measures the intensity of the incoherent nuclear forward scattering, which proceeds both elastically and inelas- tically; detector 2 measures only the intensity of the coherent nuclear forward scattering, which proceeds elastically. Figure according to Ru¨ ffer and Chumakov (224). Resonant sample Detector #2 High-heat-load monochromator FIG. 15. Basic equipment for measuring a nuclear inelastic scattering spectrum. Detector 1 measures the intensity of the incoherent nuclear forward scattering, which proceeds both elastically and inelas- tically; detector 2 measures only the intensity of the coherent nuclear forward scattering, which proceeds elastically. Figure according to Ru¨ ffer and Chumakov (224). 33 where f is the Lamb–Mo¨ ssbauer factor, d(E ) a Dirac function describing the elas- tic part of absorption (zero phonon term) and Snp(E ) a series of terms from which the npth term represents the inelastic absorption accompanied by creation - (annihilation) of np phonons. B.2. Nuclear Inelastic Scattering (NIS) of Synchrotron Radiation In the latter case, a soft phonon peak in the NIS spectra was found to be correlated with oxygen ordering in the structure. Changes were also observed upon absorption of CO2 that have unfortunately not been interpreted because of the complexity of the spectra. Nuclear resonant forward scattering and inelastic scattering of synchrotron ra- diation constitute a promising field for development of catalyst characterization in the future. For the characterization of solid catalysts, NFS has the advantage over 34 Mo¨ ssbauer spectroscopy of being a time-resolved spectroscopic technique. This characteristic opens new areas of research, particularly because it also combines in the same time all the advantages of Mo¨ ssbauer spectroscopy in terms of sensitivity (it is even more sensitive for hyperfine parameter effects than conventional Mo¨ ssbauer spectroscopy) and in terms of applicability under catalytic reaction conditions. It still appears, however, for complex systems, such as those often encountered in catalysis, difficult to analyze the time-domain spectra relative to energy-domain spectra, and for the time being NFS should be considered to be complementary to conventional Mo¨ ssbauer spectroscopy and not a replacement of it. In the future, NIS may also become an extremely efficient tool for characterizing adsorption, although the energy resolution for recording molecular vibrations, which is at present time about 5 cm1, has to be improved. V. Conclusions This review includes a short summary of the principles of Mo¨ ssbauer spectroscopy and of recent applications of the technique in heterogeneous catalysis. The fre- quency of applications of the technique in this field has remained nearly constant, but the applications to investigations of catalysts under controlled conditions and under catalytic reaction conditions, with characterizations of activation, reaction, and deactivation processes has increased significantly in the preceding few years. Although Mo¨ ssbauer spectroscopy is characterized by complexity in the experi- mental technique as well as in the interpretation and analysis of spectra, if offers for samples including those incorporating iron, cobalt, gold, and other elements of catalytic interest the capability for (i) determination of accurate oxidation states of the metals, (ii) resolution of microcrystalline or poorly crystalline phases, (iii) quantification of coordination and local symmetry site populations, (iv) character- ization of various electronic, magnetic, and (v) structural phase transitions; and quantification of chemical, structural, and magnetic disorder. A section of this review is devoted to the potential areas of likely future devel- opments in Mo¨ ssbauer spectroscopy in the field of catalysis. In view of the con- siderable value of spectroscopic measurements made of catalysts while they are working, Mo¨ ssbauer spectroscopy, which is relatively well adaptable to such con- ditions, is expected to gain interest in the catalysis community. Cells have already been adapted to conditions of high pressure and high temperature. Furthermore, these cells provide flexibility, allowing the coupling of Mo¨ ssbauer spectroscopy with other spectroscopic techniques. New experimental setups continue to be developed and should improve the precision of the measurements and reduce the acquisition time (which still remains a handicap for the technique). Synchrotron Mo¨ ssbauer spectroscopy in particular deserves more attention because it can become a useful for investigating catalytic reactions on many types of catalysts. The use of syn- chrotron radiation for Mo¨ ssbauer spectroscopy was proposed as early as 1974, and it is now becoming accessible to non-experts. NFS of synchrotron radiation allows experiments that are in many aspects very similar to conventional Mo¨ ssbauer spectroscopy, and they offer the benefit of combining all the advantages of 35 conventional Mo¨ ssbauer spectroscopy, with the time-resolution of NFS at a time scale interesting for catalyst characterizations. This latter advantage is conspicu- ously missing in conventional Mo¨ ssbauer spectroscopy when compared with other spectroscopic techniques for characterizing catalysts under working conditions. V. Conclusions In the future, NIS experiments may bring new insights in catalyst characterization, and these techniques should also broaden the field of application of Mo¨ ssbauer spectroscopy to allow the investigation of important isotopes with very short half- lifetime sources, such as 61Ni and 99Ru. 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Impact of Small-Scale Irrigation Schemes on Smallholders Household Income and Food Security in Ethiopia: Review
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1. INTRODUCTION In Ethiopia, agriculture remains among the most important economic sectors. It produces about one-third of GDP and employs 70 percent of the workforce, accounting for 80 percent of the country’s merchandise exports (EATA, 2014). Agriculture is the leading sector of Ethiopian economy as well the overall economic growth of the country largely depends on the agricultural sector. However, Ethiopian agriculture remains characterized by small-scale subsistence production systems where crop and livestock yields are very low(Desta and Almaz, 2015).Although agricultural productivity is very much affected due to variability of rainfall and drought(Sensen, 2013). Even if agriculture status is the backbone of the Ethiopian economy, agricultural practice has been traditionally dominated for centuries by small-scale farmers and its performance has long been adversely affected by shortage of rain and water that left many to sustain their lives on famine relief support(Abebe et al., 2011, Temesgen et al., 2018). From the total production of Ethiopia’s, about 97% food crops are produced by rain-fed agriculture, whereas only 3% is from irrigated agriculture (FAO, 2015). Because of high dependency on rain-fed agriculture, other topographic and low adaptive capacity along with other related factors, Ethiopia ranks the ninth most susceptible country in the world to natural disasters and weather related shocks (Tongul and Hobson, 2013). On the other hand, Ethiopia has abundant water resources, its agricultural system does not yet fully benefit from the technologies of water management and irrigation(Seleshi et al., 2010). According toNahusenay and Madhu( 2015),one of the aspects given attention by the government is to improve food and agricultural production through the strengthening small scale irrigation. The net farm income is the most watched indicator of farm sector well-being, as it captures and reflects the entirety of economic activity across the range of production processes, input expenses, and marketing conditions that have persisted during a specific time period. There are different mechanisms by which irrigated agriculture can improve household income and food security, through: increasing farm production and productivity that helps very poor households meet the basic needs, protecting against risks of crop loss due to erratic, insufficient rainwater supplies, promoting greater use of yield enhancing farm inputs and creation of additional employment(Agidew, 2017). Agricultural intensification is presumed to be a necessary pre-condition for the development of the agricultural sector in Ethiopia. Impact of Small-Scale Irrigation Schemes on Smallholders Household Income and Food Security in Ethiopia: Review Abdulsemed Abanega Abdurahman1 Ahmed Mohammed Abachebsa2 1.Department of Agricultural Economics and Agribusiness Management, Jimma University P.O. Box. 307, Jimma, Ethiopia 2.Department of Natural Resource Management; Jimma University; P.O. Box. 307, Jimma, Ethiopia Abdulsemed Abanega Abdurahman1 Ahmed Mohammed Abachebsa2 Department of Agricultural Economics and Agribusiness Management, Jimma University P.O. Box. 307, Jimma, Ethiopia 2.Department of Natural Resource Management; Jimma University; P.O. Box. 307, Jimma, Ethiopia Abstract In Ethiopia, agriculture remains among the most important economic sectors and has been traditionally dominated for centuries by small-scale farmers. Its performance has long been adversely affected by shortage of rain and water to sustain their lives on famine relief support. From the total production of Ethiopia’s, about 97% food crops are produced by rain-fed agriculture, whereas only 3% is from irrigated agriculture. On the other hand, Ethiopia has abundant water resources, but not yet fully benefited from technologies of water management and irrigation. The aim of this paper is to identify the impact of small-scale irrigation on household income and food security reviewing different literature. Different studies indicated that access to irrigation water can enable farmers to adopt new technologies and intensify cultivation, leading to increased productivity, production, and greater returns from farming. Access to irrigation enables farmer to grow crops more than once a year increasing production, income and improve their food security status. This review concludes small-scale irrigation is one of the possible solutions to increase smallholder household income and food needs in the country. But there are different challenges hindering the irrigation development by smallholder households like lack of market at the time of harvest, distance from water sources, knowledge on the efficient use of irrigation, and linkage between micro finance institutions and smallholder horticultural crop producers are some constraints. These constraints have to be solved to increase participation of stallholder household in irrigation technology to improve their living standards through this sector. Keywords: Agriculture, Ethiopia, Small scale irrigation, Water resource DOI: 10.7176/JBAH/11-1-05 Publication date: January 31st 2021 www.iiste.org www.iiste.org Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11, No.1, 2021 Impact of Small-Scale Irrigation Schemes on Smallholders Household Income and Food Security in Ethiopia: Review Abdulsemed Abanega Abdurahman1 Ahmed Mohammed Abachebsa2 1.Department of Agricultural Economics and Agribusiness Management, Jimma University P.O. Box. 307, Jimma, Ethiopia 2.Department of Natural Resource Management; Jimma University; P.O. Box. 307, Jimma, Ethiopia 1. INTRODUCTION To this end, various government and non-governmental organizations (NGOs), among others, initiated small-scale irrigation schemes throughout the country(Kinfe et al., 2012).The importance of small-scale irrigation for improving farmers’ income and reducing rural poverty has been emphasized in various literatures and small-scale irrigation schemes play a huge role to increase income of the society(Ayana, 2016). The current Ethiopian government has undertaken various activities to expand irrigation in the country. The country’s 40 Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11, No.1, 2021 Agricultural Development Led Industrialization strategy considers irrigation development as a key strategy for sustainable agricultural development. Thus, irrigation development, particularly small-scale irrigation is planned to be accelerated(MoFED, 2010). The local institutions and the rural infrastructure services can improve the awareness of people regarding irrigation and he expansion of small-scale irrigation can be an important strategy to increase income, build-up assets, increase total expenditure, and spend more income on improved agricultural technologies(Woldegebrial et al., 2015). Irrigation in Ethiopia contributes to increase farmers‟ income, household resilience and buffering livelihoods against shocks and stresses by producing higher value crops for sale at market and to harvest more than once per year. In turn, this provided them to build up their assets, buy more food and non-food household items, educate their children, and reinvest in further increasing their production by buying farm inputs or livestock. However, the benefits are very unevenly distributed among households(Eshetu et al., 2010). Therefore, the aim of this paper is to contribute literature on impact of small-scale irrigation schemes on smallholder farmers’ incomes and food security and also challenges and opportunity for smallholder farmers to use irrigation in Ethiopia. 2. Basic Concept and Definitions In general terms, irrigation can be defined as the artificial supply of water to supplement natural precipitation or substitute for it for the purpose of agricultural production(Mohamed, 2006). According to Indhumathi et al., (2017) Water is most powerful natural resource as it has great influences in health and wealth of the people and production of food. Thus, water has great role in survival and comfortable life of living things. We are depending on the water resources for development of agriculture. Besides various other uses of water, the largest use of water in the world is for irrigating land. The same report by MoWE( 2013), indicated that water is a basic need for human beings and animals. It is essential for their metabolic processes. It is used to build healthy workforce, ensuring food security, provision of clean energy for agriculture and industry. Food security is defined in different ways by different organizations around the world. For example, in 1996, the definition of food security was agreed and accepted at the world food summit in Rome and it was defined food security as a physical and economic access by all people at all times to sufficient, safe and nutritious food to meet their dietary needs and food preference for an active and healthy life(Todaro and Smith, 2011). According to FAO(2011), food security contains different features, such as food availability, accessibility, utilization and stability. Food availability refers to the existence of food from own production or on the markets. It is a combination of domestic foods production, marketable imports and food aid. Food access is guaranteed when households and all individuals within them have adequate resources, through access options. Food utilization refers to nutritional and safety aspects of food security to meet adequate diet, clean water, sanitation and health care to search a state of nutritional wellbeing. Food stability complements the previous factors by stressing that food must be available, accessible, affordable and properly utilized on a continuous basis. 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia Access to such small scale irrigation can significantly improve income level and food security status of beneficiary households(Eshetu and Young-Bohk, 2017). According to Lipton et al. (2003), irrigation increases productivity by reducing crop losses that were due to limited water supply, enabling cultivation during the lean season, and making crop production possible on lands where rain fed agriculture is infeasible. The other study shows that farm productivity is positively correlated with the availability of farm power coupled with efficient farm implements and their sustainable utilization. On the other hand the report of (MoA, 2015) shows that Agricultural mechanization not only enables efficient utilization of various inputs such as seeds, fertilizers, plant protection chemicals and water for irrigation but also it helps in poverty alleviation by making farming an attractive enterprise. In Ethiopia, nearly all small farmers are still using traditional hand tools and oxen plow, which greatly is restricting crop productivity and production. Irrigated agriculture plays a central and dynamic role in the improvement of rural livelihoods, but is often subject to criticisms of inefficiency in water use, high capital and recurrent costs and lack of sustainability associated with inequity in the distribution of both land and water(Reinders, 2011). Although irrigation leads to increased agricultural income and production diversity, and this leads to improved diets and access to irrigation has the potential to improve both household income and the diversity of crops that farmers produce. Increasing household income, in turn, leads to higher dietary diversity, while increases in production diversity do not contribute to increases in dietary diversity over and above the effect of income (Passarelli et al., 2018). y y ( ) According to Garbero and Songsermsawas (2018), Small-scale farmers in the developing world face multiple challenges that limit their opportunities to achieve higher agricultural productivity and improve their living conditions. One promising channel to help farmers attain more desirable agricultural outcomes is to increase their access to water, an important input for agricultural activities. Several studies have noted the positive and significant benefits of irrigation infrastructure on agriculture. Although, small-scale irrigation development has a positive impact on food security status of rural households and a much higher proportion of those who are poor are non- irrigating rather than irrigating households. Thus, the food insecurity occurrence in non-irrigating households is greater than in irrigating households1(Muez, 2014). 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia The main income sources of Ethiopian rural households were cropping, livestock and off-farm activities and the irrigating households have significantly larger mean annual income than non-irrigating households. Although the poverty analysis indicated that a much higher proportion of poor are non-irrigating than irrigating households, different studies revealed that there is positive significant impact on the income of participant household income as a result of participation in small-scale irrigation(Getaneh, 2011;Temesgen et al., 2018). g ) According to Awulachew (2010), with high potential, if it is successfully operated, irrigation in Ethiopia could play a significant role in the agricultural transformation of the country, contributing up to ETB 140 billion to the economy. Furthermore, another study done by Tsegazeab (2015) micro irrigation is one of the most useful irrigation systems designed to increase production and productivity; and reduces risk, related with rainfall variability and increases income of rural farm households and not surprisingly, planners, researchers, development practitioners, and donors emphasize the importance of micro irrigation in their policy recommendations and actual measures. According to Aam (2013), small-scale irrigation has immense potential to improve agricultural productivity and incomes of poor rural households. Irrigation in fact is nothing but is a continuous and reliable water supply to different crops in accordance with their water requirement. According to Seleshi et al.(2005), agricultural production in Ethiopia has affected by increasing population, deforestation and land distribution. This reflected in a decrease in household production and grazing land. Hence, it has become a common phenomenon to ask for emergency food assistance for acutely food insecure people in Ethiopia. The adoption of new technology (e.g. irrigation) is the major powerful for agricultural growth and poverty reduction(Norton et al., 2010). This indicates that investment in irrigation can increase farmer’s independence on rainfall, it increases irrigated farmland, it generates employment, it promotes farmers to produce two or three times in a year and use more of chemical inputs. Small scale irrigation in developing countries was considered as a means of increasing production, reducing the dependence on rainfall and provides jobs to the poor(Chazovachii, 2012). Although small-scale irrigation is believed in helping to address irregular rainfall pattern problem there by reducing rural poverty, food insecurity as well as improving the overall contribution of agriculture to the national economy(Tizita, 2017), it increases land productivity, crop yields, adoption of mineral fertilizers and enables to diversify into non-conventional and market-oriented products (Eshetu et al., 2010). 3. History of Irrigation development and participation condition in Ethiopia Ethiopia has a long history of traditional irrigation systems. Simple river diversion still is the dominant irrigation system in Ethiopia. According to Seleshi et al. (2010), estimates the irrigation potential of Ethiopia may be as large as 4.3 million hectares. Traditional irrigation schemes cover more than 138,000 hectares whereas modern small-scale irrigation covers about 48,000 hectares. The total current irrigation covers only about 6% of the estimated potential land area. According to Gebremedhin and Asfaw( 2015), irrigation was practiced during ancient times in Ethiopia even if its exact date of emergence is unknown. Ancient use of irrigation water was through use of surface irrigation methods and spate irrigation types. Modern irrigation was started at the Awash River basin with bilateral cooperation of Ethiopia and Dutch company. Ethiopia is a rich country in water resource and most of the time it is termed as a water tower of east Africa because of its abundant water resource availability(Adugna , 2014). Although Ethiopia has a huge potential of water resource which accounts 122 billion meter cube annual surface runoff and 2.9 billion meter cube groundwater, it is characterized by uneven spatial and temporal distributions(Tesfa, 2015). But, Ethiopia is using a very little of its abundant water resource potential for irrigated agriculture(ATA, 2016). The study taken by Gebremedhin (2015), indicated that the policies and strategies of Ethiopia strongly supports the irrigation developments especially the small scale irrigation through the Water Sector Development Programs (WSDP) and Ethiopian Irrigation Development Plan (IDP). This irrigation development is mainly expressed in the development of small-scale irrigation schemes by governments, donors and NGOs. Irrigation is believed as a key for food security and poverty reduction in Ethiopia. As a result, developments in the Ethiopian irrigation system have shown great advancements so as to assure Ethiopian livelihoods especially in the rural areas. However, the contribution of irrigation to the national economy as compared to its potentials is insignificant(Woldegebrial et al., 2017). 41 Journal of Biology, Agriculture and Healthcare www.iiste.org iste.org 5. Challenges and Opportunities of Irrigation Development in Ethiopia There are different challenges that hinder the irrigation development by sma 5. Challenges and Opportunities of Irrigation Development in Ethiopia There are different challenges that hinder the irrigation development by small holder households in Ethiopia. According to Temesgen (2017), out of the challenges pointed out by farmers, lack of market at the time of harvest holds the first rank, distance of farm land from irrigation water source, lack of knowledge on the efficient use of irrigation by the farmers, lack of market and linkage, lack of awareness on the sustainable use of water and lack of linkage between micro finance institutions and smallholder horticultural crop producers are some constraints. Although the major small-scale irrigation problems identified by different studies throughout the country indicated that, financial constraints especially for the purchase of motor pumps, shortage of agricultural inputs specially improved seed and pesticides, high cost of irrigation, shortage of water pump technologies, spare parts and gabions, technical problems such as maintenance of motor pumps, insufficient market information and market networks, shortage of ponds and diversion, infrastructure specially road and storage, theft of fruits, diseases and pests such as rust, root ruts, ball worm, blights, powdery mildew, gummosis and water borne diseases are among the problems (Nahusenay and Madhu, 2015). Another study taken by Gebremedhin and Asfaw (2015), identified the challenges for Ethiopian irrigation development are: Inadequate awareness of irrigation water management as in irrigation scheduling techniques, water saving irrigation technologies, water measurement techniques, operation and maintenance of irrigation facilities, inadequate knowledge on improved and diversified irrigation agronomic practices, shortage of basic technical knowledge on irrigation pumps, drip irrigation system, sprinkler irrigations, surface and spate irrigation methods, inadequate baseline data and information on the development of water resources, lack of experience in design, construction and supervision of quality irrigation projects, low productivity of existing irrigation schemes, inadequate community involvement and consultation in scheme planning, construction and implementation of irrigation development and poor economic background of users for irrigation, infrastructure development are some challenges. Another study taken by Tizita(2017) shows that lack of suitable land that can be used for irrigation (irrigable farm land) was the major constraints for irrigation non-user households. On the other hand, there are many opportunities that enhance irrigation development in Ethiopia. From these opportunities the policies and strategies of Ethiopia strongly supports the irrigation developments(Tadesse and Baihilu, 2017). 5. Challenges and Opportunities of Irrigation Development in Ethiopia There are different challenges that hinder the irrigation development by sma Another study taken by Amede(2014), identified the following future opportunities: high water potential, high commitment of the Ethiopia government, donors and NGOs to support irrigation management and development activity. There is also availability of favorable climate condition and ground water in Ethiopia. It can be used as an opportunity to produce more than once a year if the farmers could be able to access the ground water by means of using different types small-scale irrigation (Temesgen, 2017). 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia It also improves farm households‟ diet, incomes, health and food security(Torell and Ward, 2010). Hence irrigated agriculture is accepted as essential in increasing land productivity, enhancing food security, earning higher and more stable incomes and increasing for multiple cropping and crop diversification(Smith, 2004). Access to irrigation positively and significantly affects household food security and irrigation user households were more likely to be food secure as compared to irrigation non-users, holding other variables constant. This implies that irrigation enables households to grow food crops more than once a year, hence increased production, income and food availability 42 Journal of Biology, Agriculture and Healthcare Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11, No.1, 2021 www.iiste.org iste.org of the household(Tizita., 2017). Although irrigation expansion has been regarded as a promising approach to ensure food and livelihood security in the face of climate change and population growth while only 4% of cultivated area in sub-Saharan Africa is currently irrigated, the potential for the expansion of irrigation in the region is large and investments are accelerating(Giordano et al., 2012). Previous investments in irrigation by donors and governments have focused on developing large scale irrigation schemes, but evidence suggests that the greatest gains in terms of profitability and sustainability will come from investments in small scale irrigation (Xie et al., 2014). In general irrigation in Ethiopia is considered as a basic strategy to alleviate poverty and reduce food security. It is useful to transform the rain-fed agricultural system which depends on rainfall into the combined rain-fed and irrigation agricultural system. This is believed to be the most prominent way of sustainable development in the country. However, the development of irrigation practices in Ethiopia has to be investigated so as to seriously know the history of irrigation emergence and its subsequent developments(Gebremedhin, 2015). 6. Conclusions Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11, No.1, 2021 and linkage, lack of awareness on the sustainable use of water and lack of linkage between micro finance institutions, smallholder horticultural crop producers, shortage of agricultural inputs specially improved seed and pesticides, high cost of irrigation, shortage of water pump technologies and infrastructure specially road and storage are some constraints . On the other hand, there are different opportunities that increase irrigation development in Ethiopia. i.e. High-water potential, effective utilization of small-scale irrigation scheme infrastructure through diversification of uses to meet various needs for water such as domestic, irrigation, the interest of development agencies to give education and training, policies and strategies of Ethiopia strongly supports the irrigation developments. and linkage, lack of awareness on the sustainable use of water and lack of linkage between micro finance institutions, smallholder horticultural crop producers, shortage of agricultural inputs specially improved seed and pesticides, high cost of irrigation, shortage of water pump technologies and infrastructure specially road and storage are some constraints . On the other hand, there are different opportunities that increase irrigation development in Ethiopia. i.e. High-water potential, effective utilization of small-scale irrigation scheme infrastructure through diversification of uses to meet various needs for water such as domestic, irrigation, the interest of development agencies to give education and training, policies and strategies of Ethiopia strongly supports the irrigation developments. In general irrigation have a great role in economic development and in increments of food security and Ethiopia has high potential of water resource that can help irrigation development from African countries but, still she didn’t use this potential efficiently and the various actors of this sectors have to give attention to utilize this resource efficiently. 6. Conclusions Irrigation plays an important role in the performance of agriculture and improvement of rural livelihoods which increases income from agriculture sector and reduce the risk of crop failure as a result of erratic rainfall. It increases farm production and productivity that helps poor households to meet the basic needs, protecting against risks of crop loss due to erratic, unreliable or insufficient rainwater supplies, promote greater use of yield enhancing farm inputs and create additional employment. Also, small-scale irrigation development has a positive impact on food security status of rural households and a much higher proportion of those who are poor are non-irrigating rather than irrigating households. Ethiopia is a rich country in water resource, but using a very little of its abundant water resource potential for irrigated agriculture. While investment in irrigation can increase farmer’s independence on rainfall, it increases irrigated farmland, generates employment, promotes farmers to produce two or three times in a year and use more of chemical inputs. There are different challenges that hinder the irrigation development by small holder households in Ethiopia. Out of these challenges written in different papers, lack of market at the time of harvest holds, distance of farm land from irrigation water source, lack of knowledge on the efficient use of irrigation by the farmers, lack of market 43 Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11, No.1, 2021 and linkage, lack of awareness on the sustainable use of water and lack of linkage between micro finance institutions, smallholder horticultural crop producers, shortage of agricultural inputs specially improved seed and pesticides, high cost of irrigation, shortage of water pump technologies and infrastructure specially road and storage are some constraints . On the other hand, there are different opportunities that increase irrigation development in Ethiopia. i.e. High-water potential, effective utilization of small-scale irrigation scheme infrastructure through diversification of uses to meet various needs for water such as domestic, irrigation, the interest of development agencies to give education and training, policies and strategies of Ethiopia strongly supports the irrigation developments. References ( Aam O. (2013). The Influence of Ndanda Traditional Irrigation Scheme on Crop Productivity In Masasi District. Sokoine University. Aam O. (2013). The Influence of Ndanda Traditional Irrigation Scheme on Crop Productivity In Masasi District. Sokoine University. Abebe , E., Bihon K & Y, G. (2011). Determinants of Land Allocation to Irrigation and its Wealth Effect: E from Northern Ethiopia. Journal of the Drylands 4, 310-319. Adugna E. (2014). 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Overseas Development Institute, Research Inspired Policy and Practice Learning in Ethiopia and The Nile Region (Ripple), Working Paper 14, Ethiopia. Eshetu, T. & Young-Bohk, C. (2017). Contribution of Small Scale Irrigation to Households’ Income And Food Security: Evidence from Ketar Irrigation Scheme, Arsi Zone, Oromiya Region, Ethiopia. African Journal of Business Management, 11, 57-68. g FAO (2011). The State of Food Insecurity in The World. How Does International Price Volatility Affect Domestic Economies A Food Security? 15). References ( Water Sa 37 (5), 122-127. Seleshi, B., Merrey, A., Kamara, B., Koppen, F. & Boelee, E. (2005). Experiences and Opportunities for Promoting Small Scale/Micro Irrigation and Rainwater Harvesting for Food Security In Ethiopia. Addis Ababa: Iwrm. Seleshi , B., Teklu, E. & Regassa, E. (2010). Irrigation Potential In Ethiopia; Constraints and Opportunities For Enhancing The System. International Water Management Institute,. Addis Ababa, Ethiopia. Sensen H. (2013). The Impact of Community Managed Irrigation on Household Income and Poverty Reduction (The Case of Seharti Samre Wereda, Tigray, Ethiopia). Mekelle Smith, L. (2004). Assessment of The Contribution of Irrigation to Poverty Reduction and Sustainable Livelihoods. Int. J. Water Resourc. Dev. , 20, 243-257. Tadesse, M. & Baihilu, B. (2017). Review on Participatory Small-Scale Irrigation Schemes and Small-Scale Rainwater Harvesting Technology Development and its Contribution to Household food Security In Ethiopia. International Journal of Water Resources and Environmental Engineering, 9, 54-63. g g Temesgen, H., ., Mengistu, K., . & Fekadu, B., . (2018). Evaluating The Impact of Small-Scale Irrigation Practice on Household Income In Abay Chomen District of Oromia National Regional State, Ethiopia. Journal of Development and Agricultural Economics, 10, 384-393. Temesgen H. (2017). 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Adoption and Impact of Micro Irrigation on Households Income: The Case of Bambasi eda Research and Knowledge Management of St Mary’s University 86-96 Tsegazeab G. (2015). Adoption and Impact of Micro Irrigation on Households Income: The Case of Woreda. Research and Knowledge Management of St. Mary’s University, 86-96. Woldegebrial, Z., Assefa, H. & Gebresilassie, H. (2017). References ( & R., S. (2012). Effect of Small-Scale Irrigation on The Income of Rural Farm Households: The Case of Laelay Maichew District, Central Tigray, Ethiopia. The Journal Of Agricultural Sciences,, 7. (Ijirah), 2. Kinfe, A., Chilot Y. & R., S. (2012). Effect of Small-Scale Irrigation on The Income of Rural Farm Households: The Case of Laelay Maichew District, Central Tigray, Ethiopia. The Journal Of Agricultural Sciences,, 7. (Ijirah), 2. Kinfe, A., Chilot Y. & R., S. (2012). 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Skin sodium content as a predictor of blood pressure response to renal denervation
Hypertension research
2,023
cc-by
8,253
Abstract Patients with treatment resistant hypertension (TRH) are known to have elevated sodium (Na) content in muscle and skin. Renal denervation (RDN) emerged as an adjacent therapeutic option in this group of patients. This analysis aimed at evaluating whether tissue Na content predicts blood pressure (BP) response after RDN in patients with TRH. Radiofrequency- device based RDN was performed in 58 patients with uncontrolled TRH. Office and 24-h ambulatory BP were measured at baseline and after 6 months. To assess tissue Na content Na magnetic resonance imaging (Na-MRI) was performed at baseline prior to RDN. We splitted the study cohort into responders and non-responders based on the median of systolic 24-h ambulatory blood pressure (ABP) reduction after 6 months and evaluated the association between BP response to RDN and tissue Na content in skin and muscle. The study was registered at http://www.clinicaltrials.gov (NCT01687725). Six months after RDN 24-h ABP decreased by −8.6/−4.7 mmHg. BP-Responders were characterized by the following parameters: low tissue sodium content in the skin (p = 0.040), female gender (p = 0.027), intake of aldosterone antagonists (p = 0.032), high baseline 24-h night-time heart rate (p = 0.045) and high LDL cholesterol (p < 0.001). These results remained significant after adjustment for baseline 24-h systolic BP. Similar results were obtained when the median of day-time and night-time ABP reduction after 6 months were used as cut-off criteria for defining BP response to RDN. We conclude that in addition to clinical factors including baseline 24-h ABP Na-MRI may assist to select patients with uncontrolled TRH for RDN treatment. Keywords Renal denervation ●Tissue sodium ●Sodium MRI ARTICLE ARTICLE * Roland E. Schmieder roland.schmieder@uk-erlangen.de Merve Guenes-Altan1 ●Axel Schmid2 ●Dennis Kannenkeril1 ●Peter Linz2 ●Christian Ott1 ●Agnes Bosch1 ● Mario Schiffer1 ●Michael Uder2 ●Roland E. Schmieder1 Received: 17 July 2023 / Revised: 24 August 2023 / Accepted: 26 August 2023 / Published online: 25 October 2023 © The Author(s) 2023. This article is published with open access Hypertension Research (2024) 47:361–371 https://doi.org/10.1038/s41440-023-01450-4 Hypertension Research (2024) 47:361–371 https://doi.org/10.1038/s41440-023-01450-4 1 Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg (FAU), Erlangen, Germany 2 Institute of Radiology, University Hospital Erlangen, Friedrich- Alexander University Erlangen-Nuremberg (FAU), Erlangen, Germany Skin sodium content as a predictor of blood pressure response to renal denervation Merve Guenes-Altan1 ●Axel Schmid2 ●Dennis Kannenkeril1 ●Peter Linz2 ●Christian Ott1 ● Mario Schiffer1 ●Michael Uder2 ●Roland E. Schmieder1 * Roland E. Schmieder roland.schmieder@uk-erlangen.de 1 Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg (FAU), Erlangen, Germany 2 Institute of Radiology, University Hospital Erlangen, Friedrich- Alexander University Erlangen-Nuremberg (FAU), Erlangen, Germany Study cohort All patients who participated in our study were aged 40–77 years, had uncontrolled TRH with 3–10 antihypertensive drugs and were eligible for Na-MRI examination without MRI contraindication. True hypertension was confirmed by 24-h ambulatory blood pressure (ABP) measurement (average 24-h ABP ≥130/80 mmHg). All patients fulfilled the following exclusion criteria: No known secondary cause of HTN including hyperaldosteronism, no significant renal artery pathologies, no prior RDN and no known contra- indication for RDN procedure (e.g. renal artery stenosis > 50%, implanting of renal stents). Study design Our single-center, post-hoc study includes 58 patients with uncontrolled TRH who underwent RDN and Na-MRI. The patients participated in the “Renal Denervation in Treatment Resistant Hypertension” trial, an investigator initiated study program performed only in our Erlanger center. The study was registered at http://www.clinicaltrials. gov (NCT01687725). All patients were followed up for Several studies have documented an accumulation of Na without simultaneously commensurate water which leads to the concept that there must exist non-osmotic storage of Na in tissue, for example in skin and muscle [13–16]. Na magnetic resonance imaging (Na-MRI) can be used to visualize and quantify Na content in tissue. Using this non- invasive technique elevated Na content in muscle and skin has been detected in patients with hypertension and in particular in patients with TRH [17]. Moreover, we pre- viously analyzed the impact of RDN on tissue Na content in patients with treatment resistant hypertension and did not observe any change in muscle or skin Na content 6 months after RDN [18]. The objective of the present study was to analyze whether tissue Na content in patients with TRH serves as a determinant of BP response in RDN. 6 months at the Clinical Research Centre of the Department of Nephrology and Hypertension, University Hospital Erlangen-Nuremberg, Germany (www.crc-erlangen.de). The respective study protocol was approved by the local Ethical Review Committee (ethics committee of the Uni- versity of Erlangen-Nuremberg) and the study was con- ducted according to the Declaration of Helsinki and Good Clinical Practice. Written informed consent was obtained from all patients and prior to study inclusion. Introduction treatment resistant hypertension (TRH) among patients with HTN was approximately 10% [3]. Arterial hypertension (HTN) is highly prevalent worldwide and a major risk factor for cardiovascular disease (CVD) and stroke. In 2016, it was estimated that 46.0% of adults in the United States suffered from HTN [1]. A multinational study in adults revealed that 55.6% of patients were aware of their diagnosis with only 17.1% having adequately controlled HTN [2]. Furthermore, the prevalence of Increased activity of the sympathetic nervous system (SNS) activity and increased sodium (Na) retention have been found to play an important role in the pathogenesis of TRH while both mechanisms are interconnected to each other [4–6]. High SNS activity results in increased Na and water retention [7, 8]. This enhances vasoconstriction due to vasoactive hormones and induces hypertrophic response of the myocardium and vessels [9]. Thus, targeting increased SNS activity appears to be an attractive treatment strategy [10]. The ESH 2021 and ESC 2023 consensus statements recommend RDN as an adjacent treatment option to achieve BP control in patients with uncontrolled TRH in addition to pharmacotherapy and lifestyle changes [11, 12]. Since blood pressure (BP) response after RDN has a large variability and there exist patients with poor BP response after RDN, the need to identify determinants of BP response to RDN have been underlined in the consensus statements [11, 12]. M. Guenes-Altan et al. 362 Graphical Abstract Clinical characteristics We included 58 patients in our study with a mean age of 62 years. Most patients were male and overweight. About half of the patients had type 2 diabetes (T2D). All patients had uncontrolled TRH with 6.2 (3.0–10.0) antihypertensive drugs on average. Four patients were not on diuretic therapy because of contraindications and drug intolerance. The detailed clinical characteristics are shown in Table 1. Blood pressure The average 24-h ABP in our study cohort was 157/87 mmHg and the office BP was 158/87 mmHg. Six months after RDN 24-h ABP was reduced by −8.6/−4.7 mmHg and office BP was reduced by −12.9/−7.7 mmHg (all p < 0.001, see Table 2). The average 24-h ABP in our study cohort was 157/87 mmHg and the office BP was 158/87 mmHg. Six months after RDN 24-h ABP was reduced by −8.6/−4.7 mmHg and office BP was reduced by −12.9/−7.7 mmHg (all p < 0.001, see Table 2). Medication change (conducted according to the discre- tion of the primary care physician) did not differ between the two groups (p = 0.320): At 6 months, decrease in number of antihypertensive drugs took place in 37.0% of responders versus 23.1% in non-responders, whereas increase in medication number occurred in 25.9% in responders and 23.1% in non-responders. Assessments Baseline assessments included office and 24-h ABP mea- surements, Na-MRI examination, collection of demographic Skin sodium content as a predictor of blood pressure response to renal denervation 363 by performing Pearson’s test. Subsequently, since baseline BP predicted the BP change in many previous RDN studies related to Wilders principle “law of initial value” [24], we adjusted our univariate approach only to 24-h, day-time and night-time systolic ABP. A two-sided p value of <0.05 was considered statistically significant. data and antihypertensive medication as well as physical examination, standard blood and urine tests. Office and ambulatory BP were measured with validated devices fol- lowing the recommendations of the European Society of Hypertension/European Society of Cardiology [19, 20]. Office BP was assessed after a rest of at least 5 min and repeated twice in a sitting position with a validated auto- matic device. Ambulatory BP was conducted with a vali- dated device (Mobilograph, IEM, Aachen, Germany) and mean values for 24-h, day-time and night-time ABP were calculated according to the published recommendations. Estimated glomerular function (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula [21]. Adverse events occurring during the trial were recorded at each visit. Na-MRI measurements At baseline, skin Na content was 20.9 ± 4.3 AU and muscle Na content was 20.6 ± 4.4 AU. Six months after RDN we observed no change in skin (20.9 ± 4.3 AU versus baseline 21.1 ± 4.9 AU; p = 0.915) and muscle (20.6 ± 4.4 AU ver- sus baseline 20.9 ± 4.0 AU; p = 0.683) Na content. At baseline, skin Na content was 20.9 ± 4.3 AU and muscle Na content was 20.6 ± 4.4 AU. Six months after RDN we observed no change in skin (20.9 ± 4.3 AU versus baseline 21.1 ± 4.9 AU; p = 0.915) and muscle (20.6 ± 4.4 AU ver- sus baseline 20.9 ± 4.0 AU; p = 0.683) Na content. Na-MRI measurements Skin and muscle Na content in the left lower leg were measured non-invasively with a clinical 3.0 T MR system (Magnetom Skyra, Siemens Healthineers, Erlangen, Ger- many) using a transit/receive Na RF birdcage knee coil (32.6 MHz, Stark Contrast, Erlangen, Germany) at baseline and 6 months after RDN. A detailed description of the procedure, as well as reliability and accuracy have been shown previously [9, 23]. RDN procedure A radiofrequency-based Symplicity-Flex catheter (Sympli- city by Ardian Inc, Palo Alto, CA, USA) was used for RDN procedure. A renal angiogram was previously performed to exclude renal artery abnormalities. As previously described [22], the femoral artery was accessed with standard endo- vascular technique and renal arteries of both sides were treated in one session. Up to 6 radiofrequency ablations (energy delivery for up to 120 s and 8 watts each) were applied longitudinally and rotationally within each artery to achieve a full 4-quadrant ablation. Visceral pain during the procedure was managed with anxiolytics and narcotics and patients were given 500 IE heparin. Correlation analysis We also did not observe any correlation between baseline 24-h systolic ABP and skin Na content (r = −0.070, p = 0.601). heart rate. We also did not observe any correlation between baseline 24-h systolic ABP and skin Na content (r = −0.070, p = 0.601). heart rate. We also did not observe any correlation between baseline 24-h systolic ABP and skin Na content (r = −0.070, p = 0.601). Correlation analysis triceps surae (AU) 20.6 ± 4.4 Sodium content in skin (AU) 20.9 ± 4.3 Office BP Office systolic BP (mmHg) 158 ± 23 Office diastolic BP (mmHg) 87 ± 16 Office heart rate (bpm) 71 ± 15 24-h ABP 24-h systolic ABP (mmHg) 157 ± 16 24-h diastolic ABP (mmHg) 87 ± 13 24-h ambulatory heart rate (bpm) 67 ± 12 Day-time systolic ABP (mmHg) 159 ± 16 Day-time diastolic ABP (mmHg) 89 ± 13 Day-time heart rate (bpm) 69 ± 12 Night-time systolic ABP (mmHg) 151 ± 21 Night-time diastolic ABP (mmHg) 80 ± 13 Night-time heart rate (bpm) 62 ± 11 Systolic nocturnal dipping rate (%) 5.1 ± 11.0 Diastolic nocturnal dipping rate (%) 9.9 ± 10.1 Laboratory values HbA1c (%) 6.6 ± 1.3 Creatinine (mg/dl) 1.2 ± 0.5 eGFR, CKD-Epi formula (mL/min/1.73 m²) 69.5 ± 24.7 Triglyceride 207.2 ± 117.5 Cholesterol (mg/dl) 196.0 ± 52.0 LDL-cholesterol (mg/dl) 131.5 ± 42.0 Hemoglobin (g/dl) 13.9 ± 1.6 Hematocrit (%) 40.6 ± 4.4 Antihypertensive medication Number of antihypertensive medication, n (%) 6.2 ± 1.6 ACE inhibitors, n (%) 20 (35) ARBs, n (%) 45 (78) Direct renin inhibitors, n (%) 25 (43) Betablockers, n (%) 46 (79) Calcium-channel blockers, n (%) 49 (85) Diuretics, n (%) 54 (93) Table 1 (continued) Aldosterone antagonists, n (%) 10 (17) Vasodilators, n (%) 21 (36) Centrally acting sympatholytics, n (%) 46 (79) Data are presented as mean ± SD Na Sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, HbA1c glycated hemoglobin, eGFR estimated glomerular filtration rate, CKD-EPI Chronic Kidney Disease Epidemiology Collaboration, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker Table 2 Change of 24-h ambulatory and office blood pressure 6 months after renal denervation (n = 53) BP change 6 months Office systolic BP (mmHg) −12.9 ± 21.1* Office diastolic BP (mmHg) −7.7 ± 12.5* Office heart rate (bpm) −3.2 ± 10.0* 24-h systolic ABP (mmHg) −8.6 ± 14.0* Day-time systolic ABP (mmHg) −8.6 ± 14.3* Night-time systolic ABP (mmHg) −10.3 ± 22.6* 24-h diastolic ABP (mmHg) −4.7 ± 9.5* Day-time diastolic ABP (mmHg) −4.7 ± 12.5* Night-time diastolic ABP (mmHg) −4.6 ± 12.5* 24-h ambulatory heart rate (bpm) 1.1 ± 6.1 Day-time ambulatory heart rate (bpm) 1.4 ± 6.7 Night-time ambulatory heart rate (bpm) 0.5 ± 6.5 Data are presented as mean ± SD BP blood pressure, ABP ambulatory blood pressure, bpm beats per minute *p < 0.05 versus baseline Demographic data Age (years) 61.6 ± 9.7 Male/female (n/n) 37/14 Body mass index (kg/m2) 30.2 ± 3.9 Weight (kg) 90.0 ± 15.6 Comorbidities Diabetes mellitus, n (%) 32 (55) Coronary artery disease, n (%) 20 (35) Left ventricular hypertrophy, n (%) 15 (26) Hyperlipidaemia, n (%) 24 (41) History of stroke, TIA, n (%) 9 (16) Current smoking, n (%) 9 (16) Na-MRI Sodium content in M. Correlation analysis triceps surae (AU) 20.6 ± 4.4 Sodium content in skin (AU) 20.9 ± 4.3 Office BP Office systolic BP (mmHg) 158 ± 23 Office diastolic BP (mmHg) 87 ± 16 Office heart rate (bpm) 71 ± 15 24-h ABP 24-h systolic ABP (mmHg) 157 ± 16 24-h diastolic ABP (mmHg) 87 ± 13 24-h ambulatory heart rate (bpm) 67 ± 12 Day-time systolic ABP (mmHg) 159 ± 16 Day-time diastolic ABP (mmHg) 89 ± 13 Day-time heart rate (bpm) 69 ± 12 Night-time systolic ABP (mmHg) 151 ± 21 Night-time diastolic ABP (mmHg) 80 ± 13 Night-time heart rate (bpm) 62 ± 11 Systolic nocturnal dipping rate (%) 5.1 ± 11.0 Diastolic nocturnal dipping rate (%) 9.9 ± 10.1 Laboratory values HbA1c (%) 6.6 ± 1.3 Creatinine (mg/dl) 1.2 ± 0.5 eGFR, CKD-Epi formula (mL/min/1.73 m²) 69.5 ± 24.7 Triglyceride 207.2 ± 117.5 Cholesterol (mg/dl) 196.0 ± 52.0 LDL-cholesterol (mg/dl) 131.5 ± 42.0 Hemoglobin (g/dl) 13.9 ± 1.6 Hematocrit (%) 40.6 ± 4.4 Antihypertensive medication Number of antihypertensive medication, n (%) 6.2 ± 1.6 ACE inhibitors, n (%) 20 (35) ARBs, n (%) 45 (78) Direct renin inhibitors, n (%) 25 (43) Betablockers, n (%) 46 (79) Calcium-channel blockers, n (%) 49 (85) Diuretics, n (%) 54 (93) Na Sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, HbA1c glycated hemoglobin, eGFR estimated glomerular filtration rate, CKD-EPI Chronic Kidney Disease Epidemiology Collaboration, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker Table 2 Change of 24-h ambulatory and office blood pressure 6 months after renal denervation (n = 53) BP change 6 months Office systolic BP (mmHg) −12.9 ± 21.1* Office diastolic BP (mmHg) −7.7 ± 12.5* Office heart rate (bpm) −3.2 ± 10.0* 24-h systolic ABP (mmHg) −8.6 ± 14.0* Day-time systolic ABP (mmHg) −8.6 ± 14.3* Night-time systolic ABP (mmHg) −10.3 ± 22.6* 24-h diastolic ABP (mmHg) −4.7 ± 9.5* Day-time diastolic ABP (mmHg) −4.7 ± 12.5* Night-time diastolic ABP (mmHg) −4.6 ± 12.5* 24-h ambulatory heart rate (bpm) 1.1 ± 6.1 Day-time ambulatory heart rate (bpm) 1.4 ± 6.7 Night-time ambulatory heart rate (bpm) 0.5 ± 6.5 Data are presented as mean ± SD BP blood pressure, ABP ambulatory blood pressure, bpm beats per minute *p < 0.05 versus baseline heart rate. Correlation analysis Statistical analysis was performed using SPSS Statistics 28.0 (IBM, Armonk, NY, USA) and data were expressed as mean ± standard deviation (SD) in text and tables. Paired t- test was applied for the comparison of 6 months follow-up BP data versus baseline. Predictors of BP change were assessed by comparing responders versus non-responders defined by the median reduction of 24-h, day-time and night-time systolic ABP at 6 month follow-up visit, respectively. Bivariate correlation analyses were assessed We observed a correlation between baseline 24-h systolic ABP and BP response 6 months after RDN (r = −0.394, p = 0.003). In addition, we observed a correlation between skin sodium content and BP response (r = 0.339, p = 0.013, Fig. 1). Other than that, we did not observe any correlation between BP response and other parameters (all p > 0.1). In particular, no correlation was observed with gender, LDL- cholesterol, aldosterone-antagonist medication or night-time M. Guenes-Altan et al. 364 heart rate. We also did not observe any correlation between baseline 24-h systolic ABP and skin Na content (r = −0.070, p = 0.601). Predictors of BP response To identify any predictors of BP response after RDN we splitted the study cohort according to the median systolic 24-h ABP reduction after 6 months into responders and non-responders (median ≤−10.0 mmHg versus > −10.0 mmHg, Table 3). We identified patients with low skin Na, high BMI, female patients, patients with high LDL-cho- lesterol, medication with aldosterone antagonists and diuretics, and high night-time heart rate to be more likely to respond to RDN with >10 mmHg fall in 24-h ABP after 6 months. After adjustment for baseline 24-h systolic ABP, skin Na, gender, LDL-cholesterol, night-time heart rate and aldosterone-antagonist medication remained significantly different between responders and non responders (Table 3) Table 1 Clinical characteristics Demographic data Age (years) 61.6 ± 9.7 Male/female (n/n) 37/14 Body mass index (kg/m2) 30.2 ± 3.9 Weight (kg) 90.0 ± 15.6 Comorbidities Diabetes mellitus, n (%) 32 (55) Coronary artery disease, n (%) 20 (35) Left ventricular hypertrophy, n (%) 15 (26) Hyperlipidaemia, n (%) 24 (41) History of stroke, TIA, n (%) 9 (16) Current smoking, n (%) 9 (16) Na-MRI Sodium content in M. Predictors of BP response To identify any predictors of BP response after RDN we splitted the study cohort according to the median systolic 24-h ABP reduction after 6 months into responders and non-responders (median ≤−10.0 mmHg versus > −10.0 mmHg, Table 3). We identified patients with low skin Na, high BMI, female patients, patients with high LDL-cho- lesterol, medication with aldosterone antagonists and diuretics, and high night-time heart rate to be more likely to respond to RDN with >10 mmHg fall in 24-h ABP after 6 months. After adjustment for baseline 24-h systolic ABP, skin Na, gender, LDL-cholesterol, night-time heart rate and aldosterone-antagonist medication remained significantly different between responders and non-responders. (Table 3) Skin sodium content as a predictor of blood pressure response to renal denervation 365 Fig. 1 Correlation between skin sodium content at baseline and 24-h systolic ambulatory blood pressure change 6 months after renal denervation skin sodium content at baseline and 24-h systolic ambulatory blood pressure change 6 months after renal denervation Fig. 1 Correlation between skin sodium content at baseline and 24-h systolic ambulatory blood pressure change 6 of initial value (Wilder’s principle) [24, 26–34]. In our study we focused on the importance of skin Na content, assessed by Na-MRI, in 58 patients with TRH as a potential predictor of the BP response after RDN. The skin, like the muscles, is an important storage of extracellular Na content [16]. Na accumulation in tissue may exaggerate hyper- trophy of the myocardial and vascular smooth muscle cells and thereby augment HTN associated complications [9, 16]. It is known that patients with TRH achieve a greater reduction in BP by restricting salt compared to other hypertensive patients and that patients with TRH are more sensitive to salt intake [35]. However, the effect of RDN on sodium homeostasis is not well understood. Ott et al. per- formed Na-MRI in 41 patients who had underwent RDN and observed no change in tissue Na content at the second examination after 6 months, in accordance with the results of the current study [18]. In contrast, a post-hoc analysis of 137 patients showed an increase in urinary Na in patients with TRH 6 months after RDN, but the reliability of the measurements were questionable as they were based on the Kawasaki formula and spot urine [36]. Predictors of BP response Experimental animal studies have also shown an increased Na excretion in the acute stage after RDN and a decrease in Na excretion after renal sympathetic nerve stimulation [37–39]. We repeated the whole analysis by dividing patients into 2 groups based on the median of day-time and night-time BP change after 6 months, respectively. Besides systolic ABP, skin Na, aldosterone-antagonist medication, baseline 24-h, day-time and night-time heart rate remained significantly different between responders and non-responders (Tables 4 and 5). Age, body mass index (BMI) and renal function (i.e. eGFR) were not identified as determinants of BP response. Thus, lower skin sodium content (together with a non- significant signal for muscle sodium content) emerged as a BP independent predictor of BP fall after RDN. Discussion In our study we performed a post-hoc analysis of 58 patients with uncontrolled TRH who underwent RDN and Na-MRI. We observed a reduction of office BP by −12.9/ −7.7 mmHg and 24-h ABP by −8.6/−4.7 mmHg 6 months after RDN. Considering BP reduction, our results are con- sistent with the results of the Global Symplicity Registry. In this worldwide registry, office BP decreased by −11.6/−4.3 mmHg and 24-h ABP decreased by −6.6/ −3.9 mmHg 6 months after RDN [25]. From a clinical perspective, it is important to identify potential predictors for the efficacy of RDN procedure as guidance to personalize treatment options in hypertension. Many studies have uniformly identified a high baseline systolic ABP as a predictor for good BP response after RDN, but this phenomenon is unspecific and known as law In this study we focused on a potential role of Na as a predictor of the BP drop after RDN. We restricted our analysis to ABP measurements following the current con- sensus statement [11, 12]. According to the change of systolic 24-h, day-time and night-time ABP 6 months after RDN we splitted the study cohort into responders and 366 M. Guenes-Altan et al. non-responders by the respective median drop in ABP We according to median decrease of the following BP para- Table 3 Univariate analysis of responders versus non-responders at 6 months based on the median of the change of 24-h systolic ABP Baseline Median ≤−10 mmHg Median > −10 mmHg P value P value* Responder (n = 27) Non-Responder (n = 26) Change of systolic ABP at 6 months (mmHg) −18.7 ± 8.4 0.2 ± 8.2 <0.001 <0.001 Baseline characteristics BMI (kg/m2) 31.2 ± 4.2 28.8 ± 3.3 0.024 0.066 Gender (m/f) 17/10 23/3 0.031 0.027 Age (years) 60.4 ± 10.4 62.7 ± 8.4 0.386 0.389 Na-MRI Sodium in M. Discussion triceps surae (AU) 19.5 ± 3.3 21.6 ± 5.3 0.095 0.111 Sodium in skin (AU) 19.7 ± 3.3 22.2 ± 5.1 0.042 0.040 Baseline office BP Systolic BP (mmHg) 156.5 ± 16.9 152.2 ± 23.2 0.448 Diastolic BP (mmHg) 88.1 ± 16.6 83.6 ± 12.2 0.268 Heart rate (bpm) 73.1 ± 13.9 65.6 ± 14.0 0.059 0.086 Baseline ABP 24-h systolic BP (mmHg) 158.9 ± 12.5 151.8 ± 15.8 0.077 24-h diastolic BP (mmHg) 89.2 ± 12.9 83.7 ± 10.1 0.090 24-h heart rate (bpm) 69.2 ± 12.3 64.2 ± 9.8 0.121 0.124 Systolic day-time BP (mmHg) 161.1 ± 13.0 154.3 ± 15.5 0.091 Diastolic day-time BP (mmHg) 91.8 ± 12.9 86.2 ± 10.3 0.089 Heart rate day-time (bpm) 70.4 ± 11.7 65.8 ± 10.6 0.162 0.159 Systolic night-time BP (mmHg) 153.7 ± 17.1 145.2 ± 24.3 0.145 Diastolic night-time BP (mmHg) 83.1 ± 13.4 77.0 ± 12.7 0.099 Night-time heart rate (bpm) 65.3 ± 12.8 58.9 ± 8.0 0.045 0.047 Laboratory values Hematocrit (%) 41.9 ± 3.8 39.6 ± 4.9 0.059 0.102 LDL-cholesterol (mg/dl) 150.6 ± 39.1 114.7 ± 37.0 0.001 <0.001 Antihypertensive medication Diuretics, n (%) 23 (85.2) 26 (100) 0.041 0.053 Aldosterone-antagonist, n (%) 7 (25.9) 1 (3.8) 0.025 0.032 ACE inhibitors, n (%) 10 (37) 9 (35) 0.854 0.699 ARBs, n (%) 21(78) 21 (81) 0.788 0.691 Direct renin inhibitors, n (%) 14 (52) 8 (31) 0.119 0.182 Betablockers, n (%) 21 (78) 21 (81) 0.788 0.971 Calcium-channel blockers, n (%) 23 (85) 22 (85) 0.954 0.896 Vasodilators, n (%) 9 (33) 10 (38) 0.697 0.608 Centrally acting sympatholytics, n (%) 21 (78) 20 (77) 0.941 0.824 Data are presented as mean ± SD. Discussion Bold values represent p-values ≤0.05 Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h systolic BP ysis of responders versus non-responders at 6 months based on the median of the change of 24-h systolic ABP Table 3 Univariate analysis of responders versus non-responders at 6 months based on the median of the chang Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h systolic BP according to median decrease of the following BP para- meters: 24-h ABP, day-time ABP and night-time ABP. With respect to muscle Na content, a lower content tended to be related to better ABP response. Thus, baseline skin Na content (and to lesser extent muscle sodium content) non-responders by the respective median drop in ABP. We found patients with low skin Na content to be more likely to respond to RDN than those with high baseline skin Na. This result persisted after adjustment for baseline 24-h systolic BP and were also found after separating the study cohort Skin sodium content as a predictor of blood pressure response to renal denervation 367 emerged as a predictor for systolic ABP response to RDN in addition to and independent from baseline systolic ABP populations, such as in patients with T2D, hypertension Conn’s syndrome and CKD [17 23 40 41] We previously Table 4 Univariate analysis of responders versus non-responders at 6 months based on the median of the change of day-time systolic ABP Baseline Median ≤−12 mmHg Median > −12 mmHg P value P value* Responder (n = 27) Non-Responder (n = 26) Change of day-time systolic BP at 6 months (mmHg) −19.7 ± 7.6 1.8 ± 9.1 <0.001 <0.001 Baseline characteristics BMI (kg/m2) 30.7 ± 4.2 29.3 ± 3.7 0.182 0.392 Gender (m/w) 0.389 0.256 Age (years) 59.5 ± 10.1 63.7 ± 8.2 0.106 0.089 Na-MRI Sodium in M. Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h day-time systolic BP Data are presented as mean ± SD. Bold values represent p-values ≤0.05 Discussion triceps surae (AU) 19.5 ± 3.0 21.6 ± 5.5 0.074 0.081 Sodium in skin (AU) 19.6 ± 2.6 22.4 ± 5.4 0.021 0.015 Baseline office BP Systolic BP (mmHg) 152.4 ± 17.4 156 ± 22.8 0.481 Diastolic BP (mmHg) 87.1 ± 16.3 84.7 ± 12.9 0.550 Heart rate (bpm) 73.2 ± 14.5 65.6 ± 13.3 0.056 0.150 Baseline ABP 24-h systolic BP (mmHg) 159.8 ± 13.5 150.8 ± 14.5 0.023 24-h diastolic BP (mmHg) 89.2 ± 12.1 83.6 ± 11.0 0.081 24-h heart rate (bpm) 70.5 ± 12.1 63.0 ± 9.2 0.019 0.025 Systolic day-time BP (mmHg) 162.5 ± 13.8 152.8 ± 13.9 0.014 Diastolic day-time BP (mmHg) 91.9 ± 12.1 86.1 ± 11.2 0.074 Heart rate day-time (bpm) 71.8 ± 12.0 64.5 ± 9.6 0.025 0.030 Systolic night-time BP (mmHg) 153.6 ± 19.8 145.3 ± 22.1 0.153 Diastolic night-time BP (mmHg) 82.6 ± 13.4 77.5 ± 13.0 0.159 Night-time heart rate (bpm) 66.1 ± 12.2 58.1 ± 8.2 0.010 0.012 Laboratory values Hematocrit (%) 40.9 ± 4.1 40.7 ± 4.9 0.883 0.759 Triglyceride 233.6 ± 119.2 76.7 ± 104.7 0.071 0.071 Cholesterol (mg/dl) 204.3 ± 43.9 189.4 ± 58.7 0.299 0.286 LDL-cholesterol (mg/dl) 140.6 ± 36.0 125.0 ± 46.4 0.178 0.161 Antihypertensive medication Diuretics, n (%) 24 (88.9) 25 (96.2) 0.317 0.432 Aldosterone-antagonist, n (%) 7 (25.9) 1 (3.8) 0.025 0.036 ACE inhibitors, n (%) 10 (37.0) 9 (34.6) 0.854 0.583 ARBs, n (%) 21 (77.8) 21 (80.8) 0.788 0.681 Direct renin inhibitors, n (%) 14 (51.9) 8 (30.8) 0.119 0.201 Betablockers, n (%) 20 (74.1) 22 (84.6) 0.344 0.596 Calcium-channel blockers, n (%) 23 (85.2) 22 (84.6) 0.954 0.961 Vasodilators, n (%) 11 (40.7) 8 (30.8) 0.449 0.619 Centrally acting sympatholytics, n (%) 21 (77.8) 20 (76.9) 0.941 0.746 Data are presented as mean ± SD. Bold values represent p-values ≤0.05 Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h day-time systolic BP of responders versus non-responders at 6 months based on the median of the change of day-time systolic ABP Table 4 Univariate analysis of responders versus non-responders at 6 months based on the median of the chang Data are presented as mean ± SD. Discussion Bold values represent p-values ≤0.05 Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h day-time systolic BP populations, such as in patients with T2D, hypertension Conn’s syndrome and CKD [17, 23, 40, 41]. We previously showed that high tissue sodium content is linked to emerged as a predictor for systolic ABP response to RDN in addition to and independent from baseline systolic ABP. High tissue sodium was observed in several diseased 368 M. Guenes-Altan et al. Table 5 Univariate analysis of responders versus non-responders at 6 months based on the median of the change of night-time systolic ABP Baseline Median ≤−13 mmHg Median > −13 mmHg P value P value* Responder (n = 27) Non-Responder (n = 26) Change of night-time systolic BP at 6 months (mmHg) −26.6 ± 11.1 3.2 ± 11.9 <0.001 <0.001 Baseline characteristics BMI (kg/m2) 30.5 ± 4.6 29.5 ± 3.2 0.338 0.931 Gender (m/w) 18/9 22/4 0.129 0.150 Age (years) 60.7 ± 10.0 62.4 ± 8.9 0.524 0.351 Na-MRI Sodium in M. Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h night-time systolic BP Discussion triceps surae (AU) 19.8 ± 3.5 21.2 ± 5.3 0.257 0.251 Sodium in skin (AU) 19.7 ± 3.2 22.2 ± 5.1 0.038 0.016 Baseline office BP Systolic BP (mmHg) 157.3 ± 21.1 151.4 ± 19.1 0.290 Diastolic BP (mmHg) 89.4 ± 13.5 82.3 ± 15.1 0.078 Heart rate(bpm) 71.3 ± 12.8 67.6 ± 15.9 0.350 0.133 Baseline ABP 24-h systolic BP (mmHg) 160.7 ± 13.2 149.9 ± 14.0 0.005 24-h diastolic BP (mmHg) 90.4 ± 11.9 82.4 ± 10.5 0.011 24-h heart rate (bpm) 69.9 ± 11.2 63.6 ± 10.6 0.053 0.004 Systolic day-time BP (mmHg) 160.4 ± 13.3 155.0 ± 15.6 0.183 Diastolic day-time BP (mmHg) 91.4 ± 11.9 86.7 ± 11.7 0.148 Heart rate day-time (bpm) 70.6 ± 10.9 65.5 ± 11.3 0.117 0.017 Systolic night-time BP (mmHg) 159.9 ± 17.8 138.8 ± 19.1 <0.001 Diastolic night-time BP (mmHg) 86.6 ± 12.5 73.4 ± 10.7 <0.001 Night-time heart rate (bpm) 65.7 ± 11.6 58.5 ± 9.3 0.021 0.003 Laboratory values eGFR, CKD-EPI (ml/min/1.73 m²) 74.9 ± 25.3 62.8 ± 21.2 0.065 0.129 Hematocrit (%) 41.1 ± 3.5 40.4 ± 5.3 0.574 0.668 Triglyceride 218.8 ± 125.8 192.1 ± 103.0 0.403 0.291 Cholesterol (mg/dl) 201.9 ± 45.8 191.9 ± 57.8 0.487 0.265 LDL-cholesterol (mg/dl) 137.6 ± 34.7 128.2 ± 48.3 0.416 0.165 Antihypertensive medication Diuretics, n (%) 24 (88.9) 25 (96.2) 0.317 0.303 Aldosterone-antagonist, n (%) 5 (18.5) 3 (11.5) 0.478 0.443 ACE inhibitors, n (%) 10 (37.0) 9 (34.6) 0.854 0.720 ARBs, n (%) 21 (77.8) 21 (80.8) 0.788 0.431 Direct renin inhibitors, n (%) 13 (48.1) 9 (34.6) 0.318 0.409 Betablockers, n (%) 19 (70.4) 23 (88.5) 0.104 0.124 Calcium-channel blockers, n (%) 22 (81.5) 23 (88.5) 0.478 0.747 Vasodilators, n (%) 8 (29.6) 11 (42.3) 0.336 0.243 Centrally acting sympatholytics, n (%) 22 (81.5) 19 (73.1) 0.465 0.841 Data are presented as mean ± SD. Bold values represent p-values ≤0.05 Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h night-time systolic BP of responders versus non-responders at 6 months based on the median of the change of night-time systolic ABP Table 5 Univariate analysis of responders versus non-responders at 6 months based on the median of the chang Data are presented as mean ± SD. *p = adjusted for baseline 24-h night-time systolic BP Data are presented as mean ± SD. Bold values represent p-values ≤0.05 night-time systolic BP *p = adjusted for baseline 24-h night-time systolic BP Discussion Bold values represent p-values ≤0.05 Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker Skin sodium content as a predictor of blood pressure response to renal denervation 369 hypertrophic vascular remodeling and to left ventricular hypertrophy [40, 42]. Thus, we assume that patients with a higher skin Na content may be in a more advanced stage of arterial hypertension with greater extent of hypertrophic vascular remodeling and are therefore less likely to respond to renal denervation. Despite BP reduction, RDN may have pleiotropic effects. We defined non-responders and responders only by their BP change. However, there may be positive effects of RDN beyond BP reduction also in patients defined as non- responders. Our study has several limitations. It is a single-center and post-hoc study with a small sample size with a limited follow-up of 6 months. However, 24-h, day-time and night- time systolic BP reduction demonstrated consistent results, with low skin Na being related to greater BP reduction after RDN. Though not significant, a similar signal has been found for baseline muscle content even after adjustment for baseline 24-h ABP. Nevertheless, only prospective studies with longer follow-up are able to corroborate our observations. Our findings also suggest that gender, LDL cholesterol, aldosterone-antagonist medication, 24-h, day-time and night-time heart rate may play a role in determining BP response to RDN. Böhm et al. identified in the SPYRAL HTN-OFF MED pivotal trial a high baseline heart rate to determine the effectiveness of RDN [43]. In contrast, Esler et al. found no correlation between HR and renal sympa- thetic activation but only between HR and cardiac sympa- thetic activation [44]. It was previously shown that RDN reduces cardiac sympathetic activity [45]. We identified 24- h, day-time and night-time HR to be a predictive parameter for good BP response after RDN and this result remained significant after adjustment for baseline 24-h systolic ABP. However, we could not identify any correlation between 24-h, day-time or night-time heart rate and BP response. In addition, we identified patients with aldosterone-antagonist medication use to have a better BP response to RDN. Interestingly aldosterone-antagonist medication has also been identified as a predictor in the Symplicity HTN 3 trial [27]. Data availability The datasets used and analyzed for this register are available from the corresponding author on reasonable request. Acknowledgements We gratefully acknowledge the expert technical assistance of Dorothea Bader-Schmieder, Ingrid Fleischmann, Kerstin Fröhlich-Endreß, Ulrike Heinritz, Simone Pejkovic, Wiebke Maurer and Theresa Federlein. Despite identifying that female patients were more likely in the responder group, we did not find any correlation between sex and BP response. Funding Open Access funding enabled and organized by Projekt DEAL. Several studies also analyzed vascular parameters to identify potential predictors for BP response after RDN. Weber et al. identified pulsatile hemodynamics such as augmentation index, augmentation pressure and estimated aortic pulse wave velocity to be potential predictors for BP response after RDN [49]. In accordance, other studies analyzed the influence of invasively measured pulse wave velocity on BP response after RDN and showed that patients with lower pulse wave velocity at baseline were more likely to be responders to RDN [50, 51]. Additionally, Fengler et al. identified cardiac magnetic resonance assessment of central and peripheral vascular function as a potential predictor of RDN [52]. In accordance, we pre- viously identified lower baseline central pulse pressure (measured non-invasively with SphygmoCor™device), indicative for the degree of arterial stiffening, to be a pre- dictive value for better BP response after RDN [53]. Conclusion Our findings suggest that patients with low skin Na have a greater BP reduction after RDN, independent of baseline ABP. Utilizing Na-MRI prior to RDN might provide a valuable tool in selecting patients with TRH that have a BP reduction after RDN above the median and thereby being particularly suitable for the procedure. Discussion In accordance, a single-center trial identified aldosterone-antagonist medication in patients with TRH to be predictive for better BP response after RDN [46]. 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Conflict of interest MGA, AS, DK, PL, CO, AB, MS and MU declare that they have no conflict of interest with respect to this study. RES has conflict of interests: Grants to the institution: Medtronic, Recor Medical, Ablative Solutions. Speaker and Adviser bureau: Medtronic, Recor Medical, Ablative Solutions. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons 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 M. Guenes-Altan et al. 370 17. 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Quantitative EEG as a biomarker in mild cognitive impairment with Lewy bodies
Alzheimer's research & therapy
2,020
cc-by
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Objectives: To investigate using quantitative EEG the (1) differences between patients with mild cognitive impairment with Lewy bodies (MCI-LB) and MCI with Alzheimer’s disease (MCI-AD) and (2) its utility as a potential biomarker for early differential diagnosis. Methods: We analyzed eyes-closed, resting-state, high-density EEG data from highly phenotyped participants (39 MCI-LB, 36 MCI-AD, and 31 healthy controls). EEG measures included spectral power in different frequency bands (delta, theta, pre-alpha, alpha, and beta), theta/alpha ratio, dominant frequency, and dominant frequency variability. Receiver operating characteristic (ROC) analyses were performed to assess diagnostic accuracy. Results: There was a shift in power from beta and alpha frequency bands towards slower frequencies in the pre- alpha and theta range in MCI-LB compared to healthy controls. Additionally, the dominant frequency was slower in MCI-LB compared to controls. We found significantly increased pre-alpha power, decreased beta power, and slower dominant frequency in MCI-LB compared to MCI-AD. EEG abnormalities were more apparent in MCI-LB cases with more diagnostic features. There were no significant differences between MCI-AD and controls. In the ROC analysis to distinguish MCI-LB from MCI-AD, beta power and dominant frequency showed the highest area under the curve values of 0.71 and 0.70, respectively. While specificity was high for some measures (up to 0.97 for alpha power and 0.94 for theta/alpha ratio), sensitivity was generally much lower. Conclusions: Early EEG slowing is a specific feature of MCI-LB compared to MCI-AD. However, there is an overlap between the two MCI groups which makes it difficult to distinguish between them based on EEG alone. Keywords: Dementia with Lewy bodies, Alzheimer’s disease, Quantitative electroencephalography, Biomarker Keywords: Dementia with Lewy bodies, Alzheimer’s disease, Quantitative electroencephalograp Quantitative EEG as a biomarker in mild cognitive impairment with Lewy bodies Julia Schumacher1* , John-Paul Taylor1, Calum A. Hamilton1, Michael Firbank1, Ruth A. Cromarty1, Paul C. Donaghy1, Gemma Roberts1, Louise Allan1,3, Jim Lloyd2, Rory Durcan1, Nicola Barnett1, John T. O’Brien4 and Alan J. Thomas1 * Correspondence: julia.schumacher@newcastle.ac.uk 1Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Biomedical Research Building 3rd floor, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK Full list of author information is available at the end of the article Background stratification for clinical trials and research studies and for optimizing clinical care such as by highlighting the need to carefully avoid using medication with anticholin- ergic properties and encouraging active identification of therapeutic targets such as orthostatic hypotension and constipation [1]. The early diagnosis of dementia is becoming increasingly important as it is likely that potential disease-modifying treatments will have their greatest effect at this stage. Additionally, accurate and early differential diagnosis of dementia subtypes is crucial for correct patient The intermediate stage between normal aging and de- mentia in which cognitive decline is present, but inde- pendence in activities of daily living is still preserved, is referred to as mild cognitive impairment (MCI) [2, 3]. While much research has focused on MCI patients who * Correspondence: julia.schumacher@newcastle.ac.uk 1Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Biomedical Research Building 3rd floor, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK Full list of author information is available at the end of the article * Correspondence: julia.schumacher@newcastle.ac.uk 1Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Biomedical Research Building 3rd floor, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK Full list of author information is available at the end of the article Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 https://doi.org/10.1186/s13195-020-00650-1 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 https://doi.org/10.1186/s13195-020-00650-1 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 2 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 2 of 12 later develop Alzheimer’s disease (AD), MCI in the con- text of dementia with Lewy bodies (DLB) has only been characterized more recently with research criteria re- cently published [4–6]. Differential diagnosis is often im- peded by a substantial clinical overlap between AD and DLB, which is especially pronounced in early stages, highlighting the need for objective biomarkers. In DLB, dopaminergic brain imaging and myocardial scintigraphy are indicative biomarkers [7]; these can improve diag- nostic accuracy at the MCI stage [8] and are included in the new research criteria for MCI with Lewy bodies (MCI-LB). EEG has been suggested as a non-invasive al- ternative, and an abnormal EEG is listed as a supportive biomarker in the DLB diagnostic criteria [7]. However, its value for differential diagnosis at the MCI stage is less certain. based on the clinical history and research assessments. In addition to a detailed clinical assessment, participants had already undergone dopaminergic imaging with 123I-N- fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) single- photon emission computed tomography (FP-CIT SPECT) and 123iodine-metaiodobenzylguanidine (MIBG) myocar- dial scintigraphy through their involvement in an ongoing study investigating the diagnostic accuracy of imaging bio- markers in MCI, and this information was used to apply diagnostic criteria (see below). After the initial baseline visit, participants were followed annually for up to 3 years with a mean follow-up time of 16.5 months (standard deviation = 6.5 months). Patients who were taking dopaminergic medication were assessed in the “ON” motor state. The aims of the present study were therefore to inves- tigate changes in quantitative EEG measures in a large cohort of well-characterized patients, all of whom had biomarker assessments with both dopaminergic and car- diac MIGB imaging, and to test the utility of EEG as a biomarker for early differential diagnosis. Diagnosis Diagnoses were based on all information available at the end of the recruitment period (December 2019) includ- ing any baseline and follow-up visits. MCI diagnoses were made independently by a consensus panel of three experienced old-age psychiatrists (AJT, PCD, JPT) in ac- cordance with NIA-AA criteria [3], i.e., subjective and objective cognitive impairment with maintained inde- pendence of function with minimal aids or assistance and a CDR of 0 or 0.5. Patients with a diagnosis of dementia were excluded from the study. Furthermore, patients with possible contributing frontotemporal or vascular etiologies or with a history of parkinsonism of more than 1 year prior to the onset of cognitive impair- ment were excluded. The presence or absence of the core Lewy body symptoms was rated by the panel utiliz- ing the rating scales and all information from the clinical assessments [7]. Findings from the FP-CIT and MIBG scans were used for diagnosis (see below), but the clin- ical MCI diagnoses as well as the rating of the presence/ absence of core DLB symptoms were performed blind to these imaging findings. The secondary aim was to better understand the rela- tionship between EEG abnormalities in the MCI-LB group and the core Lewy body symptoms. We hypothe- sized that EEG abnormalities would be more severe in patients with more core symptoms. Participants All participants in this study were over 60 years of age. Patients with a clinical diagnosis of MCI in memory ser- vices in the north east of England were screened and approached for this study if they reported additional clinical symptoms suggestive of Lewy body disease (e.g., mood changes, sleep disturbances, or autonomic symp- toms) or any core DLB features (visual hallucinations, cognitive fluctuations, parkinsonism, and REM sleep behavior disorder). After obtaining written informed consent, participants underwent a baseline clinical as- sessment including the Addenbrooke’s Cognitive Exam- ination–Revised (ACE-R), from which the Mini-Mental State Examination (MMSE) score was derived; the Uni- fied Parkinson’s Disease Rating Scale motor sub-score (UPDRS-III); the Epworth Sleepiness Scale (ESS); and the Geriatric Depression Scale (GDS). Additionally, the Instrumental Activities of Daily Living (IADL) Scale, the Clinician Assessment of Fluctuations (CAF), the Dementia Cognitive Fluctuations Scale (DCFS), the North-East Vis- ual Hallucinations Interview (NEVHI), the Neuropsychi- atric Inventory (NPI), and the Mayo Sleep Questionnaire (MSQ) were administered to informants, and the Clinical Dementia Rating (CDR) Scale and the Cumulative Illness Rating Scale for Geriatrics (CIRS-G) were completed A diagnosis of MCI with probable Alzheimer’s disease (MCI-AD) was given to patients who had no core Lewy body symptoms, negative FP-CIT and MIBG findings, and evidence of cognitive decline that was characteristic of AD, i.e., they met the additional NIA-AA criterion for “etiology of MCI consistent with AD pathophysiologic process” [3]. Probable MCI with Lewy bodies (MCI-LB) was diag- nosed if a patient had two or more core Lewy body symptoms or one core symptom in addition to a positive FP-CIT or MIBG scan [6]. Out of 103 MCI participants who were included in the study, 20 had only one core Lewy body symptom with negative FP-CIT and MIBG scans or no core symptoms in addition to a positive FP-CIT or MIBG scan, i.e., they did not meet the criteria for either MCI-AD or probable Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 3 of 12 Schumacher et al. Alzheimer's Research & Therapy Fig. 1 Study flowchart MCI-LB and were therefore not included in the present analysis (Fig. 1). Additionally, eight MCI participants did not have usable EEG data available. This study therefore included 39 participants who were diagnosed with prob- able MCI-LB and 36 who were diagnosed with MCI-AD. Participants Healthy control participants (N = 31) were recruited from relatives and friends of patients and from a local research register. Control participants had the same as- sessment as the patients and had no history of psychi- atric or neurological illness and no evidence of any cognitive decline. They also had normal structural MR imaging. The study was approved by the local ethics committee, and written informed consent was obtained from all participants. EEG technician during the recording to monitor adher- ence to the protocol. The ground electrode was attached to the right clavicle, and all EEG channels were refer- enced to Fz during recording. Pre-processing of eyes-closed EEG data was performed using the EEGLAB toolbox (version 14) in MATLAB (R2017a) [9] and was blinded to group membership. First, EEG data were bandpass-filtered between 0.3 and 54 Hz using a second-order Butterworth filter and split into non-overlapping epochs of 2 s. Subsequently, EEG recordings were visually inspected to identify noisy channels and noisy epochs which were excluded prior to applying independent component analysis for further artifact removal. The resulting components were visually inspected, and components representing muscular, car- diac, ocular, or electrical (50 Hz line noise) artifacts were rejected. The previously excluded channels were then re- placed using spherical spline interpolation, and data were recomputed against the average reference. For each participant, the first 45 2-s-long artifact-free epochs were selected for further analysis. EEG acquisition and pre-processing Resting-state high-density EEG recordings were acquired from all participants using Waveguard caps (ANT Neuro, The Netherlands) comprising 128 sintered Ag/ AgCl electrodes that were placed according to the 10-5 system. Participants were seated during the recording and instructed to remain awake. Electrode impedance was kept below 5 kΩ, and continuous EEG data were re- corded at a sampling frequency of 1024 Hz. Three hun- dred seconds of eyes-closed data were recorded from each participant. Participants were supervised by the Frequency analysis For each 2-s epoch, the power spectral density (PSD) was estimated using Bartlett’s method in MATLAB (R2017a) with a frequency resolution of 0.5 Hz and a Page 4 of 12 Page 4 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 the different EEG measures to distinguish between MCI- AD and MCI-LB patients. The sensitivity/specificity cut- off was determined using Youden’s index. Hamming window across the power spectrum from 2 to 30 Hz, for each electrode separately. To compensate for inter-individual variability in brain neurophysiology, anatomy, and physical tissue properties, the PSD was normalized by the total power across the power spectrum [10]. To assess the association between the range of Lewy body symptomatology and EEG abnormalities, we per- formed an exploratory analysis in which we compared quantitative EEG measures between MCI-LB patients who had two core symptoms or one core symptom and one abnormal biomarker (N = 13) with those patients who had more than two core symptoms/abnormal bio- markers (N = 26) using two-sample t tests. Furthermore, Spearman’s correlations between symptom/biomarker count (ranging from 2 to 6) and the different EEG mea- sures were computed in the MCI-LB group. For each electrode separately, the mean power across all included epochs was estimated for different standard EEG frequency bands including delta (2–4 Hz), theta (4–5.5 Hz), pre-alpha (5.5–8 Hz), alpha (8–13 Hz), and beta (13– 30 Hz). Higher frequencies were excluded because these are particularly affected by muscular artifacts [11]. The dominant frequency was calculated as the frequency with the highest power between 4 and 15 Hz (averaged across epochs). Dominant frequency was calculated for all elec- trodes as well as from occipital electrodes only (PO9, PO7, POO9h, PO5, O1, PO3, POO3h, OI1h, POz, Oz, PO4, POO4h, PO6, O2, OI2h, PO8, POO10h, PO10). Dominant frequency variability was defined as the stand- ard deviation of dominant frequency across epochs [12]. The association between quantitative EEG measures and overall cognitive impairment was assessed using Spearman’s correlations, in the MCI-AD and MCI-LB groups separately. p values were FDR-corrected for mul- tiple comparisons. Additionally, we investigated the association between EEG characteristics and the core Lewy body symptoms of visual hallucinations and cognitive fluctuations which have been shown to be related to EEG abnormalities in dementia patients [10, 13, 14]. Frequency analysis To this end, two-sample t tests were performed, dichotomizing the MCI-LB group according to the presence/absence of visual hallucina- tions and cognitive fluctuations. Statistics Statistical analyses were performed in SPSS and R (https://www.r-project.org/). Relative power within the different frequency bands was compared between the three groups using a multivariate ANOVA with a within-subject factor of frequency band and a between- subject factor of diagnosis, followed by univariate ANO- VAs and post hoc tests, Bonferroni-corrected for mul- tiple comparisons. Since relative power was not normally distributed in all groups, the variables were log-transformed before applying the ANOVA. Theta/ alpha ratio, dominant frequency, and dominant fre- quency variability were compared between the groups using univariate ANOVAs followed by post hoc tests (Bonferroni-corrected). To account for differences in the number of male and female participants in the three groups, sex was included as a covariate in all analyses. Given previous reports of an effect of acetylcholin- esterase inhibitors on the EEG signal [15, 16], a two- sample t test was performed in the MCI-LB group to compare the EEG characteristics between patients who were taking acetylcholinesterase inhibitors and those pa- tients not taking these medications. Demographics All groups were similar in age (Table 1). The proportion of male participants was higher in the MCI-LB group whereas more MCI-AD patients were female. MCI-LB patients had significantly lower years of education com- pared to controls, but there was no significant difference between the two MCI groups in terms of education. More MCI-LB patients were taking cholinesterase inhib- itors and Parkinson’s disease medication compared to MCI-AD patients. The two MCI groups were matched in terms of overall cognitive impairment. As expected, the MCI-LB group had more parkinsonism, higher cog- nitive fluctuation, and visual hallucination scores com- pared to the MCI-AD group. To test the applicability of this analysis to a clinical setting, the group comparison of relative power within the different frequency bands was repeated only includ- ing the 21 electrodes that are part of the 10-20 system that is routinely used in clinical practice. Additionally, to test whether EEG changes have a differ- ent spatial distribution in the different groups, we con- ducted a supplementary analysis splitting the whole set of electrodes into four macroscopic regions (frontal, central, lateral, and posterior) and applying a repeated measures ANOVA with region as the within-subject factor and diagnosis as the between-subject factor [10]. If the inter- action between region and diagnosis was significant, this was followed up by post hoc univariate ANOVAs. EEG frequency analysis Alzheimer's Research & Therapy (2020) 12:82 Page 5 of 12 Table 1 Demographic and clinical variables, mean (standard deviation) HC (N = 31) MCI-AD (N = 36) MCI-LB (N = 39) Group differences Male to female 22:9 15:21 35:4 χ2 = 20.0, p < 0.001a p(HC,MCI-AD) = 0.02 p(HC,MCI-LB) = 0.045 p(MCI-AD,MCI-LB) < 0.001 Age 73.7 (7.3) 76.1 (7.7) 74.7 (6.4) F(2, 103) = 1.0, p = 0.38b AChEI – 7 (19%)e 18 (46%)f χ2 = 5.7, p = 0.02c PD meds – 0e 4 (10%)f χ2 = 3.8, p = 0.052c Years of education 14.7 (4.0)g 12.9 (3.4)h 12.1 (2.8) F(2, 100) = 5.1, p = 0.008c p(HC,MCI-AD) = 0.12 p(HC,MCI-LB) = 0.006 p(MCI-AD,MCI-LB) = 0.87 ACE-R 92.7 (4.2) 82.4 (8.5) 83.8 (9.2) t73 = 0.7, p = 0.50d MMSE 28.5 (1.1) 26.9 (2.1) 26.6 (2.5) t73 = 0.7, p = 0.51d UPDRS III 5.5 (4.4) 15.1 (13.8) 23.3 (14.2) t73 = 2.5, p = 0.01d DCFS – 6.9 (1.9)i 8.5 (3.3)k t61 = 2.4, p = 0.02d CAF total – 1.4 (2.7)i 3.7 (4.2)k t61 = 2.5, p = 0.02d NPI total – 8.6 (9.3)i 16.4 (12.9)k t61 = 2.7, p = 0.01d NEVHI – 0.8 (1.5)e 2.7 (4.1) t71 = 2.6, p = 0.01d GDS 1.3 (1.8) 3.5 (2.5) 5.1 (4.1) F(2, 103) = 13.4, p < 0.001b p(HC,MCI-AD) = 0.01 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.08 ACE-R Addenbrooke’s Cognitive Examination–Revised, AChEI number of patients taking acetylcholinesterase inhibitors, CAF total clinician assessment of fluctuation total score, DCFS Dementia Cognitive Fluctuation Scale, GDS Geriatric Depression Scale, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies, MMSE Mini-Mental State Examination, NEVHI North-East Visual Hallucinations Interview, NPI Neuropsychiatric Inventory, PD meds number of patients taking dopaminergic medication for the management of Parkinson’s disease symptoms, UPDRS III Unified Parkinson’s Disease Rating Scale III (motor subsection) aChi-square test HC, MCI-AD, MCI-LB bUnivariate ANOVA HC, MCI-AD, MCI-LB cChi-square test MCI-AD, MCI-LB dStudent’s t test MCI-AD, MCI-LB eN = 34 f ACE-R Addenbrooke’s Cognitive Examination–Revised, AChEI number of patients taking acetylcholinesterase inhibitors, CAF total clinician assessment of fluctuation total score, DCFS Dementia Cognitive Fluctuation Scale, GDS Geriatric Depression Scale, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies, MMSE Mini-Mental State Examination, NEVHI North-East Visual Hallucinations Interview, NPI Neuropsychiatric Inventory, PD meds number of patients taking dopaminergic medication for the management of Parkinson’s disease symptoms, UPDRS III Unified Parkinson’s Disease Rating Scale III (motor subsection) aChi-square test HC, MCI-AD, MCI-LB bUnivariate ANOVA HC, MCI-AD, MCI-LB cChi-square test MCI-AD, MCI-LB dStudent’s t test MCI-AD, MCI-LB eN = 34 fN = 38 gN = 29 hN = 35 iN = 27 kN = 36 Figs. EEG frequency analysis 2 and 3): There were no group differences in terms of delta power. Theta power was increased in MCI-LB (mean = 8.8, standard deviation (SD) = 4.8) compared to controls (mean = 5.7, SD = 3.2) with no significant differ- ence between controls and MCI-AD (mean = 7.0, SD = 3.1) or between the two MCI groups. Pre-alpha power was increased in MCI-LB (mean = 28.8, SD = 14.2) and MCI-AD (mean = 19.6, SD = 10.2) compared to controls (mean = 13.3, SD = 9.2), and it was further increased in MCI-LB compared to MCI-AD. Alpha power was de- creased in MCI-LB (mean = 28.3, SD = 14.7) compared to controls (mean = 39.9, SD = 15.8) with no difference between controls and MCI-AD (mean = 35.6, SD = 12.7) or between the two MCI groups. However, when the alpha band was split further into low-alpha (8–10 Hz) and high-alpha (10–13 Hz), it became evident that high- alpha power was significantly reduced in MCI-LB com- pared to both controls (p < 0.001) and MCI-AD (p < 0.001) with no difference between MCI-AD and controls (p = 1.0) while there were no group differences in terms of low-alpha power (F(2, 102) = 1.7, p = 0.2). Beta power was reduced in MCI-LB (mean = 18.9, SD = 11.0) com- pared to both controls (mean = 26.7, SD = 10.3) and MCI-AD (mean = 25.4, SD = 8.7), but not different be- tween MCI-AD and controls. Figs. 2 and 3): There were no group differences in terms of delta power. Theta power was increased in MCI-LB (mean = 8.8, standard deviation (SD) = 4.8) compared to controls (mean = 5.7, SD = 3.2) with no significant differ- ence between controls and MCI-AD (mean = 7.0, SD = 3.1) or between the two MCI groups. Pre-alpha power was increased in MCI-LB (mean = 28.8, SD = 14.2) and MCI-AD (mean = 19.6, SD = 10.2) compared to controls (mean = 13.3, SD = 9.2), and it was further increased in MCI-LB compared to MCI-AD. Alpha power was de- creased in MCI-LB (mean = 28.3, SD = 14.7) compared to controls (mean = 39.9, SD = 15.8) with no difference between controls and MCI-AD (mean = 35.6, SD = 12.7) or between the two MCI groups. EEG frequency analysis The multivariate ANOVA revealed an overall effect of diagnosis: F(10, 196) = 5.1, p < 0.001. Follow-up univari- ate ANOVAs showed the following (see Table 2 and A receiver operating characteristic (ROC) analysis was conducted in R to assess the sensitivity and specificity of Schumacher et al. EEG frequency analysis Sex was included as a covariate DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies of using all electrodes. Dominant frequency variability was not significantly different between the groups. y g Area under the ROC curve (AUC) values, sensitivity, and specificity for the different EEG measures for differ- entiating between MCI-LB and MCI-AD are shown in Table 3. Beta power and dominant frequency achieved the highest AUC values of 0.71 and 0.70, respectively. Specificity was high for some measures (up to 0.97 for alpha power); however, sensitivity was generally much lower. The results did not change when restricting the ana- lysis to the 21 electrodes that are part of the 10-20 sys- tem (see Supplementary Table S1). The regional analysis showed very similar results compared to the analysis where all electrodes were combined (see Supplementary Table S2). The only slight difference was that alpha power was signifi- cantly reduced in MCI-LB compared to MCI-AD pa- tients in posterior (p = 0.047) and lateral (p = 0.01) regions whereas mean alpha power from all electrodes was not significantly different between the two MCI groups (p = 0.07). EEG frequency analysis Alzheimer's Research & Therapy (2020) 12:82 Page 6 of 12 Table 2 Group comparison of quantitative EEG characteristics HC MCI-AD MCI-LB Group comparison Delta power 14.1 [11.3, 17.0] 14.1 [11.8, 16.3] 14.9 [12.7, 17.1] F(2, 102) = 0.8, p = 0.47 Theta power 5.7 [4.6, 6.9] 7.0 [6.0, 8.1] 8.8 [7.2, 10.3] F(2, 102) = 4.8, p = 0.01 p(HC,MCI-AD) = 0.19 p(HC,MCI-LB) = 0.004 p(MCI-AD,MCI-LB) = 0.46 Pre-alpha power 13.3 [9.9, 16.7] 19.6 [16.1, 23.1] 28.8 [24.2, 33.4] F(2, 102) = 16.0, p < 0.001 p(HC,MCI-AD) = 0.006 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.02 Alpha power 39.9 [34.1, 45.6] 33.6 [29.3, 37.9] 28.3 [23.6, 33.1] F(2, 102) = 5.5, p = 0.005 p(HC,MCI-AD) = 0.39 p(HC,MCI-LB) = 0.001 p(MCI-AD,MCI-LB) = 0.07 Beta power 26.7 [22.9, 30.4] 25.4 [22.4, 28.4] 18.9 [15.3, 22.5] F(2, 102) = 8.1, p = 0.001 p(HC,MCI-AD) = 1.0 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.001 Theta/alpha ratio 0.34 [0.27, 0.40] 0.42 [0.36, 0.47] 0.51 [0.44, 0.57] F(2, 102) = 7.5, p < 0.001 p(HC,MCI-AD) = 0.22 p(HC,MCI-LB) = 0.001 p(MCI-AD,MCI-LB) = 0.10 DF, all electrodes 8.4 [8.0, 8.9] 8.0 [7.6, 8.3] 7.2 [6.8, 7.6] F(2, 102) = 8.7, p < 0.001 p(HC,MCI-AD) = 0.29 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.01 DF, occipital electrodes 8.5 [8.1, 9.0] 8.0 [7.6, 8.3] 7.3 [6.9, 7.7] F(2, 102) = 8.7, p < 0.001 p(HC,MCI-AD) = 0.16 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.03 DFV, occipital electrodes 1.3 [0.9, 1.6] 1.4 [1.1, 1.7] 1.0 [0.8, 1.2] F(2, 102) = 3.6, p = 0.03 p(HC,MCI-AD) = 0.54 p(HC,MCI-LB) = 1.0 p(MCI-AD,MCI-LB) = 0.17 Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc tests, Bonferroni-corrected for multiple comparisons. EEG frequency analysis Sex was included as a covariate DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies Table 2 Group comparison of quantitative EEG characteristics HC MCI-AD MCI-LB Group comparison Delta power 14.1 [11.3, 17.0] 14.1 [11.8, 16.3] 14.9 [12.7, 17.1] F(2, 102) = 0.8, p = 0.47 Theta power 5.7 [4.6, 6.9] 7.0 [6.0, 8.1] 8.8 [7.2, 10.3] F(2, 102) = 4.8, p = 0.01 p(HC,MCI-AD) = 0.19 p(HC,MCI-LB) = 0.004 p(MCI-AD,MCI-LB) = 0.46 Pre-alpha power 13.3 [9.9, 16.7] 19.6 [16.1, 23.1] 28.8 [24.2, 33.4] F(2, 102) = 16.0, p < 0.001 p(HC,MCI-AD) = 0.006 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.02 Alpha power 39.9 [34.1, 45.6] 33.6 [29.3, 37.9] 28.3 [23.6, 33.1] F(2, 102) = 5.5, p = 0.005 p(HC,MCI-AD) = 0.39 p(HC,MCI-LB) = 0.001 p(MCI-AD,MCI-LB) = 0.07 Beta power 26.7 [22.9, 30.4] 25.4 [22.4, 28.4] 18.9 [15.3, 22.5] F(2, 102) = 8.1, p = 0.001 p(HC,MCI-AD) = 1.0 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.001 Theta/alpha ratio 0.34 [0.27, 0.40] 0.42 [0.36, 0.47] 0.51 [0.44, 0.57] F(2, 102) = 7.5, p < 0.001 p(HC,MCI-AD) = 0.22 p(HC,MCI-LB) = 0.001 p(MCI-AD,MCI-LB) = 0.10 DF, all electrodes 8.4 [8.0, 8.9] 8.0 [7.6, 8.3] 7.2 [6.8, 7.6] F(2, 102) = 8.7, p < 0.001 p(HC,MCI-AD) = 0.29 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.01 DF, occipital electrodes 8.5 [8.1, 9.0] 8.0 [7.6, 8.3] 7.3 [6.9, 7.7] F(2, 102) = 8.7, p < 0.001 p(HC,MCI-AD) = 0.16 p(HC,MCI-LB) < 0.001 p(MCI-AD,MCI-LB) = 0.03 DFV, occipital electrodes 1.3 [0.9, 1.6] 1.4 [1.1, 1.7] 1.0 [0.8, 1.2] F(2, 102) = 3.6, p = 0.03 p(HC,MCI-AD) = 0.54 p(HC,MCI-LB) = 1.0 p(MCI-AD,MCI-LB) = 0.17 Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc tests, Bonferroni-corrected for multiple comparisons. Sex was included as a covariate DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies DFV, occipital electrodes 1.3 [0.9, 1.6] 1.4 [1.1, 1.7] 1.0 [0.8, 1.2] Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc tests, Bonferroni-corrected for multiple comparisons. Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc tests, Bonferroni-corrected for multiple comparisons. Sex was included as a covariate DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies EEG frequency analysis However, when the alpha band was split further into low-alpha (8–10 Hz) and high-alpha (10–13 Hz), it became evident that high- alpha power was significantly reduced in MCI-LB com- pared to both controls (p < 0.001) and MCI-AD (p < The theta/alpha ratio was increased in MCI-LB (mean = 0.51, SD = 0.21) compared to controls (mean = 0.34, SD = 0.17), but not significantly different between MCI-AD (mean = 0.42, SD = 0.16) and controls or be- tween the two MCI groups. Dominant frequency was slower in MCI-LB (mean = 7.2, SD = 1.1) compared to both MCI-AD (mean = 8.0, SD = 1.1) and controls (mean = 8.4, SD = 1.2), but there was no significant difference between controls and MCI- AD. These results did not change when calculating the dominant frequency from the occipital electrodes instead Schumacher et al. Discussion In this study, we investigated the differences in quantita- tive EEG measures between highly phenotyped patients with MCI-AD and MCI-LB and in comparison with similarly aged healthy controls. We showed that there are significant differences between the two MCI groups at the group level with more severe EEG abnormalities in MCI-LB compared to MCI-AD and that these EEG changes correlated with the burden of Lewy body fea- tures, supporting their high specificity for Lewy body disease. However, their diagnostic accuracy for differen- tiating MCI-LB from MCI-AD is modest and not con- sistent with previous studies [17]. Fig. 2 Mean power spectra for the three diagnostic groups. Shaded areas indicate standard errors. HC, healthy controls; MCI-AD, mild cognitive impairment with Alzheimer’s disease; MCI-LB, probable mild cognitive impairment with Lewy bodies hallucinations (N = 30, mean (SD) = 31.1 (15.4); t37 = 2.5, p = 0.02; Fig. 4a). Additionally, the theta/alpha ratio was increased in patients with visual hallucinations (mean (SD) = 0.61 (0.13)) compared to patients without visual hallucinations (mean (SD) = 0.47 (0.22), t24 = 2.4, p = 0.02, Fig. 4b). Overall, the findings suggest a slowing of the EEG in MCI-LB patients compared to healthy controls by a shift in power from beta and alpha frequency bands towards slower frequencies in the pre-alpha and theta range. This was also reflected by a shift of the dominant frequency towards slower frequencies. These findings are consist- ent with the results from previous EEG studies at the de- mentia stage which generally report an increase in slow- wave activity and slowing of the dominant EEG rhythm in DLB [10, 18–20]. A slowing of the dominant fre- quency has also been reported in MCI-LB patients be- fore [21]. None of the EEG measures showed a significant differ- ence between those MCI-LB patients with cognitive fluc- tuations (N = 21) compared to those without cognitive fluctuations (N = 18) or between MCI-LB patients taking cholinesterase inhibitors (N = 18) compared to those pa- tients not taking cholinesterase inhibitors (N = 20, all p > 0.1). Similarly, EEG measures were not different be- tween MCI-AD patients taking cholinesterase inhibitors (N = 7) compared to those MCI-AD patients not taking these medications (N = 27). We did not observe any group differences in dominant frequency variability which has previously been found to be increased in DLB patients compared to controls [12, 17, 19]. Association with clinical symptoms There was a significant reduction in alpha power in MCI-LB patients with visual hallucinations (N = 9, mean (SD) = 19.1 (6.4)) compared to those without visual Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 7 of 12 Page 7 of 12 Schumacher et al. Alzheimer's Research & Therapy Fig. 2 Mean power spectra for the three diagnostic groups. Shaded areas indicate standard errors. HC, healthy controls; MCI-AD, mild cognitive impairment with Alzheimer’s disease; MCI-LB, probable mild cognitive impairment with Lewy bodies in the MCI-AD group (Supplementary Table S4). In the MCI-LB group, more severe cognitive impairment corre- lated with higher theta power, higher pre-alpha power, lower alpha power, higher theta/alpha ratio, and de- creased dominant frequency (Supplementary Table S4). Discussion In each boxplot, the central line corresponds to the sample median; the upper and lower border of the box represent the 25th and 75th percentile, respectively; and the length of the whiskers is 1.5 times the interquartile range. Corresponding results from statistical comparisons between the groups are presented in Table 2. DF, dominant frequency; DFV, dominant frequency variability; HC, healthy controls; MCI-AD, mild cognitive impairment with Alzheimer’s disease; MCI-LB, probable mild cognitive impairment with Lewy bodies In a previous EEG study in MCI patients, Bonanni et al. [17] found that all MCI patients who converted to DLB within a 3-year follow-up period had an abnormal EEG whereas 93% of MCI patients who developed AD within the follow-up period had normal EEGs. The high specificity of EEG abnormalities for MCI-LB in this re- port is in line with our findings whereas the high sensi- tivity stands in contrast to the findings of the present study. However, the sample in this previous study was more selective by only including patients who developed dementia within 3 years and might therefore be biased towards MCI patients with more severe disease and thus having a rapid progression to dementia. When also tak- ing into account those MCI patients who did not con- vert to dementia within 3 years, diagnostic accuracy was reduced to an overall predictive value of 76% [17]. Fur- thermore, in a multi-center study in dementia patients, the diagnostic accuracy of EEG was also found to be much lower [19], indicating that heterogeneity in EEG acquisition and analysis protocols across different cen- ters might be an important limiting factor. between more widespread Lewy body disease and more severe EEG abnormalities and suggests that Lewy body disease might affect electro-cortical activity in a dose- dependent manner. While there were significant group-level differences in EEG characteristics between the two MCI groups, it is difficult to translate this, at present, into a measure for use in early clinical diagnosis. Power in the beta fre- quency range and dominant frequency had the highest diagnostic accuracy; however, they only reached moder- ate AUC values of around 0.7. Discussion However, other studies have failed to replicate these findings [10, 18, 22], and our results further indi- cate that an increase in dominant frequency variability may not be a reliable feature of Lewy body disease. MCI-LB patients with more than two core symptoms/ abnormal biomarkers had significantly higher pre-alpha power, lower alpha power, and a higher theta/alpha ratio compared to MCI-LB patients with only two core symp- toms or one core symptom and one abnormal biomarker (Supplementary Table S3). Higher delta power, theta power, pre-alpha power, and theta/alpha ratio correlated with higher Lewy body symptom/biomarker count whereas higher alpha power, beta power, and dominant frequency correlated with lower Lewy body symptom/ biomarker count (Supplementary Table S3). There were no significant differences in overall cognition (ACE-R scores) between MCI-LB patients with more than two core symptoms/abnormal biomarkers compared to pa- tients with two symptoms, and the Lewy body symptom/ biomarker count was not significantly correlated with the ACE-R score (see Supplementary Table S3). The only difference between MCI-AD patients and healthy controls was an increase in pre-alpha power, suggesting only a small degree of EEG slowing. This is again consistent with previous reports at the dementia stage which have generally found less severe EEG abnor- malities in AD compared to DLB [10, 12, 19]. Addition- ally, we found significantly increased pre-alpha power, decreased beta power, and slower dominant frequency in the MCI-LB compared to the MCI-AD group, which indicates more severe EEG slowing in MCI-LB than in MCI-AD patients and mirrors the findings in dementia patients. Furthermore, we found more severe EEG ab- normalities in MCI-LB patients with more core Lewy body symptoms and/or abnormal biomarkers and a higher symptom count correlated with more severe EEG slowing. This indicates that there is an association More severe cognitive impairment (as measured by the ACE-R) was correlated with higher pre-alpha power, lower alpha power, and decreased dominant frequency Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 8 of 12 Schumacher et al. Alzheimer's Research & Therapy Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page ) Fig. 3 (See legend on next page.) Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 9 of 12 (See figure on previous page.) Fig. 3 Group comparison of quantitative EEG characteristics. a under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheime e mild cognitive impairment with Lewy bodies The sensitivity/specificity cutoff was determined using Youden s index AUC area under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies Discussion The specificity for MCI- LB was very high for certain EEG measures (up to 0.97 for alpha power with a cutoff of < 20.5) which suggests that if a substantial shift of power towards slower fre- quencies is observed, i.e., in the case of a very abnormal EEG, a diagnosis of MCI-LB over MCI-AD becomes highly likely. The relationship between EEG slowing and Lewy body symptom count (see above) suggests that these might be the MCI-LB patients with more wide- spread Lewy body disease and these are easier to distin- guish from MCI-AD patients based on their more severe EEG slowing. However, sensitivity was generally much lower, i.e., in the case of a more normal EEG, differenti- ating between MCI-AD and MCI-LB is difficult and many MCI-LB cases would be missed by applying these measures for diagnostic purposes. These findings thus show that early changes in quantitative EEG characteris- tics are specific, but not sensitive to Lewy body disease which is in alignment with the diagnostic performance of other biomarkers such as FP-CIT and MIBG in early Lewy body disease [8]. Another previous EEG study that attempted to differ- entiate MCI-AD from MCI-LB did not find any EEG measures that reliably distinguished between the two MCI subgroups [21]. However, this was a retrospective study of selected MCI-AD and MCI-LB patients who were recruited using clinical criteria which differed across the several centers who supplied patient data. Furthermore, participants from the different centers were also assessed using different EEG protocols which again makes the comparison difficult [21]. Conclusions In conclusion, the present study suggests that early EEG slowing is specific to MCI-LB and, from a diagnos- tic point of view, a very abnormal EEG favors MCI-LB over MCI-AD. However, the overlap between the two MCI groups is large, and for patients with a more nor- mal EEG, it is difficult to distinguish between the two MCI groups based on EEG characteristics alone. Given its specificity for Lewy body disease burden, EEG may be a promising future diagnostic biomarker and may have value as part of a panel of other biomarkers. To investigate this further, there needs to be a consensus regarding the standardization of acquisition protocols, analysis approaches, and choice of EEG parameters. Work in this field will undoubtedly yield benefits simi- lar to those seen with CSF biomarkers for example [23]. More complex EEG analyses such as functional con- nectivity or source reconstruction methods may pro- vide better sensitivity for detecting differences between the two MCI groups as well as between MCI-AD pa- tients and controls [21, 24]. Furthermore, multimodal approaches including EEG features in addition to MIBG/FP-CIT or structural MRI markers may be an- other route by which it will be possible to achieve bet- ter accuracy for the early distinction between AD and DLB at the MCI stage. Fig. 4 Comparison of quantitative EEG characteristics between patients with and without visual hallucinations. a Comparison of alpha power and b theta/alpha ratio between MCI-LB patients with visual hallucinations (VH+, N = 9) and without visual hallucinations (VH−, N = 30). In each boxplot, the central line corresponds to the sample median; the upper and lower border of the box represent the 25th and 75th percentile, respectively; and the length of the whiskers is 1.5 times the interquartile range We explored the influence of the presence of visual hallucinations on quantitative EEG characteristics in MCI-LB patients and found that early EEG slowing, both in terms of a reduction of power in the alpha frequency range and a shift of power towards slower frequencies, might be more severe in patients who experience visual hallucinations compared to those patients who do not have visual hallucinations, a finding which has previously been reported in dementia patients [14]. Conclusions However, the number of patients experiencing visual hallucinations in our cohort was small (N = 9) and these findings should therefore be interpreted with caution, and replication in a larger group of patients with a higher occurrence of visual hallucinations is needed. Discussion Table 3 Results from receiver operating characteristic (ROC) analysis to distinguish MCI-AD from MCI-LB EEG measure AUC [95% CI] Cutoff for MCI-LB Sensitivity Specificity Delta power 0.54 [0.41, 0.67] > 21.9 0.23 0.89 Theta power 0.60 [0.47, 0.73] > 10.7 0.33 0.89 Pre-alpha power 0.68 [0.56, 0.81] > 28.1 0.56 0.83 Alpha power 0.66 [0.53, 0.78] < 20.5 0.41 0.97 Beta power 0.71 [0.59, 0.83] < 19.0 0.61 0.81 Theta/alpha ratio 0.64 [0.51, 0.77] > 0.56 0.49 0.83 DF, all electrodes 0.70 [0.58, 0.82] < 7.1 0.51 0.86 DF, occipital electrodes 0.69 [0.57, 0.81] < 7.1 0.51 0.86 The sensitivity/specificity cutoff was determined using Youden’s index AUC area under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies AUC area under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable mild cognitive impairment with Lewy bodies Page 10 of 12 Page 10 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Schumacher et al. Alzheimer's Research & Therapy Fig. 4 Comparison of quantitative EEG characteristics between patients with and without visual hallucinations. a Comparison of alpha power and b theta/alpha ratio between MCI-LB patients with visual hallucinations (VH+, N = 9) and without visual hallucinations (VH−, N = 30). In each boxplot, the central line corresponds to the sample median; the upper and lower border of the box represent the 25th and 75th percentile, respectively; and the length of the whiskers is 1.5 times the interquartile range MCI-LB patients who were taking acetylcholinesterase inhibitors to those who were not, we did not find any significant differences in any EEG measures. The same was found in the MCI-AD patients although this result should be interpreted with caution because of the small number of MCI-AD patients who were taking acetyl- cholinesterase inhibitors (N = 7). Furthermore, it would be unethical to withdraw medication, and including medicated patients is more reflective of clinical practice. Another potential limitation is the lack of AD bio- markers in our study. Additional file 1: Table S1. Group comparison of quantitative EEG characteristics, restricting the analysis to 21 electrodes from the 10-20 sys- tem. Table S2. Group comparison of quantitative EEG characteristics, splitting the set of electrodes into four macroscopic regions. Table S3. Association between Lewy body symptom severity and EEG characteris- tics in the MCI-LB group. Two-sample t-tests comparing EEG measures between MCI-LB patients with two symptoms/biomarkers (N=13) and MCI-LB patients with more than two symptoms/biomarkers (N=26) and Spearman’s correlations between EEG characteristics and symptom/bio- marker count (ranging from 2 to 6). P-values are FDR-corrected for mul- tiple comparisons. Table S4. Association between the severity of overall cognitive impairment (ACE-R scores) and EEG characteristics in the MCI- LB and MCI-AD groups using Spearman’s correlations. P-values are FDR- corrected for multiple comparisons. Limitations Strengths of the present study include consistent and ro- bust clinical assessments, diagnoses by a consensus panel, the use of two diagnostic biomarkers, and the prospective longitudinal design of the study. A potential limitation is the use of acetylcholinesterase inhibitors in the MCI patients as more patients in the MCI-LB group were taking these medications compared to the MCI-AD group. In the north east of England, services implement expert advice and thus have high prescription rates for DLB/MCI-LB patients because neuropsychiatric symp- toms are treated using anti-dementia drugs early on. It has been shown that cholinergic medication can normalize EEG measures [15, 16], and it is therefore possible that differences between the two MCI groups were occluded by the higher number of MCI-LB patients taking these medications. However, when comparing Author details 1 Author details 1Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Biomedical Research Building 3rd floor, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK. 2Nuclear Medicine Department, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. 3Institute of Health Research, University of Exeter, Exeter, UK. 4Department of Psychiatry, University of Cambridge School of Medicine, Cambridge CB2 0SP, UK. 16. Babiloni C, Del Percio C, Bordet R, Bourriez JL, Bentivoglio M, Payoux P, et al. Effects of acetylcholinesterase inhibitors and memantine on resting-state electroencephalographic rhythms in Alzheimer’s disease patients. Clin Neurophysiol. 2013;124:837–50. https://doi.org/10.1016/j.clinph.2012.09.017. 1Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Biomedical Research Building 3rd floor, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK. 2Nuclear Medicine Department, Newcastle upon Tyne Hospitals NHS Foundation Trust, 3 17. Bonanni L, Perfetti B, Bifolchetti S, Taylor J-P, Franciotti R, Parnetti L, et al. Quantitative electroencephalogram utility in predicting conversion of mild cognitive impairment to dementia with Lewy bodies. Neurobiol Aging. 2015;36:434–45 Available from: http://linkinghub.elsevier.com/retrieve/pii/ S0197458014004783. Funding h This research was funded by the Alzheimer’s Research UK (ARUK-PG2015-13) and supported by the NIHR Newcastle Biomedical Research Centre (grant numbers BH120812 and BH120878). GE Healthcare provided the FP-CIT radi- oligand for this investigator-led study. 12. Bonanni L, Thomas A, Tiraboschi P, Perfetti B, Varanese S, Onofrj M. EEG comparisons in early Alzheimer’s disease, dementia with Lewy bodies and Parkinson’s disease with dementia patients with a 2-year follow-up. Brain. 2008;131:690–705 Available from: http://www.brain.oxfordjournals.org/cgi/ doi/10.1093/brain/awm322. Consent for publication Consent for publication Not applicable Not applicable 15. Kai T, Asai Y, Sakuma K, Koeda T, Nakashima K. Quantitative electroencephalogram analysis in dementia with Lewy bodies and Alzheimer’s disease. J Neurol Sci. 2005;237:89–95 Available from: http:// linkinghub.elsevier.com/retrieve/pii/S0022510X05002005. 15. Kai T, Asai Y, Sakuma K, Koeda T, Nakashima K. Quantitative electroencephalogram analysis in dementia with Lewy bodies and Alzheimer’s disease. J Neurol Sci. 2005;237:89–95 Available from: http:// linkinghub.elsevier.com/retrieve/pii/S0022510X05002005. Competing interests p g The authors declare that they have no competing interests. Received: 13 May 2020 Accepted: 2 July 2020 18. Peraza LR, Cromarty RA, Kobeleva X, Firbank MJ, Killen A, Graziadio S, et al. Electroencephalographic derived network differences in Lewy body dementia compared to Alzheimer’s disease patients. Sci Rep. 2018;8:4637 Available from: http://www.nature.com/articles/s41598-018-22984-5. Supplementary information f Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s13195-020-00650-1. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13195-020-00650-1. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13195-020-00650-1. Additional file 1: Table S1. Group comparison of quantitative EEG characteristics, restricting the analysis to 21 electrodes from the 10-20 sys- tem. Table S2. Group comparison of quantitative EEG characteristics, splitting the set of electrodes into four macroscopic regions. Table S3. Association between Lewy body symptom severity and EEG characteris- tics in the MCI-LB group. Two-sample t-tests comparing EEG measures between MCI-LB patients with two symptoms/biomarkers (N=13) and MCI-LB patients with more than two symptoms/biomarkers (N=26) and Spearman’s correlations between EEG characteristics and symptom/bio- marker count (ranging from 2 to 6). P-values are FDR-corrected for mul- tiple comparisons. Table S4. Association between the severity of overall cognitive impairment (ACE-R scores) and EEG characteristics in the MCI- LB and MCI-AD groups using Spearman’s correlations. P-values are FDR- corrected for multiple comparisons. Page 11 of 12 Page 11 of 12 Page 11 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Authors’ contributions 9. Delorme A, Makeig S. EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods. 2004;134:9–21 Available from: https://linkinghub.elsevier. com/retrieve/pii/S0160791X13000638. 9. Delorme A, Makeig S. EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods. 2004;134:9–21 Available from: https://linkinghub.elsevier. com/retrieve/pii/S0160791X13000638. JS analyzed and interpreted the data, performed the statistical analyses, and wrote the manuscript. CAH, MF, RAC, GR, RD, and NB acquired the data and revised the manuscript. PCD acquired the data, diagnosed the patients, and revised the manuscript. MF, LA, JL, and JTOB conceived the study, participated in its design and coordination, and revised the manuscript. JPT and AJT conceived the study, participated in its design and coordination, diagnosed the patients, interpreted the data, and revised the manuscript. All authors read and approved the final version of the manuscript. JS analyzed and interpreted the data, performed the statistical analyses, and wrote the manuscript. CAH, MF, RAC, GR, RD, and NB acquired the data and revised the manuscript. PCD acquired the data, diagnosed the patients, and revised the manuscript. MF, LA, JL, and JTOB conceived the study, 10. Stylianou M, Murphy N, Peraza LR, Graziadio S, Cromarty RA, Killen A, et al. Quantitative electroencephalography as a marker of cognitive fluctuations in dementia with Lewy bodies and an aid to differential diagnosis. Clin Neurophysiol. 2018;129:1209–20 Available from: http://linkinghub.elsevier. com/retrieve/pii/S1388245718302670. 10. Stylianou M, Murphy N, Peraza LR, Graziadio S, Cromarty RA, Killen A, et al. Quantitative electroencephalography as a marker of cognitive fluctuations in dementia with Lewy bodies and an aid to differential diagnosis. Clin Neurophysiol. 2018;129:1209–20 Available from: http://linkinghub.elsevier. com/retrieve/pii/S1388245718302670. diagnosed the patients, interpreted the data, and revised the manuscript. All authors read and approved the final version of the manuscript. 11. Whitham EM, Pope KJ, Fitzgibbon SP, Lewis T, Clark CR, Loveless S, et al. Scalp electrical recording during paralysis: quantitative evidence that EEG frequencies above 20Hz are contaminated by EMG. Clin Neurophysiol. 2007; 118:1877–88 Available from: https://linkinghub.elsevier.com/retrieve/pii/S13 88245707001988. 11. Whitham EM, Pope KJ, Fitzgibbon SP, Lewis T, Clark CR, Loveless S, et al. Scalp electrical recording during paralysis: quantitative evidence that EEG frequencies above 20Hz are contaminated by EMG. Clin Neurophysiol. 2007; 118:1877–88 Available from: https://linkinghub.elsevier.com/retrieve/pii/S13 88245707001988. Abbreviations characteristic; UPDRS-III: Unified Parkinson’s Disease Rating Scale motor sub- score 8. Thomas AJ, Donaghy P, Roberts G, Colloby SJ, Barnett NA, Petrides G, et al. Diagnostic accuracy of dopaminergic imaging in prodromal dementia with Lewy bodies. Psychol Med. 2019;49:396–402 Available from: https://www. cambridge.org/core/product/identifier/S0033291718000995/type/journal_ article. Availability of data and materials The data that support the findings of this study are available from the corresponding author, upon reasonable request. 13. Walker MP, Ayre GA, Cummings JL, Wesnes KA, McKeith IG, O’Brien JT, et al. Quantifying fluctuation in dementia with Lewy bodies, Alzheimer’s disease, and vascular dementia. Neurol Int. 2000;54:1616–25. https://doi.org/10.1212/ WNL.54.8.1616. 13. Walker MP, Ayre GA, Cummings JL, Wesnes KA, McKeith IG, O’Brien JT, et al. Quantifying fluctuation in dementia with Lewy bodies, Alzheimer’s disease, and vascular dementia. Neurol Int. 2000;54:1616–25. https://doi.org/10.1212/ WNL.54.8.1616. Ethics approval and consent to participate Ethics approval and consent to participate The study was approved by the Newcastle & North Tyneside 2 Research Ethics Committee (reference number: 15/NE/0420). Written informed consent was obtained from all participants prior to study participation. Ethics Committee (reference number: 15/NE/0420). Written informed consent was obtained from all participants prior to study participation. Ethics Committee (reference number: 15/NE/0420). Written informed consent was obtained from all participants prior to study participation. 14. Dauwan M, Linszen MMJ, Lemstra AW, Scheltens P, Stam CJ, Sommer IE. EEG-based neurophysiological indicators of hallucinations in Alzheimer’s disease: comparison with dementia with Lewy bodies. Neurobiol Aging. 2018;67:75–83 Available from: https://linkinghub.elsevier.com/retrieve/pii/ S0197458018300915. 14. Dauwan M, Linszen MMJ, Lemstra AW, Scheltens P, Stam CJ, Sommer IE. EEG-based neurophysiological indicators of hallucinations in Alzheimer’s disease: comparison with dementia with Lewy bodies. Neurobiol Aging. 2018;67:75–83 Available from: https://linkinghub.elsevier.com/retrieve/pii/ S0197458018300915. Abbreviations 3. Albert MS, DeKosky ST, Dickson D, Dubois B, Feldman HH, Fox NC, et al. The diagnosis of mild cognitive impairment due to Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimer’s Dement. 2011;7:270–9. https://doi.org/10.1016/j.jalz.2011.03.008. ACE-R: Addenbrooke’s Cognitive Examination–Revised; AD: Alzheimer’s disease; AUC: Area under the ROC curve; CAF: Clinician assessment of fluctuations; CDR: Clinical Dementia Rating; CIRS-G: Cumulative Illness Rating Scale for Geriatrics; DCFS: Dementia Cognitive Fluctuations Scale; DLB: Dementia with Lewy bodies; EEG: Electroencephalography; ESS: Epworth Sleepiness Scale; FDR: False discovery rate; FP-CIT: 123I-N- fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) single-photon emission computed tomography; GDS: Geriatric Depression Scale; IADL: Instrumental Activities of Daily Living; MCI-AD: Mild cognitive impairment with Alzheimer’s disease; MCI-LB: Mild cognitive impairment with Lewy bodies; MIBG: 123Iodine-metaiodobenzylguanidine myocardial scintigraphy; MSQ: Mayo Sleep Questionnaire; MMSE: Mini-Mental State Examination; NEVHI: North-East Visual Hallucinations Interview; NPI: Neuropsychiatric Inventory; PSD: Power spectral density; ROC: Receiver operating characteristic; UPDRS-III: Unified Parkinson’s Disease Rating Scale motor sub- score 4. Donaghy PC, O’Brien JT, Thomas AJ. Prodromal dementia with Lewy bodies Psychol Med. 2015;45:259–68. 4. Donaghy PC, O’Brien JT, Thomas AJ. Prodromal dementia with Lewy bodies. Psychol Med. 2015;45:259–68. 5. Donaghy PC, Taylor JP, O’Brien JT, Barnett N, Olsen K, Colloby SJ, et al. Neuropsychiatric symptoms and cognitive profile in mild cognitive impairment with Lewy bodies. Psychol Med. 2018;48:2384–90. 5. Donaghy PC, Taylor JP, O’Brien JT, Barnett N, Olsen K, Colloby SJ, et al. Neuropsychiatric symptoms and cognitive profile in mild cognitive impairment with Lewy bodies. Psychol Med. 2018;48:2384–90. 6. McKeith IG, Ferman TJ, Thomas AJ, Blanc F, Boeve BF, Fujishiro H, et al. Research criteria for the diagnosis of prodromal dementia with Lewy bodies. Neurology. 2020;94(17):743–755. 6. McKeith IG, Ferman TJ, Thomas AJ, Blanc F, Boeve BF, Fujishiro H, et al. Research criteria for the diagnosis of prodromal dementia with Lewy bodies. Neurology. 2020;94(17):743–755. 6. McKeith IG, Ferman TJ, Thomas AJ, Blanc F, Boeve BF, Fujishiro H, et al. Research criteria for the diagnosis of prodromal dementia with Lewy bodies. Neurology. 2020;94(17):743–755. 7. McKeith IG, Boeve BF, Dickson DW, Halliday G, Aarsland D, Attems J, et al. Diagnosis and management of dementia with Lewy bodies fourth consensus report of the DLB Consortium. Neurology. 2017;0:1–13. 7. McKeith IG, Boeve BF, Dickson DW, Halliday G, Aarsland D, Attems J, et al. Diagnosis and management of dementia with Lewy bodies fourth consensus report of the DLB Consortium. Neurology. 2017;0:1–13. 2016;127:349–59 Available from: http://linkinghub.elsevier.com/retrieve/pii/ S1388245715006768. 2016;127:349–59 Available from: http://linkinghub.elsevier.com/retrieve/pii/ S1388245715006768. 21. Babiloni C, Del Percio C, Lizio R, Noce G, Lopez S, Soricelli A, et al. Abnormalities of resting state cortical EEG rhythms in subjects with mild cognitive impairment due to Alzheimer’s and Lewy body diseases. J Alzheimer’s Dis. 2018;62:247–68 Available from: http://www.medra.org/ servlet/aliasResolver?alias=iospress&doi=10.3233/JAD-170703. 22. van der Zande JJ, Gouw AA, van Steenoven I, Scheltens P, Stam CJ, Lemstra AW. EEG characteristics of dementia with Lewy bodies, Alzheimer’s disease and mixed pathology. Front Aging Neurosci. 2018;10:1–10 Available from: https://www.frontiersin.org/article/10.3389/fnagi.2018.00190/full. 23. Sonnen JA, Montine KS, Quinn JF, Breitner JCS, Montine TJ. Cerebrospinal fluid biomarkers in mild cognitive impairment and dementia. J Alzheimers Dis. 2010;19:301–9. 24. Babiloni C, Del Percio C, Lizio R, Noce G, Lopez S, Soricelli A, et al. Functional cortical source connectivity of resting state electroencephalographic alpha rhythms shows similar abnormalities in patients with mild cognitive impairment due to Alzheimer’s and Parkinson’s diseases. Clin Neurophysiol. 2018;129:766–82. 24. Babiloni C, Del Percio C, Lizio R, Noce G, Lopez S, Soricelli A, et al. Functional cortical source connectivity of resting state electroencephalographic alpha rhythms shows similar abnormalities in patients with mild cognitive impairment due to Alzheimer’s and Parkinson’s diseases. Clin Neurophysiol. 2018;129:766–82. References 1. Taylor J-P, McKeith IG, Burn DJ, Boeve BF, Weintraub D, Bamford C, et al. New evidence on the management of Lewy body dementia. Lancet Neurol. 2020;19:157–69. https://doi.org/10.1016/S1474-4422(19)30153-X. 19. Bonanni L, Franciotti R, Nobili F, Kramberger MG, Taylor JP, Garcia-Ptacek S, et al. EEG markers of dementia with Lewy bodies: a multicenter cohort study. J Alzheimers Dis. 2016;54:1649–57. 2. Petersen RC, Smith GE, Waring SC, Ivnik RJ, Tangalos EG, Kokmen E. Mild cognitive impairment. Arch Neurol. 1999;56:303 Available from: http:// archneur.jamanetwork.com/article.aspx?doi=10.1001/archneur.56.3.303. 20. Cromarty RA, Elder GJ, Graziadio S, Baker M, Bonanni L, Onofrj M, et al. Neurophysiological biomarkers for Lewy body dementias. Clin Neurophysiol. Page 12 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 (2020) 12:82 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.
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Evaluating the Genetics of Common Variable Immunodeficiency: Monogenetic Model and Beyond
Frontiers in immunology
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Specialty section: Specialty section: This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and poor response to vaccines. Its diagnosis is made based on clinical and immunological criteria, after exclusion of other diseases that can cause similar phenotypes. Currently, less than 20% of cases of CVID have a known underlying genetic cause. We have analyzed whole-exome sequencing and copy number variants data of 36 children and adolescents diagnosed with CVID and healthy relatives to estimate the propor- tion of monogenic cases. We have replicated an association of CVID to p.C104R in TNFRSF13B and reported the second case of homozygous patient to date. Our results also identify five causative genetic variants in LRBA, CTLA4, NFKB1, and PIK3R1, as well as other very likely causative variants in PRKCD, MAPK8, or DOCK8 among others. We experimentally validate the effect of the LRBA stop-gain mutation which abolishes protein production and downregulates the expression of CTLA4, and of the frameshift indel in CTLA4 producing expression downregulation of the protein. Our results indicate a monogenic origin of at least 15–24% of the CVID cases included in the study. The pro- portion of monogenic patients seems to be lower in CVID than in other PID that have also been analyzed by whole exome or targeted gene panels sequencing. Regardless of the Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and poor response to vaccines. Its diagnosis is made based on clinical and immunological criteria, after exclusion of other diseases that can cause similar phenotypes. Currently, less than 20% of cases of CVID have a known underlying genetic cause. We have analyzed whole-exome sequencing and copy number variants data of 36 children and adolescents diagnosed with CVID and healthy relatives to estimate the propor- tion of monogenic cases. We have replicated an association of CVID to p.C104R in TNFRSF13B and reported the second case of homozygous patient to date. Our results also identify five causative genetic variants in LRBA, CTLA4, NFKB1, and PIK3R1, as well as other very likely causative variants in PRKCD, MAPK8, or DOCK8 among others. Original Research published: 14 May 2018 doi: 10.3389/fimmu.2018.00636 Edited by: Frédéric Rieux-Laucat, INSERM UMR1163 Institut Imagine, France Edited by: Frédéric Rieux-Laucat, INSERM UMR1163 Institut Imagine, France Edited by: Frédéric Rieux-Laucat, INSERM UMR1163 Institut Imagine, France Guillem de Valles-Ibáñez1†, Ana Esteve-Solé2,3‡, Mònica Piquer 2,3‡, E. Azucena González-Navarro3,4, Jessica Hernandez-Rodriguez1, Hafid Laayouni1,5, Eva González-Roca3,4, Ana María Plaza-Martin2,3, Ángela Deyà-Martínez2,3, Andrea Martín-Nalda6,7, Mónica Martínez-Gallo7,8,9, Marina García-Prat6,7, Lucía del Pino-Molina10, Ivón Cuscó11,12, Marta Codina-Solà11,12, Laura Batlle-Masó1,13, Manuel Solís-Moruno1,13, Tomàs Marquès-Bonet1,14,15, Elena Bosch1, Eduardo López-Granados10, Juan Ignacio Aróstegui3,4, Pere Soler-Palacín6,7, Roger Colobran7,8,9, Jordi Yagüe3,4, Laia Alsina2,3, Manel Juan3,4* and Ferran Casals13* Reviewed by: Markus G. Seidel, Medizinische Universität Graz, Austria Yuval Itan, Icahn School of Medicine at Mount Sinai, United States Reviewed by: Markus G. Specialty section: We experimentally validate the effect of the LRBA stop-gain mutation which abolishes protein production and downregulates the expression of CTLA4, and of the frameshift indel in CTLA4 producing expression downregulation of the protein. Our results indicate a monogenic origin of at least 15–24% of the CVID cases included in the study. The pro- portion of monogenic patients seems to be lower in CVID than in other PID that have also been analyzed by whole exome or targeted gene panels sequencing. Regardless of the Received: 12 December 2017 Accepted: 14 March 2018 Published: 14 May 2018 Seidel, Medizinische Universität Graz, Austria Yuval Itan, Icahn School of Medicine at Mount Sinai, United States *Correspondence: Manel Juan mjuan@clinic.cat; Ferran Casals ferran.casals@upf.edu *Correspondence: Manel Juan mjuan@clinic.cat; Ferran Casals ferran.casals@upf.edu 1 Institut de Biologia Evolutiva (UPF-CSIC), Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain, 2 Allergy and Clinical Immunology Department, Hospital Sant Joan de Déu, Institut de Recerca Pediàtrica Hospital Sant Joan de Déu, Barcelona, Spain, 3 Functional Unit of Clinical Immunology Hospital Sant Joan de Déu-Hospital Clinic, Barcelona, Spain, 4 Servei d’Immunologia, Centre de Diagnòstic Biomèdic, Hospital Clinic-IDIBAPS, Barcelona, Spain, 5 Bioinformatics Studies, ESCI-UPF, Barcelona, Spain, 6 Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d’Hebron (HUVH), Vall d’Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain, 7 Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Barcelona, Spain, 8 Immunology Division, Department of Clinical and Molecular Genetics, Hospital Universitari Vall d’Hebron (HUVH), Vall d’Hebron Research Institute (VHIR), Barcelona, Spain, 9 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain, 10 Clinical Immunology Department, University Hospital La Paz and Physiopathology of Lymphocytes in Immunodeficiencies Group, IdiPAZ Institute for Health Research, Madrid, Spain, 11 Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain, 12 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain, 13 Servei de Genòmica, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain, 14 Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain, 15 CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Spain †Present address: Guillem de Valles-Ibáñez, Department of Paediatrics and Child Health, University of Otago, Wellington, New Zealand ‡These authors have contributed equally to this work. INTRODUCTION we have analyzed high coverage whole-exome sequencing and copy number variants data for 36 CVID pediatric patients. We hypothesize that focusing on pediatric cases will allow us to esti- mate the maximum proportion of monogenic CVID cases, based on the higher incidence of infectious disease in childhood and theoretical and molecular evidence of higher impact of inborn single gene defects in childhood than in adults, which tend to present more complex genetics of predisposition to infection (15, 16). Because of the heterogeneity of CVID etiology and mani- festations, we first examined the role of known genetic variants and candidate genes for CVID, and then expanded the analysis to interacting proteins and genes in the same pathway, and finally to the rest of the genome. We propose single candidate genes for the CVID patients according to different models of inheritance and by considering both genetic variants properties such as the allele frequency, bioinformatic predictions of the phenotypic effect or evolutionary conservation rates, as well as gene features such as haploinsufficiency and essentiality predictors. In addition, beyond the estimation of the proportion of patients under a monogenic model, we also propose exploring other possible disease models such us the oligogenic or polygenic by considering the presence of mutations in interacting proteins or the accumulation of func- tional variants in immunological pathways, as well as the disease association with rare functional genetic variants by comparison to healthy controls (17). Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary humoral immunodeficiency with a prevalence from 1:10,000 to 1:50,000 in North America and Europe (1). The diagnosis criteria consist in low serum con- centrations of IgG, IgA and/or IgM, recurrent bacterial infections and poor antibody response to vaccines, in addition to the exclu- sion of other known causes of hypogammaglobulinemia (1–4). Patients’ phenotypes are highly heterogeneous due to different time onsets and to a high variety of related complications, such as autoimmune manifestations, lymphoproliferation, enteropathy, and lymphoid malignancies, suggesting that CVID could be a common outcome of diverse immune system failures.h The clinical heterogeneity of CVID has hindered both the diagnostic and the identification of the underlying genetic defect of the disease, allowing a molecular characterization of the origin in less than 20% of the patients, and usually in familiar forms of the disease which constitute only a small fraction of the CVID cases (1, 5–7). INTRODUCTION Despite that, mutations in the genes CR2, LRBA, NFKB1, NFKB2, IL21, TNFRSF13B, TNFRSF13C, CD81, IKZF1, PRKCD, MS4A1, and CD19 are listed in the OMIM database1 as causative of disease, inducing reclassification of CVID in these new diagnostics, and establishing new therapeutic approaches based on the affected pathways that have markedly improved affected patients’ prognoses (8). Specific variants in these genes as well as in others not listed in the OMIM database (NOD2, MSH5, TNFRSF13B, HLA) have been reported to confer susceptibility to the disease or to originate similar phenotypes to CVID (CTLA4, PLCG2, PIK3CD, PIK3R1), blurring even more the boundaries that define this disorder. Furthermore, some of the mutations have incomplete penetrance (9, 10) and many sporadic cases remain unexplained after deep genetic analyzes, suggesting that an important fraction of CVID cases might not follow a mono- genic Mendelian pattern of inheritance (11). Citation: de Valles-Ibáñez G, Esteve-Solé A, Piquer M, González-Navarro EA, Hernandez-Rodriguez J, Laayouni H, González-Roca E, Plaza-Martin AM, Deyà-Martínez Á, Martín-Nalda A, Martínez-Gallo M, García-Prat M, del Pino-Molina L, Cuscó I, Codina-Solà M, Batlle-Masó L, Solís-Moruno M, Marquès-Bonet T, Bosch E, López-Granados E, Aróstegui JI, Soler-Palacín P, Colobran R, Yagüe J, Alsina L, Juan M and Casals F (2018) Evaluating the Genetics of Common Variable Immunodeficiency: Monogenetic Model and Beyond. Front. Immunol. 9:636. doi: 10.3389/fimmu.2018.00636 May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 1 de Valles-Ibáñez et al. WES for CVID exact proportion of CVID monogenic cases, other genetic models have to be considered for CVID. We propose that because of its prevalence and other features as intermediate penetrancies and phenotypic variation within families, CVID could fit with other more complex genetic scenarios. In particular, in this work, we explore the possibility of CVID being originated by an oligogenic model with the presence of heterozygous mutations in interacting proteins or by the accumulation of detrimental variants in particular immu- nological pathways, as well as perform association tests to detect association with rare genetic functional variation in the CVID cohort compared to healthy controls. Keywords: common variable immunodeficiency, primary immunodeficiency, exome sequencing, loss-of-function, rare disease genetics Keywords: common variable immunodeficiency, primary immunodeficiency, exome sequencing, loss-of-function, rare disease genetics Keywords: common variable immunodeficiency, primary immunodeficiency, exome sequencing, loss-of-function, rare disease genetics 1 https://www.omim.org/ (Accessed: September, 2015). Frontiers in Immunology  |  www.frontiersin.org Individuals Included in the Studyh Individuals Included in the Study This study includes 36 patients diagnosed with CVID, includ- ing both sporadic and familiar cases, without any genetically confirmed primary immunodeficiency (PID), and completing the conventional criteria for CVID classification: (1) from 2 to 18 years old at the age of diagnosis; (2) lack of antibody produc- tion after immunization of antigen exposure in at least two assays; (3) 2 years post-diagnosis to exclude lymphoid malignancy; (4) IgG levels 2.5th centile for age and low IgA or/and IgM levels. CVID patients presenting one of the following features were excluded from the study: (a) well-known gene-identified PID such as hyper IgM; CD19+ or CD20+ B cell deficiency; ICOS or transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) gene mutation already diagnosed; (b) secondary immunodeficiencies such as those due to complica- tions such as associated tumors and lymphomas or from other In recent studies using whole-genome and exome sequencing to study CVID, 15–30% of CVID patients have been proposed to have a monogenic origin (12–14), with genetic variants both at candidate or new genes for CVID, although not all of these mutations have been functionally validated. In this work, we aim to estimate the proportion of monogenic cases in CVID and to explore other possible genetic models for CVID. For that, May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 2 WES for CVID de Valles-Ibáñez et al. used predicted haploinsufficiency (31), intolerance to functional variation (32), and essentiality (33) scores to infer the possible model of the disease and prioritize candidate genes in the differ- ent patients.i therapies (side-effects following splenectomy, corticosteroid, or immune suppressive therapies). Patients L283, L286, and N216 were reported to be consanguineous. In addition, parents and siblings have also been included in the study, when available. Written informed consent for genetic analysis and research was obtained from all participants and ethical approval for the project was obtained from the institutional ethical committees.f We used the Fisher’s exact test to assess the statistical signifi- cance of an excess of rare functional variants in cases compared to controls, from two by two tables with the total number of rare functional variants, and the total number of synonymous variants in patients and controls. In both cases, variants present in more than 10 individuals were excluded from the analysis to exclude false positives produced by sequencing artifacts. Genetic Analyses The protein–protein interaction (PPI) data was obtained from the Human Protein Reference Database (35) considering the whole set of non-redundant interactions between two proteins. Gene lists for each pathway were extracted from the KEGG data- base (36–38). We considered the 25 pathways shown in Table S1 in Supplementary Material. DNA was extracted from blood samples. CNV analysis was performed with the CytoScanHD array (Affymetrix) according to the manufacturer’s protocol. The CytoScanHD array contains 743,304 SNPs and 2,696,550 CNV markers. The obtained cychp files were analyzed with Chromosome Analysis Suite v.2.1.0.16 software and NetAffx na33 annotation version. For CNV detec- tion and to prevent false positives, we considered alterations involving at least 25 markers and more than 150 Kb in length for gains, and 35 markers and more than 75 Kb for losses. For detection of loss of heterozygosity (LOH) regions, we considered alterations of at least 50 markers in more than 5 Mb. Exome cap- ture was performed with the Agilent SureSelect XT enrichment system. DNA was sequenced in an Illumina HiSeq 2000 platform in a 2 × 75 paired-end cycles run. PCR duplicates were removed with Picard.2 Sequence reads were mapped to the human refer- ence genome (hg19) using GEM (20). Variant calling was per- formed using GATK (21) and SNP annotation with SnpEff (22) and SnpSift (23). Candidate mutations were visually inspected with the Integrative Genomics Viewer (24) and, when required, validated by Sanger sequencing. Somatic variants analysis was performed with VarScan2 (25), considering the high impact variants predicted with SnpEff (22), P-value <0.05, present in less than 40% of the reads and in a maximum of two patients. 2 http://www.picard.sourceforge.net (Accessed: January, 2016). 3 http://evs.gs.washington.edu/EVS/ (Accessed: April, 2016). Individuals Included in the Studyh We applied the Li and Leal’s collapsing method (34) to detect an excess of CVID patients with rare functional variation when compared to con- trols. Statistical significance was also assessed using the Fisher’s exact test. For these two analyses, only nucleotide substitutions were considered. We used two different sets of controls: whole-exome sequences from 36 individuals from a Spanish cohort diagnosed with autism spectrum disorders (ASD) (18) and 267 whole-exome sequences from healthy controls from a Spanish cohort (19). In the case where no data were available for the 267 whole-exome sequences, we retrieved data from the CIBERER Spanish Variant Server (csvs.babelomics.org) and used data for individuals with different syndromes not related to primary immunodeficiencies. Functional Validationsfi To assess the effect of specific gene alterations, additional functional tests were performed. Mainly with peripheral blood mononuclear cells (PBMCs) or Epstein–Barr transformed B cells (EBV-B), including lymphocyte phenotyping and western-blot Ficoll–Hypaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation of heparinized blood was used for PBMC isolation. Cells were cultured with complete medium [RPMI (Gibco, Grand Island, NY, USA) supplemented with 10% heat- inactivated fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 1 µg/ml penicillin and 1 µg/ml streptomycin (Invitrogen, Grand Island, NY, USA)]. Viable cells were counted using a hemocytom- eter in an inverted microscope. CTLA-4 expression detection was performed as described elsewhere (39–42). Specifically, for Treg cell phenotyping and CTLA-4 expression PBMCs were left with medium (resting) or stimulated with PHA (5μg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Treg intracellular staining was performed with Treg Detection Kit (CD4/CD25/FoxP3) kit (Miltenyi Biotec, Germany) following manufacturer’s instructions. CD3 BV421 (BD bioscience, San Jose, CA, USA), CD4 FITC, and CD25 APC (Miltenyi) were used for extracellular staining and FoxP3 APC (Miltenyi) and CTLA4 PE (BD biosciences) for intracellular staining and then acquired with the cytometer (FACS Canto II, BD biosciences).l Genetic Data and Statistical Analyses y Only functional variants were considered, including missense, stop-gain and stop-loss, splice donor or acceptor sites mutations, and frameshift insertions and deletions. In addition to standard filters for mapping and variant calling and annotation we also discarded indels clustering within 10 base pairs of another indel and for most of the analyses we excluded those variants present in 10 or more individuals in our dataset. We used allele frequen- cies from The 1000 Genomes Project (26) and the NHLBI and Exome Sequencing Project.3 We used GERP (27, 28) to asses for evolutionary conservation and Polyphen (29) and SIFT (30) to predict the phenotypic impact of missense variants. We have also Lymphocyte stimulation capacity was assessed by flow cyto- metric detection of activation markers. PBMCs were stimulated for 7 days and then surface-stained with the following antibodies against activation markers: CD62L, CD25, HLA-DR, CD69, and CD40-L (BD Biosciences) and then acquired with the cytometer (FACS Canto II, BD biosciences). Protein extraction and Western Blot: LRBA determination was performed in EBV-B cells (43). EBV-B cells were lysed with 1% NP-40 buffer. Protein concentration was normalized between May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 3 WES for CVID de Valles-Ibáñez et al. Four of them were found in the CVID patients of this study (Table 1). control and patient. Products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. A nitrocellulose membrane was blocked with a 2% milk TBS, then incubated overnight with primary antibodies anti-LRBA (1:500, polyclonal, Abcam, United Kingdom) and anti-GAPDH (1:1000, polyclonal, Bio-Rad, United Kingdom) then the membrane was washed with TTBS and incubated for 1,5  h with Goat Anti- Rabbit IgG H&L (HRP) (1:5000, Abcam). It was then developed with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) following the manu- facturer’s instructions and acquired with ImageQuant LAS-4000 (GE Healthcare Life Sciences, Buckinghamshire, England, UK) equipment. Two of the reported variants are included in the TNFRSF13B gene (TACI), which is known to harbor functional mutations in 5–10% of patients diagnosed with CVID (48, 49). However, the existence of healthy controls with heterozygous mutations in this gene and the lack of a clear Mendelian pattern of inheritance in families have led to consider some of the mutations at TNFRSF13B as risk factors (9, 10) which could be determinant only in the case of homozygous individuals (50). Genetic Data and Statistical Analyses Thus, TNFRSF13B would be considered a modifier gene rather than a causal gene in monogenic cases (51). The p.C104R variant is the most common TNFRSF13B functional mutation found in CVID patients (51). Three of the patients in this study present this mutation, in one case in homozygous state, being the second case found to date (52). This mutation is significantly more frequent in our CVID patients compared to the Spanish cohort controls (19) (P = 0.003, Fisher’s exact test) and absent in the ASD controls (18) (Table 1). In the same gene, we report nine samples with the protein change P251L, although in this case the proportion is not significantly higher than in controls. In addition, a direct causal role for this variant can probably be discarded because of its high frequency in the reference populations (14% in the ExAc database, 11% for the European population). On the other hand the P21R variant of the TNFRSF13C gene found in four patients, and also one healthy parent, shows a higher frequency when compared to controls (P = 0.003, Fisher’s exact test). However, this vari- ant (rs77874543) has also been found in non-CVID exomes in homozygosity, and has a population frequency higher than 5%. Finally, we also detected two patients with the L85F substitution in the MSH5 gene (47). The same aminoacid substitution was also present in the mother of one these patients, not diagnosed with CVID but with some of the clinical features described in the patient. Nonetheless, this genetic variant has been found at lower frequencies in CVID patients compared to controls, and has a population frequency of 2% or higher in some populations (7% in Africans), which suggests that it does not have a determinant role in CVID. OMIM CVID-Causing Mutationsh g The OMIM database4 includes known variants originating CVID in 13 genes: ICOS, TNFRSF13B, TNFRSF13C, CD19, CR2, MS4A1, CD81, IL21, LRBA, NFKB1, NFKB2, PRKCD, and IKZF1. There is also evidence that defects in other genes (CTLA4, PLCG2) can cause a similar phenotype or modify the severity of the disease with comorbidities (MSH5). These genes are mainly related to T-cell and B-cell defects leading to a deficiency in antibody production. In these 16 genes, we found a total of 96 nucleotide variants and 6 CNVs previously described to be putatively related to CVID in the literature (Table S3 in Supplementary Material). RESULTS We generated whole-exome sequencing data for the 36 CVID patients included in the study, as well as for eight relatives, with an average coverage of 120×. In addition, we also generated CNV data for all the samples except in one case where DNA was not available. Table S2 in Supplementary Material shows the number of functional genetic variants described in each sample, classified in different annotation categories: missense, stop-gain (or nonsense), start-gain, splice site, and inframe and frameshift indels, with total numbers similar to what has been previously reported (44). Table S2 in Supplementary Material also contains the number of structural variants and LOH regions detected in the genotyping analysis with the CytoScanHD array. Loss-of-Function (LoF) Variants Furthermore, the expression of CTLA4 is downregulated in Treg cells of the LRBA-deficient patient (Figure  1C), in agreement with the previous description of CTLA4 detection in Treg cells from LRBA-deficient patients (39).h in Supplementary Material shows the number of LoF variants identified in each individual of the study. The number of LoF vari- ants ranges from 78 to 153, similar to what has been previously described (44, 53, 54). Applying different frequency thresholds substantially reduces the number of LoF variants per individual (54, 55). We established a permissive allele frequency threshold of 1%, and first focused the analysis on the LoF variants described in candidate genes for CVID (Table 2). With this aim, we generated a list of 97 candidate genes for CVID (Table S4 in Supplementary Material), including genes in the OMIM database,5 genes defined in a review by Bogaert and colleagues (51), and others from the literature. Second, we also analyzed the presence of LoF variants in proteins interacting with the proteins encoded by candidate genes (see Materials and Methods) (Table  2). Finally, we also report all the genes with LoF variants using a very low frequency threshold (0.001) (Table S5 in Supplementary material). Eight patients harbor a LoF variant at a frequency less than 1% in CVID candidate genes (Table  2). Among them, L283 presents a new homozygous nonsense variant at the exon 4 of the LRBA gene [chr4:151392836G > A (hg19)]. This stop codon at LRBA (R2214*) is introduced at the beginning of the BEACH domain (IPR000409 in InterPro), a highly conserved domain with known crystal structure but unknown function (56). This i The N211 patient presents a new LoF genetic variant located at the CTLA4 gene, which has already been reported to harbor causal heterozygous CVID variants (41, 42). The mutation causes a frameshift deletion not previously described and absent in the reference databases. We performed Sanger sequencing of this mutation and confirmed that it is a de novo mutation absent in the parents (Figure S1 in Supplementary Material) and, therefore, a strong candidate to originate CVID. We per- formed functional analyses to study the expression of CTLA4 in Treg cells and we found that it is downregulated before and after stimulation with PHA. CTLA4 detection was lower than in the case of the aforementioned LRBA-deficient patient after PHA stimulation (Figure  1C). Finally, we also analyzed the lymphocyte stimulation in the patient. Loss-of-Function (LoF) Variants Loss-of-Function variants include stop-gain and loss muta- tions, splice-site mutations, and frameshift indels, which are predicted to disrupt proteins and, therefore, could likely relate to disease phenotypes, and in fact account for approximately 20% of the coding variants associated with disease (53). Table S2 4 https://omim.org/ (Accessed: September, 2015). Table 1 | Known common variable immunodeficiency (CVID) variants detected in CVID patients in this study. Gene cDNA Aa change Genotypea (reference) hg19_pos CVID (N = 36)b Controls (literature) Controls (Autism, N = 36)b Controls (Spain, N = 267)b TNFRSF13B c.752C > T p.P251L 0/1 (10) 17:16842991 9 Yes 0 36 (3) TNFRSF13B c.310T > C p.C104R */1 (45) 17:16852187 3 (1) Yes 0 2 TNFRSF13C c.62G > C p.P21R 2*0/1 (46) 22:42322716 4 Yes 0 16c MSH5 c.253C > T p.L85F 2*0/1 (47) 6:31709045 2 Yes 0 55 (2) a0/1 heterozygotes; 1/1 homozygotes; */1 heterozygotes and homozygotes; 2*0/1 compound heterozygotes. bHomozygous individuals are shown in brackets. cNo data available for the 267 controls. Instead, we used data from 578 whole-exome sequences at the CIBERER Spanish Variant Server (csvs.babelomics.org). yg cNo data available for the 267 controls. Instead, we used data from 578 whole-exome sequences at the CIBERER Spanish Variant Server (csvs.babelomics.org). May 2018  |  Volume 9  |  Article 636 May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org de Valles-Ibáñez et al. WES for CVID mutation was validated by Sanger sequencing in the patient, and also detected in heterozygosis in both parents and three healthy siblings (Figure 1A). Copy number and SNP analyses confirmed the existence of consanguinity in this patient. We estimated a consanguinity index of 0.058 compatible with descendants from third degree kinship marriages, based in the total of 174  Mb included in LOH regions (57), with 10 LOH regions of more than 5 Mb. We then performed assays with the patient cells to test the effect of the variant on the protein. The western blot gel electrophoresis separation (Figure 1B) shows that the cells of the patient do not produce any detectable amount of LRBA protein, thus validating the deleterious effect of the mutation abolishing protein production probably through nonsense-mediated decay. PPI, protein–protein interaction. Loss-of-Function (LoF) Variants Figure 1 | (A) Chromatograms corresponding to the Sanger sequencing of the LRBA nonsense mutation region in L283 and five healthy relatives. (B) Western blot analysis of LRBA and GAPDH for L283 patient (P) and a healthy control C+. LRBA protein is not detectable in the LRBA-deficient patient. (C) CTLA4 expression is downregulated in LRBA- and CTLA4-deficient patients. CTLA4 expression was assessed in Treg cells (CD3+CD4+CD25hiFoxP3+ cells) in resting and in PHA-stimulated cells (24 h). Bars represent mean values and error bars represent SE of the mean values for adult healthy controls (n = 5). Figure 1 | (A) Chromatograms corresponding to the Sanger sequencing of the LRBA nonsense mutation region in L283 and five healthy relatives. (B) Western blot analysis of LRBA and GAPDH for L283 patient (P) and a healthy control C+. LRBA protein is not detectable in the LRBA-deficient patient. (C) CTLA4 expression is downregulated in LRBA- and CTLA4-deficient patients. CTLA4 expression was assessed in Treg cells (CD3+CD4+CD25hiFoxP3+ cells) in resting and in PHA-stimulated cells (24 h). Bars represent mean values and error bars represent SE of the mean values for adult healthy controls (n = 5). Loss-of-Function (LoF) Variants After 7 days stimulation with PHA, the stimulation ratio of different lymphocyte stimula- tion markers was increased in the patient compared to a healthy control (Figure 2). Table 2 | Genes with Loss-of-Function (LoF) homozygous or heterozygous variants in common variable immunodeficiency (CVID) candidate genes and interacting proteins. Individual CVID < 0.01 PPI < 0.01 L283 LRBA(hom) L287 C7orf64(het), PDGFRB(hom), RIPK4(het) L289 HDAC1(het) L291 GP6(het) L292 NOD2(het) SLA2(het), ZNF655(het) L297 NFKB1(het) L298 MAPK8(het) L299 FGFR3(het) N202 PIK3R1(het) FHOD1(het) N204 HP(het), PLSCR1(het) N205 HNF1A (comp_het) N206 R PA 2(het) N207 NFKB1(het) N208 EEF1G(het) N210 DERL3(het), HP(het), PDGFRB(het) N211 CTLA4(het) N213 IBTK(het), PDGFRB(het) N216 CASP1(het), HCLS1(het), NCOR2(het) N223 BCAP31(het), SLC6A8(het), TNFRSF12A(het) N224 BCAP31(hom), CASP1(het), SLC6A8(hom), TNFRSF12A(het) N227 BCAP31(het), SLC6A8(het) N229 CR1(het), SPI1(het) N231 PML(het), TNFRSF12A(het) N232 TNFRSF12A(het) N233 NOD2(het) TNFRSF12A(het) N234 IL10RA(het), NFKB1(het) TNFRSF12A(het) N235 C9(het), PIAS1(het), TRPV1(het) PPI, protein–protein interaction. 5 http://omim.org (Accessed: September, 2015). N202 presents a heterozygous splicing variant in PIK3R1. This variant has been previously reported to originate an immu- nodeficiency because of its dominant gain of function effect on PI3K signaling (58) in agreement with its high haploinsufficiency prediction value of 0.89 (31). For the remaining five patients presenting a low frequency heterozygous LoF variant in a CVID candidate gene (Table 2), three of them have a variant in NFKB1, which has also been reported to harbor heterozygous mutations originating CVID (7). Two of them share a start loss variant affecting one of the transcripts, although its frequency of 0.002 makes it unlikely to have a causal (monogenic) role in the disease. By contrast, a new splice-site mutation in NFKB1 is described in N234, being a good candidate to originate the disease. In addition, N227 presents a 13 MB heterozygous deletion (chr4: 94,135,868–107,295,574) not present in parents which includes the NFKB1 gene among others (Table S2 in Supplementary Material). Finally, although the variants described at NOD2 and IL10RA are not present in any database, no CVID cases with heterozygous variants at these genes have been described, in agreement with their low haploinsufficiency values (0.119 and 0.173, respectively). In addition, Table 2 also includes low frequency LoF variants of genes interacting with candidate genes related with CVID. May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 5 WES for CVID de Valles-Ibáñez et al. Functional Genetic Variation at Candidate Genes for CVID Figure 2 | Stimulation ratio of different lymphocyte activation markers after PHA stimulation (7 days). Stimulation ratio: mean fluorescence intensity of PHA-stimulated/basal conditions. Figure 2 | Stimulation ratio of different lymphocyte activation markers after PHA stimulation (7 days). Stimulation ratio: mean fluorescence intensity of PHA-stimulated/basal conditions. We then explored the presence of functional variants, other than LoF described above, in candidate genes for CVID. The final number of variants with frequency less than 1% in each individual is shown in Table S6 in Supplementary Material, differentiating variants in candidate genes, variants in interacting proteins and in other genes. We excluded from this, and subsequent analyses, the two individuals with a functionally validated LoF candidate (L283 and N211, see above), and the variants also present in healthy relatives (when this information is available from exome sequencing). We first analyzed the presence of single variants in CVID genes that could originate the disease following a dominant Figure 2 | Stimulation ratio of different lymphocyte activation markers after PHA stimulation (7 days). Stimulation ratio: mean fluorescence intensity of PHA-stimulated/basal conditions. May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 6 de Valles-Ibáñez et al. WES for CVID gene names are shown in Table S7 in Supplementary Material. The number of genes per patient can be reduced using addi- tional filters based in evolutionary conservation or predicted phenotypic effect. We established a threshold of a GERP > 2 for the functional variants, since positions with values greater than 2 are considered to be conserved among mammals and, therefore, more to prone to be of functional importance (28). On the functional effect, we used the Polyphen prediction and established a threshold value of 0.5 (60) (Table 5). Table S8 in Supplementary Material also shows additional information on gene properties which might aid the prioritization of candidate genes. Four genes are detected as compound heterozygotes in more than one patient (with GERP > 2 and Polyphen > 0.5): SLC25A5 (eight), ACOT4 (7), KMT2C (two), and OR10X1 (two). However, SLC25A5 and OR10X1 are two genes which have been recurrently reported in next-generation sequenc- ing studies (61), probably because of being prone to mapping artifacts and, thus, to accumulating false variants. On the other hand, ACOT4 (with a function apparently not related to the immune function), is also a paralog of ACOT1. Functional Genetic Variation at Candidate Genes for CVID Finally, KMT2C is also present in two patients, although one of them is N227 which harbors a large deletion encompassing NFKB1 among other genes. model. Because the number of genes with one or more functional variants is too high we applied stringent filters to produce a short list of candidate genes. We selected the variants with a GERP conservation score higher than 2 (28), a Polyphen score higher than 0.5 (for nucleotide variants) and a frequency in the ExAC and GMAF databases below 0.001. The nine variants at CVID genes fulfilling these conditions are shown in Table 3. Two of them are in frame indels and, therefore, less prone to have an effect on the protein. Among the heterozygous missense variants, PRKCD, CLEC16A, and DOCK8 (this latter absent in the healthy sister N209) are the more interesting candidates, considering their haploinsufficiency predictions (31) and essentiality values estimated from network and evolutionary properties (59). We then considered the recessive genetic model with the disease being originated by two rare functional variants in the same gene. We analyzed the presence of homozygous variants or compound heterozygotes in CVID candidate genes, at frequencies below 0.01 (Table 4). Interestingly, two candidate genes (CR2 and PLCG2) are found as compound heterozygotes in patients N233 and N212, respectively. Oligogenic Disease For the patients without a clear candidate gene for a monogenic origin of the disease, we then considered an oligogenic model of inheritance. In particular, we considered the digenic model. DIDA, a database of digenic diseases, included 44 diseases with 213 digenic combinations collected from the literature until June 2015 (62). This form of disease refers to both situations with a primary and a secondary locus or cases where two loci contribute to the disease with roughly the same importance (63). Modifier Table 3 | Functional heterozygous genetic variants with high predicted phenotypic effect at common variable immunodeficiency (CVID) candidate genes. Patient chr Position Gene Function Polyphen rs GERP esp5400_all HI1 HI2 RVIS Essent L294 chr9 100,774,719 ANP32B Inframe indel – – – 0 0.808 0.655 –0.16 (41.25%) 0.89 N214 chr7 2,976,742 CARD11 Missense 0.654 – 2.18 0 0.181 0.517 –1.39 (4.33%) 0.81 N212 chr11 chr1 60,892,540 CD5 Missense 0.936 – 3.08 0 0.284 0.402 0.8 (87.66%) 0.666 N232 160,523,750 CD84 Missense 0.999 rs146076557 5.25 0.000279 0.132 0.488 0.04 (57.15%) 0.077 L292 chr16 11,073,195 CLEC16A Missense 0.857 rs74163607 5.3 0.000201 NA 0.578 –1.01 (8.2%) 0.547 N201 chr1 207,651,294 CR2 Missense 0.659 rs146465618 5.59 0.000093 0.234 NA 0.06 (57.56%) 0.558 N210 chr9 chr1 377,046 DOCK8 Missense 0.868 rs148693111 5.71 0.000186 0.535 0.57 –1.94 (1.9%) 0.845 L287 234,744,945 IRF2BP2 Inframe indel – – 2.62 0 0.852 0.626 – 0.992 N213 chr1 234,744,945 IRF2BP2 Inframe indel – – 2.62 0 0.852 0.626 – 0.992 N216 chr1 234,744,945 IRF2BP2 Inframe indel – – 2.62 0 0.852 0.626 – 0.992 L288 chr3 53,218,928 PRKCD Missense 0.733 – 5.91 0 0.636 0.553 –1.04 (7.77%) 0.966 HI1 and HI2 haploinsufficiency predictions (24); RVIS, residual variation score of genetic variation intolerance (25) with the percentile of intolerant human genes in parentheses; Essent, essentiality index estimated from network and evolutionary properties (43). HI1 and HI2 haploinsufficiency predictions (24); RVIS, residual variation score of genetic variation intolerance (25) with the percentile of intolerant human genes in parentheses; Essent, essentiality index estimated from network and evolutionary properties (43). Table 4 | Compound heterozygotes at common variable immunodeficiency (CVID) genes. Compound Heterozygotes at Non-CVID Genes We expanded the analysis beyond the list of CVID candidate genes to the rest of the genome. We based our approach on the use of stringent filters (frequency, conservation, predicted effect) and the consideration of predictors of the degree of essentiality of the gene. This approach produces a list of new candidate genes in each CVID patient which can be ranked using the different variant and gene properties. We produced a list of genes harbor- ing compound heterozygotes in each patient and applied two different allele frequency thresholds of 0.01 and 0.001. Table 5 shows the number of compound heterozygotes per patient, and May 2018  |  Volume 9  |  Article 636 Oligogenic Disease Patient CVID gene Variants Interacting protein Variants L288 PRKCD 1 het RUNX2 1 hom L293 DOCK8 1 het CDC42 1 het L299 STAT1 1 het FGFR3 1 hom L299 STAT1 1 het FGFR4 1 hom, 1 het N205 PIK3R1 1 het AXL 1 hom N205 PIK3R1 1 het TYK2 1 het N206 PIK3R1 1 het AXL 1 hom N207 PIK3CD 1 het RALY 1 het N208 PIK3CD 1 het RALY 1 hom N210 DOCK8 1 het CDC42 1 het N233 CR2 1 het FHOD1 1 het N234 RAD50 1 het NBN 1 het N234 NFKB1 1 het NCOR2 2 het considering a maximum frequency of 0.05. Interestingly, two pairs of related patients (sisters N205 and N206, and brothers N207 and N208) share the presence of variants at the interacting proteins PIK3R1-AXL and PIK3CD-RALY, respectively. In four more patients (Table 6), the CVID genes had already been sug- gested as probably causal (Tables 2–4) following recessive (N233) or dominant models (L288, N210, N234). considering a maximum frequency of 0.05. Interestingly, two pairs of related patients (sisters N205 and N206, and brothers N207 and N208) share the presence of variants at the interacting proteins PIK3R1-AXL and PIK3CD-RALY, respectively. In four more patients (Table 6), the CVID genes had already been sug- gested as probably causal (Tables 2–4) following recessive (N233) or dominant models (L288, N210, N234). We then expanded the analysis to a scenario where variants in several genes of an individual might contribute to the disease. For this purpose, we assessed the presence of particular CVID patients which compared to the rest of the patients in the study harbors an excess of rare functional variants at any of 25 KEGG pathways related to the immune function (36) (Figure 3). We used a frequency threshold of 1% and estimated the ratios of functional to synonymous variants in each sample, to correct for possible differences in coverage across samples. We considered as outliers those individuals departing from twice the SD of the average number of rare functional variants (Figure 3). Table 7 shows the CVID patients with an excess of rare functional genetic variants in a particular pathway. The presence of more than one pathway in three of the patients is mostly due to the fact that these patients have genetic variants in genes with a role in several path- ways. Oligogenic Disease chr Position ref alt Polyphen rs GERP esp5400 all GMAF Effect Gene idsample Genotype chr1 207643100 C A 0.002 – −3.25 – – Non-synonymous CR2 N233 0/1 chr1 207648456 G T 0.05 rs144572703 4.47 0.005763 0.0018 Non-synonymous CR2 N233 0/1 chr16 81939089 T C 0.598 rs187956469 5.18 0.002838 0.0032 Non-synonymous PLCG2 N212 0/1 chr16 81942175 A G 0.005 rs75472618 6.5 0.007067 0.0064 Non-synonymous PLCG2 N212 0/1 7 WES for CVID de Valles-Ibáñez et al. Table 5 | Number of genes harboring compound heterozygotes mutations in the patients included in this study. Sample Genes 1% Genes 0.1% Genes 1% filtereda Genes 0.1% filtereda L283 53 21 FAM186B, MYH11, SLC25A5, SDK1 FAM186B L287 26 11 CBS, TRIB3 0 L288 29 18 0 0 L289 39 19 FHL3, SLC25A5, FMN2 0 L290 31 15 0 0 L291 27 18 CAMPSAP3, VPS13C CAMPSAP3 L292 26 16 PKHD1L1, PLEC, MLH1 PKHD1L1 L294 31 20 PRSS16 PRSS16 L295 34 15 0 0 L296 25 13 0 0 L297 30 17 SLC25A5 0 L298 28 7 BMP1 0 L299 27 14 ACOT4, GPR112, SLC25A5, UNC13C ACOT4, GPR112 N201 33 23 ACOT4, SLC25A5, Z FYVE26 ACOT4 N202 27 13 ACOT4, WFS1, SLC25A5 ACOT4 N203 33 13 SLC25A5, SEPT1 0 N204 34 14 ACOT4 ACOT4 N205 28 15 0 0 N206 34 18 ACOT4 ACOT4 N207 29 17 0 0 N208 23 12 0 0 N210 33 18 EPPK1 0 N211 25 13 GLTSCR1 0 N212 25 11 ACOT4 ACOT4 N213 27 10 ACOT4, SLCS5A5 ACOT4 N214 19 6 SLC25A5 0 N216 75 21 PENK, NUP214, KDM4C PENK N223 35 19 OR10X1 0 N224 38 22 OR10X1 0 N227 37 25 KMT2C 0 N229 39 20 0 0 N231 35 20 BAI1, TTN, MTDH BAI1 N232 18 12 KMT2C, PMFBP1 0 N233 35 19 0 0 N234 37 22 CMYA5 0 N235 42 22 0 0 aVariants with GERP > 2 and Polyphen > 0.5. Table 6 | Patients with rare functional variants (MAF < 0.01) and GERP > 2 in a common variable immunodeficiency (CVID) candidate gene and interacting proteins. Table 5 | Number of genes harboring compound heterozygotes mutations in the patients included in this study. Table 6 | Patients with rare functional variants (MAF < 0.01) and GERP > 2 in a common variable immunodeficiency (CVID) candidate gene and interacting proteins. proteins. Oligogenic Disease In the case of patient N208, it shows an excess of variants in five pathways that share the presence of three MAP Kinases (MAPK14, MAP2K2, and MAP2K3). Of interest, we found four patients with an excess of rare functional genetic variants in the B cell signaling pathway and three in the T cell signaling pathway, in addition to another two in the tumor necrosis factor and Fc epsilon RI signaling pathways (Table 7). aVariants with GERP > 2 and Polyphen > 0.5. genes, affecting the severity of the disease, can also be considered a type of digenic inheritance (64).h The case of TNFRSF13B, with several common variants related to CVID but with reported healthy carriers, could fit with this digenic model where additional variants would be needed to develop the disease. We analyzed the two patients with variants in this gene (Table 1), and searched for variants in genes inter- acting with TNFRSF13B. Patient L297 harboring the C104R change in homozygosis, also has a heterozygous missense variant with a 2% frequency in TNFRSF13C, which directly interacts with TNFRSF13B. No other variants in interacting proteins were described in the patients with known CVID variants in TNFRSF13B, TNFRSF13C, or MSH5 (Table 1). We expanded this analysis by assessing the presence of heterozygous rare functional variants in a CVID gene and in an interacting protein in the same patient. Table 6 shows the 10 patients in which this situation has been found, considering variants with GERP  >  2 and below 0.01 frequency (see Table S9 in Supplementary Material) when Frontiers in Immunology  |  www.frontiersin.org Association to Rare Variants Table 9 | Common variable immunodeficiency (CVID) genes with an excess of patients harboring rare functional genetic variants in patients compared to controls. Table 7 | Pathways with an excess of genes with rare functional variants in common variable immunodeficiency (CVID) patients. Table 7 | Pathways with an excess of genes with rare functional variants in common variable immunodeficiency (CVID) patients. Patient Pathway Genea L296 BCSP CD79A, CR2, DAPP1, PLCG2 L297 TNFSP CREB3, CREB3L1, NFKB1, PIK3CG, TNFRSF13B N207 BCSP PIK3CD, DAPP1, NFKB1, CD81, NFATC3 TCSP PIK3CD, NFKB1, MAP14, NFATC3 N208 BCSP PIK3CD, IKBKB, CD81, NFATC3, MAP2K2 FCERISP PIK3CD, MAPK14, MAP2K3, MPA2K2 TCSP PIK3CD, MAPK14, IKBKB, NFATC3, MAP2K2 TLRS PIK3CD, MAPK14, IKBKB, IFNA14, MAP2K2, MAP2K3 TNFSP PIK3CD, MAPK14, IKBKB, MAP2K3 N210 TCSP CDC42, MAP3K8, PAK6, SOS2 N212 BCSP PIK3CG, PLCG2(2), NFATC2, MAPK1, RAC2 FCERISP PIK3CG, PLCG2(2), MAPK1, RAC2 NKCMC PIK3CG, PTK2B, PTPN11, PLCG2(2), NFATC2, IFNAR1, MAPK1, RAC2 NLRSP CASP5, NLRP1(4), MAPK1 N216 MRP MLH3, MSH2, PMS2 N229 PAP PRKCZ, GUCY1B3, LCP2, ITPR2, ORAI1, PLA2G4B, ROCK1, TBXA2R, PIK3R2 N231 IINIgA HLA-DRB1(2), TNFRSF13B, ICOSLG aNumber of genetic variants at this gene when is greater than one, indicated in brackets. after applying Bonferroni’s multiple test correction. Table S10 in Supplementary Material reports these functional variants and their properties. Second, we used the Li and Leal’s collapsing method (34) to detect an excess of CVID patients harboring rare functional genetic variants. In this method, individuals with and without at least one functional rare variant are compared between CVID patients and controls. This test has been performed only for those genes with similar lengths for the targeted regions to avoid false positives with more functional variants because of a larger scanned region in CVID patients. Table 9 presents six genes (PIK3CD, ICOSLG, TNFRSF13B, PIK3R1, CD84, and PRKCD) showing a statistically significant excess of individuals with rare functional variants in CVID cases when compared to controls. Interestingly, PRKCD showed also a significant excess of functional variation in cases in the previous analysis (Table 8), although only PIK3CD remains significant after Bonferroni’s correction. Genetic variants in each gene are shown in Table S10 in Supplementary Material. Finally, we assessed a possible excess of functional variants in the 25 KEGG pathways by comparing our CVID patients to a set of controls (see Materials and Methods), by comparing the ratios of rare (<1%) functional to synonymous variants in each sample. Association to Rare Variants Next, we assessed the association of rare functional genetic variation to CVID. In this case, analyses are performed to detect an excess of rare functional variation in a particular gene or pathway in CVID patients compared to controls, rather than the detection of the causal genetic variant(s) in particular indi- viduals. To analyze the presence of genes harboring an excess of rare functional variants in the CVID patients compared to healthy controls, we first compared the ratio of rare functional to synonymous variants for each gene in cases compared to May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 8 WES for CVID de Valles-Ibáñez et al. Table 8 | Excess of rare functional variants in common variable immunodeficiency (CVID) patients. Gene Rare Funct CVID Syn CVID Rare Funct Controls Syn Controls P-value PRKCD 3 4 5 1693 2.36e−06 CLEC16A 6 11 8 321 1.30e−05 DOCK8 3 11 0 347 4.68e−05 PLCG2 4 14 18 1465 9.28e−05 Rare Funct, number of rare genetic variants; Syn, number of synonymous variants. Table 9 | Common variable immunodeficiency (CVID) genes with an excess of patients harboring rare functional genetic variants in patients compared to controls. Gene Patients functa Patients no functb Controls functc Controls no functd P-value PIK3CD 9 25 3 264 1.84E−07 ICOSLG 4 30 6 261 0.018 TNFRSF13B 3 31 4 263 0.034 PIK3R1 2 32 1 266 0.035 CD84 3 31 5 262 0.050 PRKCD 3 31 5 262 0.050 aNumber of patients with at least one rare functional genetic variant. bNumber of patients with no rare functional genetic variants. cNumber of controls with at least one rare functional genetic variant. dNumber of controls with no rare functional genetic variants. Table 8 | Excess of rare functional variants in common variable immunodeficiency (CVID) patients. Gene Rare Funct CVID Syn CVID Rare Funct Controls Syn Controls P-value PRKCD 3 4 5 1693 2.36e−06 CLEC16A 6 11 8 321 1.30e−05 DOCK8 3 11 0 347 4.68e−05 PLCG2 4 14 18 1465 9.28e−05 Rare Funct, number of rare genetic variants; Syn, number of synonymous variants. Figure 3 | Number of functional genetic variants in common variable immunodeficiency (CVID) patients and controls in immunological pathways. Abbreviations and the number of genes in each pathway are shown in the Section “Materials and Methods.” Table 8 | Excess of rare functional variants in common variable immunodeficiency (CVID) patients. DISCUSSION sisters (N205 and N206) harbor a new missense variant in PIK3R1. This variant has not been previously reported and is located in a conserved nucleotide according to its GERP value (3.24), although it is not predicted to be damaging using SIFT and Polyphen. On the situations fitting a recessive model, for the PLCG2 gene, one of the variants is predicted to be damaging with Polyphen and also shows a very high level of evolutionary conservation, although for the second variant both the evolutionary conservation and predicted phenotypic effect are low. Similarly, only one of the variants at CR2 in patient N212 shows a high level of evolutionary conservation, and none of the two variants is predicted to be damaging with Polyphen, being therefore a less promising candidate to originate CVID. Finally, KMT2C encodes for a nuclear methyltransferase (MLL3) of the mixed-lineage leukemia family the genes of which are among the more frequently mutated in cancer (74); somatic mutations at MLL3 have been related to different types of cancer (75), while in activated B-cells, deficiencies in the MLL3–MLL4 complex have been shown to manifest defective immunoglobulin class switching (76).h In this work, we first approach the proportion of monogenic cases in CVID by using deep whole-exome sequencing combined with CNV analysis, in a cohort with mostly early diagnosis patients (and all of them less than 18 years old), which is expected to optimize the probability of including monogenic cases (15). We propose candidate genetic variants and genes with different levels of confidence (Figure 4). The higher confidence cases are the five LoF variants very likely to originate CVID: one in LRBA and CTLA4 (both functionally validated), two in NFKB1 (a large deletion and a new splice-site variant), and one in PIK3R1 (a known splice-site variant causing disease). Thus, a minimum of 15% of the 33 cases included in this study (the 36 patients include three pairs of relatives) would have a monogenic origin of CVID. Among the LoF variants described in proteins interacting with CVID candidate genes (Table 2), a new LoF variant in MAPK8 is also a good candidate variant. MAPK8 shows high essentiality and haploinsufficiency prediction scores and is thought to play a key role in T cell proliferation, apoptosis, and differentiation (65–67). This stop gain variant is not found in genetic databases although it affects a base with a very low GERP value. DISCUSSION We have also described the presence of LoF variants in the genes CR1, IBTK, and NCOR2 (Table  2) that have been related to B  cell development and activation (68), agammaglobulinemia (69), and lymphoma (70, 71), respectively. However, CR1 and IBTK show low predicted haploinsufficiency values and the cases described at NCOR2 follow a recessive model for the disease. Thus, the proportion of CVID monogenic cases described in this work would rank from 15 to 24% or higher (Figure 4), similar to what has been described in previous studies (12–14) although lower than the 40% proposed in a recent analysis of 278 PID families including 20 CVID cases (77) (Table 10). However, these studies follow differing filtering strategies and stringency criteria making the results to be only roughly comparable between them. Overall, the fraction of monogenic CVID cases seems to be slightly lower to that described in other PID (78, 79), with some recent analyses showing considerably higher detection rates of PID monogenic cases (77, 80) which is especially high in a study of severe combined immunodeficiency (SCID) (81) (Table 10). The higher percentage of Mendelian patients described in some other PID (77, 80) and especially SCID (81) is probably because of a higher severity which is also expected to correlate with the number of Mendelian cases (15). However, it is important to highlight that different factors can contribute to an underestimation of the Mendelian cases in CVID in comparison to other PID. First, because of the clinical heterogeneity of CVID it is not recommended to apply the standard exome sequencing strategy where candidate genes are In addition to these LoF mutations at candidate CVID genes and interacting proteins, we propose other possible monogenic cases produced by missense variants at CVID candidate genes follow- ing a dominant (PRKCD, CLEC16A, DOCK8) or recessive models (CR2, PLCG2), as well as in other genes not previously associated with CVID (KMT2C). Of interest, missense variants and deletions in PLCG2 with dominant inheritance have been related to PID in previous studies (72, 73). RVIS scores are also negative in these three genes which suggests a certain level of intolerance to mutations, although in the case of immunological diseases this value seems to be less indicative than for other diseases (32). Finally, two affected Figure 4 | Scheme of the approaches and main results of this study. Association to Rare Variants We detected a significant excess of variants in two of the pathways in CVID patients when compared to controls: Fc epsilon RI signaling and cytokine–cytokine receptor interaction pathways (P < 0.001 and 0.002, respectively), plus two other marginally significant pathways after applying multiple test correction: cytosolic-DNA sensing, and NFKB signaling (P = 0.002 and 0.001, respectively). The four pathways also show a significant excess of functional vari- ation in CVID patients when compared to the ASD controls set. aNumber of genetic variants at this gene when is greater than one, indicated in brackets. healthy controls. Table 8 shows the results of the analysis for the 60 genes analyzed (with at least one synonymous variant in each cohort), for the 34 patients without a validated can- didate gene for a monogenic origin of the disease. Four genes (PRKCD, CLEC16A, DOCK8, and PLCG2) show a statistically significant excess of rare functional variants in CVID patients, healthy controls. Table 8 shows the results of the analysis for the 60 genes analyzed (with at least one synonymous variant in each cohort), for the 34 patients without a validated can- didate gene for a monogenic origin of the disease. Four genes (PRKCD, CLEC16A, DOCK8, and PLCG2) show a statistically significant excess of rare functional variants in CVID patients, May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 9 WES for CVID de Valles-Ibáñez et al. DISCUSSION And third, exome and even genome sequencing have some limitations that may produce false negatives because of the difficulties to detect structural variation. However, based on our results, the contribution of CNVs to monogenic CVID cases would be quite limited, in contrast to a more important role for common CNVs proposed in previous studies (84, 85). We have used one of the highest density array optimized for CNV detection (86), and detected only a candidate CNV consisting of one big deletion including, among others, the NFKB1 gene. Similarly, in the recent whole-exome sequencing analysis of 278 PID families CNV represented 8% of the likely causing mutations, but no causal CNV was found among the 20 CVID patients (77). g Considering the digenic model, we have combined exome sequencing with PPI data, and described cases of patients with rare functional variants in CVID candidate genes and an interact- ing protein. Although promising, to date the number of reported examples in the literature with pieces of evidence of digenic inheritance remains quite low (62), probably because of difficul- ties in statistical and mainly functional analyses to demonstrate a real role in the disease (63). We have used a prudent approach based on the existence of physical interactions between proteins, to produce a reduced number of candidate interactions. Other tools to identify related genes, as the human genome connectome (92, 93) or GIANT (94) could also be used. However, since inter- actions predicted by these tools are based both in physical and functional associations, the number of candidate protein pairs would be higher. Still at the individual level, we have considered a polygenic model and hypothesized that CVID in a particular patient might be produced by an accumulation of rare functional genetic variants in genes related to the same function, producing a list of patients with an excess of genes with functional variants in the same immunological pathway. Finally, we have performed tests of association of rare genetic variants to disease. In this case, the goal is not proposing candidate gene(s) in a particular patient but to detect genes enriched for rare functional varia- tion in the cohort of CVID cases compared to healthy controls. DISCUSSION Candidate genes in bold are those with more evidences of being causal. heme of the approaches and main results of this study. Candidate genes in bold are those with more evidences of being causal. May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 10 WES for CVID de Valles-Ibáñez et al. Table 10 | NGS studies on common variable immunodeficiency (CVID) and other primary immunodeficiency (PID). Study Syndrome Approach (Coverage) N Mendelian casesa Functional study Maffucci et al. (13) CVID WES (NA), 269 genes(NA) 50 15 No van Schouwenburg et al. (12) CVID WGS (27-40X) 34 NA RNAseq This work (2018) CVID WES (120X), CNV 36 5–8 CTLA4, LRBA Gallo et al. (79) PID 571 genes (580X), WES (>10X) 45 27, 18 10 Genes Stoddard et al. (78) PID 173 genes (305X) 120 18 No Stray-Pedersen et al. (77) PID, CVID WES (>100X) 278, 20 110, 8 No Al-Mouse et al. (80) PID 162 genes (461X) 139 35 No Yu et al. (81) SCID 196 genes (1000X) 20 14 No aReported in the original study. NA, not available. Table 10 | NGS studies on common variable immunodeficiency (CVID) and other primary immunodeficiency (PID). of many genes and environmental factors (90). Other features, such as different penetrancies and severities or the phenotypic variation in affected families, could also suggest an oligogenic origin for CVID, where the disease is caused or modulated by a few genes (91). Thus, we have performed different approaches to explore the possibility of CVID cases being originated by genetic variants in two or several genes. compared across patients to identify as causal the gene present in several patients (82). Because of that, we have used a conservative approach by mainly considering a list of candidate genes, and used genetic variants characteristics (evolutionary conservation, Polyphen values) and gene features (haploinsufficiency, essential- ity or tolerance to functional variation) mainly to indicate but not conclusively exclude a given candidate gene. For example, filtering by genic intolerance to functional variation is more effective in detecting false-positive rather than identifying the causal gene since it is known that genes producing Mendelian diseases show from medium to high intolerance values (83). Second, because of the higher prevalence of CVID compared to other PID, the use of too stringent frequency filters is not recommended, which hinders the identification of causal genes by increasing the number of candidates. Frontiers in Immunology  |  www.frontiersin.org FUNDING This study was funded by grants SAF2012-35025 and SAF2015- 68472-C2-2-R from the Ministerio de Economía y Compe­titividad (Spain) and FEDER (EU) to FC; by Direcció General de Recerca, Generalitat de Catalunya (2014SGR-866 and 2017SGR-702) to FC and EB; to EB by grant BFU2016-77961-P from Ministerio de Economía, Industria y Competitividad (Spain) AEI (Spain) and FEDER (EU); by Instituto de Salud Carlos III, grant PI14/00405, cofinanced by the European Regional Development Fund (ERDF) to RC; partially funded by CERCA Programme/Generalitat de Catalunya (JIA), and SAF2015-68472-C2-1-R grant from the Spanish Ministry of Economy and Competitiveness co-financed by European Regional Development Fund (ERDF) to JIA; GV-I was supported by grant BES-2012-051794; JH-R was supported by grant BES-2013-064333. TMB is supported by U01 MH106874 grant, Howard Hughes International Early Career, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya. All phases of this study were supported by the projects PI12/01990 and PI15/01094 to LA and PI13/00676 to MJ. This work was also supported by the Jeffrey Modell Foundation. This study makes use of data generated by the Medical Genome Project. A full list of the investigators who contributed to the generation of the data is avail- able from http://www.medicalgenomeproject.com/en. Funding for the project was provided by the Spanish Ministry of Economy and Competitiveness, projects I + D + i 2008, Subprograma de actuaciones Científicas y Tecnológi­cas en Parques Científicos y Tecnológicos (ACTEPARQ 2009) and ERFD. We think that CVID is a main example of rare disease where it is possible to arrive at similar phenotypes by several different genetic defects, either by mutations in different genes or by differ- ent genetic mechanisms including from monogenic to epigenetic scenarios. After the success of new sequencing technologies, and in particular of whole-exome sequencing in unraveling the molecular mechanisms of many rare syndromes, rare diseases such as CVID that do not completely fit with a Mendelian model represent a new challenge for medical genomics. In this manu- script, we have proposed different approaches to the analysis of CVID from whole-exome sequencing data, and have shown its power and limitations as a diagnostic tool for the study of these diseases. DISCUSSION Interestingly, most of the genes with significant results in these analyses (Figure 4) are among the ones with more pieces of evi- dence of being related to primary immunodeficiencies (51), thus supporting their role in the etiology of CVID. However, the appli- cation of these cohort approaches can be limited to syndromes as CVID because of its genetic heterogeneity. Instead, the use of higher levels of association such as pathways or functionally related genes can reduce the genetic heterogeneity and increase the detection power. Independently of the exact proportion of monogenic cases in CVID, in an important percentage of patients the disorder remains genetically uncharacterized, and it seems clear than other possible models beyond the monogenic scenario should be considered. A genome-wide association study performed on 363 CVID patients has revealed susceptibility factors in MHC and ADAM, among others (84), but association with common variation seems to be far from explaining all non-monogenic situations. As has been proposed for complex disease, this CVID missing heritability (87) must be hidden under other models that have not been deeply explored, as oligogenic, accumulation of rare functional variation, epigenetic (11, 88) or even somatic (89). In fact, the prevalence of CVID would fit with a model where the disease is produced by mutations in two or in a few genes, an intermediate scenario between the very rare disorders originated by a single locus and common disease produced by the interaction The detection of somatic genetic variants from exome sequ­encing data is not straightforward. The detection power May 2018  |  Volume 9  |  Article 636 Frontiers in Immunology  |  www.frontiersin.org 11 WES for CVID de Valles-Ibáñez et al. AUTHOR CONTRIBUTIONS FC and MJ conceived the project. LA, MJ, and FC designed the study. GV-I coordinated the bioinformatic analysis. GV-I, AE-S, and EAG-N performed the functional validation experiments. All the authors participated in the analysis of the data. GV-I, AE-S, MP, PS-P, RC, LA, MJ, and FC wrote the manuscript. ETHICS STATEMENT All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Comitè ètic d’Investigació Clínica-Parc de salut Mar (Barcelona). FUNDING Beyond the identification of the causal gene in some patients, we hope that these kinds of studies can also be used to help detect key pathways related to the development of the disease, thus contributing to a better understanding of its etiology. From our and previous results, we conclude that in an important proportion of patients it will be essential to integrate data from different omic approaches to solve the genetic origin of the disease. ACKNOWLEDGMENTS We thank all participants in the study. Exome sequencing was performed at the CNAG (Barcelona). ETHICS STATEMENT ultimately depends on the mutation frequency in the tissue, which is conditioned by the cell populations affected by the mutation and their relative abundance in blood, and will be, therefore, practically undetectable if present in low-frequency cell populations. On the other hand, high-frequency mutations present in more than 40% of the reads cannot be differentiated from germline mutations unless very high coverages are achieved. In addition to high coverages, the modification of standard NGS data analysis pipelines, which by default discard genetic variants in allelic imbalance, is required. We have tentatively analyzed exome sequencing data generated in this study (with 120X is the higher for CVID produced to date) scanning for low frequency variants with predicted high impact in our set of candidate genes. Not one of the patients presented a candidate somatic variant in any of the 97 CVID genes. A previous study proposed no role for somatic CNV in CVID, based on the sta- bility of the overall CNV burden over time (85). However, for a proper analysis of the role of somatic variation much higher sequencing coverage would be needed, and the possibility of sequencing different cell populations or tissues with different origin could be also considered since variant callers for somatic variant calling are optimized for the comparison between healthy and affected tissue (tumor). We also propose that, as a change to the experimental design of our study, late onset CVID cases should be included in a study targeting somatic variation. Finally, epigenetics is also suspected to contribute to CVID. Altered epigenetic profiles are known to be related both to common and rare genetic disease (95). 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Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract
Electronic Journal of Biotechnology
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⁎ Corresponding author. E-mail address: rodrigoleite@ufgd.edu.br (R.S.R. Leite). Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. a r t i c l e i n f o Background: β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Article history: Received 18 February 2015 Accepted 15 May 2015 Available online 27 June 2015 Keywords: Cellobiase Cellulases and hemicellulases Industrial enzymes Microbial enzymes Article history: Received 18 February 2015 Accepted 15 May 2015 Available online 27 June 2015 Keywords: Cellobiase Cellulases and hemicellulases Industrial enzymes Microbial enzymes g g y p p y Results: A high production of β-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The optimal activity of β-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5–10.5. The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 10% of ethanol. Conclusions: β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the utility of this enzyme in diverse industrial processes. © 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract Nayara Fernanda Lisboa Garcia a, Flávia Regina da Silva Santos a, Fabiano Avelino Gonçalves b, Marcelo Fossa da Paz a, Gustavo Graciano Fonseca b, Rodrigo Simões Ribeiro Leite a,⁎ a Laboratório de Enzimologia e Processos Fermentativos, Faculdade de Ciências Biológicas e Ambientais, Universidade Federal da Grande Dourad b Laboratório de Bioengenharia, Faculdade de Engenharia, Universidade Federal da Grande Dourados, Dourados, MS, Brazil Electronic Journal of Biotechnology 18 (2015) 314–319 Electronic Journal of Biotechnology 18 (2015) 314–319 Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.ejbt.2015.05.007 0717-3458/© 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction enzymatic hydrolysis of cellulose in order to obtain fermentable sugars and the production of functional foods derived from soy. The enzyme is also used in the juice and beverage industry, where it can improve the aromatic quality of wine and other grape derivatives [3]. The pronounced scarcity of fossil fuels related to environmental problems resulting from their processing and consumption has prompted the search for alternative sources of biofuels and renewable energy. This in turn, has generated significant interest in the use of cellulases and other enzymes to convert vegetal biomass into fermentable sugars [1]. The obtainment of industrial enzymes in a sustainable and economically viable manner requires the pursuit of renewable raw materials and processes at low cost. The use of solid-state fermentation (SSF) can reduce the environmental impact and add value to the by-products of agroindustry [4]. The iterative improvement and advantages of SSF have been described in several reports, which studied the influence of different cultivation parameters on the production of microbial enzymes [5,6]. The advantages of SSF include the simplicity of growth conditions, because they are very similar to the environmental systems where many microorganisms develop (especially filamentous fungi); the reduced energy consumption, and that complex equipment or sophisticated control systems are not required. The method also results in higher levels of productivity and low catabolite repression, and favors increased stability of the secreted enzymes [7]. Enzymatic hydrolysis of cellulose to glucose requires at least three different enzymes including endo-glucanases (EC 3.2.1.4), that internally hydrolyze cellulose chains, reducing its degree of polymerization; exo-glucanases (EC 3.2.1.91) that attack the non-reducing and reducing extremities of cellulose, releasing cellobiose; and β-glucosidases (EC 3.2.1.21) that hydrolyze cellobiose and oligosaccharides, thereby releasing glucose [2]. The ability of β-glucosidase to utilize different glycosidic substrates renders it suitable for several industrial processes, including the In general, the industrial applicability of an enzyme is closely related to the cost of its production and physicochemical characteristics. The N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 315 2.6. Characterization of β-glucosidase produced by the fungus L. ramosa 2.6. Characterization of β-glucosidase produced by the fungus L. ramosa production costs can be reduced by screening hyper producer strains, associated with the cultivation process optimized in low-cost mediums [3]. Previous work conducted by our Research Group revealed high β-glucosidase production by the fungus Lichtheimia ramosa by SSF using several lignocellulosic materials [6,8]. 2.1. Microorganism The filamentous fungus L. ramosa was isolated from sugarcane bagasse provided by São Fernando Açúcar e Álcool Ltda., Dourados, MS, Brazil [8]. The microorganism was maintained on Sabouraud Dextrose Agar medium; after growth at 28°C for 48 h, the strain was stored at 4°C. 1. Introduction This study aimed to optimize the β-glucosidase production by this fungus by SSF. The β-glucosidase produced was biochemically characterized and the catalytic properties of the enzymatic extract were evaluated. 3.1. Production of β-glucosidase by solid-state fermentation The extraction of the enzyme from the fermented substrate was carried out by adding 50 mL of distilled water, and constantly shaking at 100 rpm for 1 h. The sample was filtered and centrifuged at 3000 × g for 5 min. The supernatant was considered the enzymatic extract and was used in the following steps. 3.1.1. Selection of substrates for β-glucosidase production Among the tested substrates, the cultivation of the fungus L. ramosa in wheat bran provided higher β-glucosidase production (162.2 U/g or 16.22 U/mL) (Table 1). The wheat bran has suitable nutritional composition as a substrate for microbial growth; it contains 2.3. Solid-state fermentation (SSF) The CMCase and xylanase activities were quantified using 3% carboxymethylcellulose (Sigma C5678) and 1% xylan (Sigma Birch-Wood), respectively. The reducing sugar released was quantified by the DNS method [11]. The β-xylosidase activity was measured with the synthetic substrate p-nitrophenyl-β-D-xylopyranoside (4 mM, Sigma), following the methodology described in Section 2.5. The potential to hydrolyze cellobiose was evaluated with a glucose-oxidase kit (Glucose-PP Analisa). Specifically, 100 μL of the enzymatic extract was added to 0.9 mL of 50 mM sodium acetate buffer containing 0.5% cellobiose (Fluka). One unit of enzymatic activity was defined as the amount of enzyme capable of producing 1 μmol of product per min of reaction. The enzyme was produced by cultivating the fungus in 250 mL Erlenmeyer flasks containing 5 g of substrate (wheat bran, soy bran, corn cob, corn straw, rice peel, or sugarcane bagasse), previously washed and dried at 60°C for 24 h. The optimal substrate for enzyme production was used in subsequent steps to evaluate the effects of varying the pH of cultivation medium, moisture content, temperature, and time of cultivation. The parameter selected in each step was used for further cultivation, in an iterative strategy designed to optimize the fermentation process for β-glucosidase production. All material was previously autoclaved for 20 min at 121°C, and the experiments were performed in duplicate. 2.6.1. Effect of pH The optimum pH for β-glucosidase activity was determined by measuring the activity at 50°C at different pH values (3.0–8.0), with increments of 0.5, using 0.1 M citrate–phosphate buffer solution. The pH stability was determined incubating the enzyme for 24 h at 25°C at different pH values, appropriately diluted with buffer solutions: 0.1 M citrate–phosphate (pH 3.0–8.0), 0.1 M Tris–HCl (pH 8.0–8.5), and 0.1 M glycine NaOH (pH 8.5–10.5), with increments of 0.5, adopting as 100% the highest value of residual activity obtained after the samples treatment. The residual activity was determined under optimal conditions of pH and temperature. 2.6.3. Effect of glucose and ethanol on β-glucosidase activity .3. Effect of glucose and ethanol on β-glucosidase activity The enzymatic activity was quantified with the addition of glucose or ethanol at different concentrations in the reaction mixture (0–200 mM glucose or 0–30% of ethanol). The activities were measured under optimal conditions of pH and temperature. 2.6.2. Effect of temperature The optimum temperature for β-glucosidase activity was obtained by determining the enzymatic activity over a temperature range of 30°C–75°C, with increments of 5°C, at the respective optimum pH. Thermostability was determined by incubating the enzyme for 1 h at different temperatures (30°C–70°C), with increments of 5°C, adopting as 100% the highest value of residual activity obtained after the samples treatment. The residual activities were measured under optimal conditions of pH and temperature. 2.5. Determination of β-glucosidase activity Table 1 β-Glucosidase production in different agroindustrial substrates (75% of initial moisture) by L. ramosa by solid state fermentation, at 96 h of incubation, under 28°C. Substrate U/mL U/g Wheat bran 16.22 ± 0.42 162.2 ± 4.2 Soy bran 1.15 ± 0.07 11.5 ± 0.7 Corn cob 0.35 ± 0.04 3.5 ± 0.45 Corn straw 0.27 ± 0.02 2.7 ± 0.2 Rice peel 0.068 ± 0.00 0.68 ± 0.00 Sugarcane bagasse 1.11 ± 0.025 11.1 ± 0.25 2.4. Enzyme extraction 3.1. Production of β-glucosidase by solid-state fermentation 2.2. Inoculum The organism was cultivated in inclined 250 mL Erlenmeyer flasks containing 40 mL of Sabouraud Dextrose Agar and maintained for 48°C h at 28°C. A fungal suspension was obtained by adding 25 mL of nutrient solution and gently scraping the surface of the culture. The nutrient solution was composed of 0.1% ammonium sulfate, 0.1% magnesium sulfate heptahydrate, and 0.1% ammonium nitrate (w/v) [9]. As inoculum, 5 mL of this suspension was transferred to each 250 mL Erlenmeyer flask containing lignocellulosic material as substrates. Table 1 Table 1 β-Glucosidase production in different agroindustrial substrates (75% of initial moisture) by L. ramosa by solid state fermentation, at 96 h of incubation, under 28°C. The β-glucosidase activity was determined with 50 μL of enzymatic extract, 250 μL of sodium acetate buffer (0.1 M, pH 4.5), and 250 μL of p-nitrophenyl-β-D-glucopyranoside (4 mM, pNPβG, Sigma) during a 10 min reaction at 50°C. The enzymatic reaction was stopped with 2 mL of sodium carbonate (2 M), and the liberated product was spectrophotometrically quantified at 410 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of nitrophenol per minute of reaction [10]. N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 316 appropriate quantities of carbohydrates, proteins, fats, fiber and ashes (Ca, Mg, P, K, S), favoring enzymes production [12]. medium, interfering with the gas transfer and temperature. On the other hand, the low moisture content can decrease the solubility of nutrients, disfavoring microbial metabolic activity [17]. High level β-glucosidase production can be achieved during the culture of microorganisms by solid-state fermentation, using wheat bran as either the main substrate, or as a substantial component of the mixture [3,8,12,13]. Thus, wheat bran was selected for subsequent assays in order to optimize the cultivation process for β-glucosidase production. The ideal temperature for β-glucosidase production by L. ramosa was 35°C, about 249.0 U/g (24.9 U/mL) (Fig. 1c). Fig. 1c reveals that a higher amount of enzyme was produced in cultures carried out at 30°C–40°C. This optimal temperature for enzyme production is higher than the range most commonly considered optimal for the cultivation of mesophilic microorganisms, which is usually between 28°C and 30°C [13,18]. This characteristic favors the use of this strain in industrial processes, where variations in process temperature are acceptable, considering that the control of fermentation parameters on a large scale is not as precise as in laboratory conditions. 3.1.2. Influence of fermentative parameters on β-glucosidase production using wheat bran as substrate 3.1.2. Influence of fermentative parameters on β-glucosidase production using wheat bran as substrate The greatest β-glucosidase production by the fungus was obtained from the cultivation where the initial pH of the nutrient solution was adjusted to 5.0 (Fig. 1a). However, the microorganism showed considerable enzyme production for all pH values evaluated. Previous reports indicate that filamentous fungi produce cellulases at pH values below neutrality [14]. Table 1 Most filamentous fungi show optimal growth in slightly acidic pH. In general, values of pH higher than 7.0 reduce fungal growth and, thereby reducing the enzyme production [15]. A considerable reduction in enzyme production was evident in cultures performed at 25°C and 45°C (Fig. 1c). Temperatures that are significantly lower than the optimal for microbial growth disfavor nutrient transport and the exchange of products between the intracellular and extracellular environment. This is because low temperatures reduce both the permeability of the plasma membrane and the speed of the metabolic reactions. On the other hand, very high temperatures cause the collapse of membranous structures and denature structural proteins and enzymes. Accordingly, both cases result in reduced enzyme production [12]. The pH was not controlled during the cultivation process due to the heterogeneity of the process of solid-state fermentation. According to Pandey et al. [16], the difficulty of monitoring and controlling fermentation parameters in solid-state fermentation is perhaps, the main drawback of this process. Variations of pH during the fermentation process are due to the metabolic activity of the microorganisms, and may be increased or decreased according to the by-products released or the nutrients consumed during the process. Finally, the influence of cultivation time was investigated. The highest enzyme production was obtained at 96 h of incubation at 35°C using wheat bran as the substrate with 65% of moisture and pH 5.0 (Fig. 1d). The overall optimization of the process increased the β-glucosidase production from 162.2 U/g (16.22 U/mL) to 274.0 U/g (27.4 U/mL) (Table 1 and Fig. 1d, respectively). In addition to increased enzyme production, the optimization permitted a reduction in cultivation time to less than the duration used in preliminary assays carried out by our research group [4,8]. Gonçalves et al. [8] reported the production of 17.26 U/mL of β-glucosidase in cultivation of the fungus L. ramosa for 120 h by solid-state fermentation. Our current results reinforce the importance of optimizing the culture parameters, as evidenced by a 68.9% increase in the β-glucosidase production when compared to the initial values. Among the moisture values evaluated, the highest enzyme production was obtained in wheat bran with 65% of initial moisture (Fig. 1b). Values between 60 and 70% of moisture are often used for cultivation of filamentous fungi when the aim is to produce β-glucosidase. Leite et al. Table 1 [3] reported the β-glucosidase production (70 U/g of substrate) by the cultivation of Thermoascus aurantiacus in wheat bran with 60% of moisture. Brijwani et al. [13] obtained higher production of β-glucosidase (10.71 U/g) using soybean peel and wheat bran with 70% of moisture, during co-cultivation of Trichoderma reesei and Aspergillus oryzae by solid-state fermentation. The moisture in solid-state fermentation can influence the synthesis and secretion of extracellular enzymes. The presence of free water between the particles of the substrate reduces the porosity of the The reduced cultivation time, achieved in the present work, is also a key improvement for fermentation techniques that use L. ramosa, since the cost of enzyme production is proportional to incubation time. A Fig. 1. Influence of fermentation parameters on β-glucosidase production by the fungus L. ramosa, by SSF in wheat bran. (a) Influence of initial cultivation pH; (b) Influence of initial substrate moisture; (c) Influence of cultivation temperature; (d) Influence of cultivation time. Conditions: pH 5.0; moisture 65%; temperature 35°C. Fig. 1. Influence of fermentation parameters on β-glucosidase production by the fungus L. ramosa, by SSF in wheat bran. (a) Influence of initial cultivation pH; (b) Influence of initial substrate moisture; (c) Influence of cultivation temperature; (d) Influence of cultivation time. Conditions: pH 5.0; moisture 65%; temperature 35°C. N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 317 the production of extremely stable β-glucosidase by mesophilic strains [3]. reduced amount of enzyme was found in extracts obtained after 96 h of cultivation (Fig. 1d). This is likely explained by the consumption of the culture medium nutrients and the excretion of by-products by the microorganism used for fermentation. Such by-products, which may interfere with protein synthesis as well as enzymatic activity, include proteases and substances that reduce macro and micronutrients, alter the pH, and decrease water availability [19]. The β-glucosidase produced by the fungus L. ramosa showed remarkable structural stability. The enzyme retained its original activity after 24 h of incubation over a pH range of 3.5–10.5 (Fig. 2c). Regarding the thermal stability of the enzyme, about 90% of the catalytic activity was recovered after 1 h at 55°C (Fig. 2d). The level of β-glucosidase production obtained during the current study is significantly higher than that described by other groups. Leite et al. [3] obtained 7.0 U/mL of β-glucosidase with 72 h of T. aurantiacus cultivation in wheat bran. Delabona et al. 3.2.2. Effect of glucose and ethanol on β-glucosidase activity Evaluation of the effect of ethanol on enzymatic activity is essential in studies with β-glucosidases, since these enzymes are frequently exposed to substantial concentrations of alcohol in many industrial applications [29]. The enzymatic extract obtained, under optimal culture conditions, was used in subsequent steps for the biochemical characterization of β-glucosidase. Fig. 3a shows the effect of different concentrations of ethanol (0– 30%) on the enzymatic activity. Ethanol concentrations up to 5% potentiated the enzymatic activity, and elicited an increase of up to 20% compared to the initial activity. Increasing ethanol concentration to 15% dramatically reduced enzymatic activity to only 22% of the original level. However, at a concentration of 10% of ethanol, β-glucosidase retained a level of catalytic activity similar to the control. Considering that the final ethanol concentration in fermented broths obtained by traditional processes is around 10% [30], we infer that the enzyme is sufficiently stable to be applied in industrial fermentation processes containing ethanol. Table 1 [20] obtained 105.82 U/g of substrate by cultivation of the fungus Aspergillus fumigatus in wheat bran for 96 h. Xin and Geng [21] obtained 61.6 U/g of substrate with T. reesei cultivated at 26°C for 192 h on woodchips. Ng et al. [2], reported the production of 159.1 U/g of substrate by the fungus Penicillium citrinum YS40-5 after cultivation in rice bran for 96 h. Zimbardi et al. [22] optimized the β-glucosidase production by the fungus Colletotrichum graminicola in wheat bran, with a maximum production substrate of 159.3 U/g, after 168 h. Silva et al. [6] reported the production of 0.061 U/mL of β-glucosidase by the fungus L. ramosa in pequi residue (typical fruit of the Cerrado vegetation), after 48 h in solid-state fermentation. The results are more significant when compared with previously published data. β-glucosidase produced by different species of Penicillium showed stability from pH 4.0 to 6.0 [26,27]. The enzyme produced by the fungus Trichoderma harzianum was stable at temperatures below 55°C for 15 min, maintaining only 36% of initial activity after 15 min at 60°C [28]. Delabona et al. [5] describe the stability of β-glucosidase produced by the fungus A. fumigatus P40M2 at temperatures from 40°C to 60°C and at pH 3.0 to 5.5. The β-glucosidase produced by the yeast Sporidiobolus pararoseus maintained its catalytic activity for 1 h at 40°C; at higher temperatures, only 30% of the initial activity was recovered [24]. 3.2. Characterization of β-glucosidase produced by the fungus L. ramosa 3.2. Characterization of β-glucosidase produced by the fungus L. ramosa 3.2.1. Effect of pH and temperature However, relatively little is known regarding the characteristics of these enzymes, and thus further studies in this area are required. Moreover, there is the possibility to improve these enzymes production in new works of culture optimization. Silva et al. [36] reported the production of 60 U/mL of CMCase and 107 U/mL by the fungus T. aurantiacus by solid-state fermentation, using corncob as substrate. Delabona et al. [20] report the production of 160.1 U/g of CMCase and 1055.62 U/g of xylanase by the fungus A. fumigatus cultivated in agroindustrial residue products. Different concentrations of glucose were added to the reaction mixture to evaluate the behavior of β-glucosidase in the presence of this inhibitor. The enzyme was strongly inhibited by glucose, maintaining approximately 30% of its original activity in the reaction carried out with 100 mM glucose (Fig. 3b). The majority of microbial β-glucosidases are inhibited by glucose, which is a major limitation of the use of these enzymes in industrial processes [3]. High glucose concentrations can interfere directly or indirectly with substrate binding to the enzyme active site, reducing the reaction rate [33]. The inhibition of β-glucosidase produced by L. ramosa was completely reversed when the substrate concentration was increased to the same glucose concentration, indicating that the interaction of the enzyme with the inhibitor is competitive. Another interesting aspect of the current study, shown in Table 2, is the impressive potential of the enzyme to hydrolyze cellobiose, as it yields values similar to those obtained with a synthetic substrate (pNPG). The microbial β-glucosidases can be classified into three major groups: (1) Aryl β-glucosidases, which exhibit high specificity to hydrolyze aryl-glycosides substrates, (2) true cellobiases, which are enzymes with that hydrolyze cellobiose with high specificity and (3) enzymes with low specificity: enzymes that act on different types of glycosides substrates [25,31]. Apparently, β-glucosidase expressed from the fungus L. ramosa has low specificity; that is, it has the potential to hydrolyze different glycosides substrates. However, to confirm this hypothesis, further studies should be performed with the purified enzyme. According to Bhatia et al. [25], most of microbial β-glucosidases are classified in the third group. Competitive inhibition can be reversed by increasing substrate concentration; the same fact is not observed in non-competitive inhibition. In competitive inhibition, the inhibitor and the substrate compete for the same binding site of the enzyme (the active site, in this case). 3.2.1. Effect of pH and temperature 3.2.1. Effect of pH and temperature The β-glucosidase produced by the fungus L. ramosa showed higher catalytic activity at pH 5.5 and temperature of 65°C (Fig. 2a and Fig. 2b). Surprisingly, the enzyme showed higher catalytic activity at temperatures above 50°C, not observed routinely in enzymes produced by mesophilic microorganisms. Belancic et al. [23] obtained the best activity at pH 5.0 and optimum temperature of 40°C for β-glucosidase produced by Debaryomyces vanrijiae. Most fungal β-glucosidases show optimum activity between 40°C and 50°C and at pH between 4.0 and 6.0 [24,25]. However, some studies have reported The increase in the catalytic potential of β-glucosidases observed by the ethanol addition is related to the glucosyltransferase activity [31]. Ethanol can increase the rate of reaction by acting as preferred acceptor of glycosyl residues during enzymatic catalysis [32]. The Fig. 2. Effect of pH and temperature on the activity (A and B) and stability (C and D) of β-glucosidase produced by the fungus L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. Fig. 2. Effect of pH and temperature on the activity (A and B) and stability (C and D) of β-glucosidase produced by the fungus L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 318 J Fig. 3. (a) effect of ethanol, and (b) glucose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. Fig. 3. (a) effect of ethanol, and (b) glucose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. g. 3. (a) effect of ethanol, and (b) glucose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and ose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35 hydrolysis and transglycosylation occur through the same biochemical pathway, differing only in the nature of the final acceptor [25]. 3.2.1. Effect of pH and temperature Thus, increasing the substrate concentration to equal or greater values than those of the inhibitor, favors the binding of the enzyme to the substrate, which is reflected in the reversibility of enzymatic inhibition [3]. The reversibility of inhibition by glucose and stability to ethanol confirm the potential of this β-glucosidase for applications that require simultaneous saccharification and fermentation processes [3], where the monosaccharides released by enzymatic hydrolysis are simultaneously converted to ethanol by fermenting microorganisms [34,35]. Table 2 Table 2 Catalytic potential of the enzymatic extract obtained by solid-state fermentation by L. ramosa in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. Enzyme Substrate U/mL U/g CMCase Carboxymethylcellulose 15.21 152.1 β-Glucosidase p-nitrophenyl-β-D-glucopyranoside (pNPG) 23.47 237.7 β-Glucosidase Cellobiose 23.45 234.5 Xylanase Xylan 2.85 28.5 β-Xylosidase Xylopyranoside p-nitrophenyl-β-D (pNPX) 11.57 115.7 Catalytic potential of the enzymatic extract obtained by solid-state fermentation by L. ramosa in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. Catalytic potential of the enzymatic extract obtained by solid-state fermentation by L. ramosa in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. 4. Conclusions L. ramosa has proven to be a remarkable fungus for use in β-glucosidase production when cultivated by solid-state fermentation using wheat bran as the substrate. This fungus is capable of producing several enzymes, including CMCase and β-xylosidase. β-Glucosidase was highly stable across a range of pH and temperatures and retained its original activity in solutions containing 10% of ethanol. Furthermore, the inhibitory effects of glucose were completely reversed at high substrate concentrations. The enzyme can hydrolyze different glycoside substrates; due to these characteristics, the β-glucosidase produced by L. ramosa can be used for the production of second-generation ethanol as well as for the improvement of food and beverage quality. 3.3. Catalytic potential of the enzymatic extract 3.3. Catalytic potential of the enzymatic extract The production of other cellulases and also hemicellulases by L. ramosa, in optimized culture conditions, was evaluated in this work (Table 2). The enzymatic extract exhibited CMCase (152.1 U/g or 15.21 U/mL), xylanase (28.5 U/g or 2.85 U/mL), and β-xylosidase (115.7 U/g or 11.57 U/mL) activity. The production of the CMCase and hemicellulases by L. ramosa is not very impressive when compared with hyper-producing strains. Financial support The authors gratefully acknowledge the financial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (444630/2014-7), the "Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul-FUNDECT (23/200.211/2014), and the Coordenação de Aperfeiçoamento Pessoal de Nível Superior-CAPES. Enzyme Substrate U/mL U/g CMCase Carboxymethylcellulose 15.21 152.1 β-Glucosidase p-nitrophenyl-β-D-glucopyranoside (pNPG) 23.47 237.7 β-Glucosidase Cellobiose 23.45 234.5 Xylanase Xylan 2.85 28.5 β-Xylosidase Xylopyranoside p-nitrophenyl-β-D (pNPX) 11.57 115.7 N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 319 [18] Deswal D, Khasa YP, Kuhad RC. Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010 under solid state fermentation. Bioresour Technol 2011;102:6065–72. http://dx.doi.org/10.1016/j.biortech.2011.03.032. The authors declare that there is no conflict of interests. p g j [19] Haq I, Javed MM, Khan TS. An innovative approach for hyperproduction of cellulolytic and hemicellulolytic enzymes by consortium of Aspergillus niger MSK-7 and Trichoderma viride MSK-10. Afr J Biotechnol 2006;5:609–14. References [20] Delabona PDS, Pirota RDPB, Codima CA, Tremacoldi CR, Rodrigues A, Farinas CS. Using Amazon forest fungi and agricultural residues as a strategy to produce cellulolytic enzymes. Biomass Bioenergy 2012;37:243–50. http://dx.doi.org/10.1016/j.biombioe.2011.12.006. [1] Singhania RR, Sukumaran RK, Patel AK, Larroche C, Pandey A. Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases. Enzyme Microb Technol 2010; 46:541–9. http://dx.doi.org/10.1016/j.enzmictec.2010.03.010. 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Quantification of avian hazards to military aircraft and implications for wildlife management
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Quantification of avian hazards to military aircraft and implications for wildlife Morgan B. PfeifferID1,2*, Bradley F. Blackwell1, Travis L. DeVault1 Morgan B. PfeifferID1,2*, Bradley F. Blackwell1, Travis L. DeVault1 1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio, United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela University, George, South Africa 1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio, United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela University, George, South Africa 1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio, United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela University, George, South Africa 1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio, United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela University, George, South Africa a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * morgan.b.pfeiffer@aphis.usda.gov * morgan.b.pfeiffer@aphis.usda.gov Editor: Ludek Bartos, Institute of Animal Science, CZECH REPUBLIC Received: May 21, 2018 Accepted: October 16, 2018 Published: November 1, 2018 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are available from Dryad, DOI: https://doi.org/10.5061/ dryad.s0n4n05. Funding: This study was funded by the Comannder, Navy Installations Command (CNIC) Airfield Operations Program. No additional external funding was received for this study. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Collisions between birds and military aircraft are common and can have catastrophic effects. Knowledge of relative wildlife hazards to aircraft (the likelihood of aircraft damage when a species is struck) is needed before estimating wildlife strike risk (combined frequency and severity component) at military airfields. Despite annual reviews of wildlife strike trends with civil aviation since the 1990s, little is known about wildlife strike trends for military aircraft. We hypothesized that species relative hazard scores would correlate positively with aircraft type and avian body mass. Only strike records identified to species that occurred within the U.S. (n = 36,979) and involved United States Navy or United States Air Force aircraft were used to calculate relative hazard scores. The most hazardous species to military aircraft was the snow goose (Anser caerulescens), followed by the common loon (Gavia immer), and a tie between Canada goose (Branta canadensis) and black vulture (Coragyps atratus). We found an association between avian body mass and relative hazard score (r2 = 0.76) for all military airframes. In general, relative hazard scores per species were higher for military than civil airframes. An important consideration is that hazard scores can vary depending on aircraft type. We found that avian body mass affected the probability of damage differentially per airframe. In the development of an airfield wildlife management plan, and absent esti- mates of species strike risk, airport wildlife biologists should prioritize management of spe- cies with high relative hazard scores. RESEARCH ARTICLE OPEN ACCESS Citation: Pfeiffer MB, Blackwell BF, DeVault TL (2018) Quantification of avian hazards to military aircraft and implications for wildlife management. PLoS ONE 13(11): e0206599. https://doi.org/ 10.1371/journal.pone.0206599 Citation: Pfeiffer MB, Blackwell BF, DeVault TL (2018) Quantification of avian hazards to military aircraft and implications for wildlife management. PLoS ONE 13(11): e0206599. https://doi.org/ 10.1371/journal.pone.0206599 Editor: Ludek Bartos, Institute of Animal Science, CZECH REPUBLIC Received: May 21, 2018 Accepted: October 16, 2018 Published: November 1, 2018 Introduction One of the most ubiquitous human-wildlife conflicts involves wildlife colliding with transpor- tation [1]. These collisions usually result in the death of the animal, damage to the vehicle, and even injuries to humans or human fatalities [2]. Compared to other countries, the United States of America (U.S.) has the most reported wildlife collisions (strikes) with aircraft, likely because of numerous aircraft operations, mandatory strike reporting in some sectors, and an abundance of large-bodied birds [3, 4]. Ranking of wildlife hazards to civil aircraft has been Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 1 / 16 Military avian hazard scores conducted regularly since the 1990s [5–7], but rarely for military aviation. Furthermore, to our knowledge, relative hazard scores (RHS) for military aircraft have not been calculated or pub- lished [8]. According to the United States Air Force (USAF) wildlife strike database from 1985–1998, an average of $35 million (U.S.) in damage was attributed to wildlife strikes annu- ally [8]. To mitigate this risk of wildlife strikes, most United States military bases with a flying mission require a Bird/Wildlife/Animal Aircraft Strike Hazard program [9,10]. A necessary component of estimating strike risk (see below), and thus prioritizing resources and time for mitigating wildlife strikes, is the ranking of wildlife by the hazard they pose to avi- ation. Hazard is defined in terms of the likelihood of damage, substantial damage, or negative effect-on-flight procedures when a strike event occurs [11]. This hazard, or severity, of a strike with an animal is one component used to estimate strike risk, along with a measure of the fre- quency of the event (i.e. how often strikes occur per species) [12]. Because RHS have never been calculated for military aircraft, our objective was to calculate these scores as the first step in estimating risk, which is dependent upon a number of local-level variables [12], in addition to mission type. Wildlife hazard ranking has been conducted for civil aviation in a number of ways [6, 11, 13]. A previous study on USAF wildlife strikes ranked species groups (i.e. species that are closely related phylogenetically) based on the number of strikes within each damage class (i.e. categories of monetary damage costs to aircraft) and found that vultures (Cathartidae), geese (Anatidae), pelicans (Pelecanidae), blackbirds/starlings (Icteridae/Sturnidae), and buteo hawks (Accipitridae) were ranked as most hazardous, in that order [8]. Introduction However, not all wild- life strikes result in aircraft damage; therefore, damaging or negative effect-on-flight strikes per species should be considered as a proportion of all strikes (including non-damaging strikes) for that species or group [11]. In recent wildlife hazard rankings for civil aviation, a composite hazard score was calculated that included the sum of the ranks of strikes with dam- age, substantial damage, and strikes that had a negative effect-on-flight [11, 12, 14]. Replicating the methods of the previous examples must be done with special consideration of the differ- ences between civil and military aviation and the assumed variation in the bird strike risk. Military and civil operations differ in their mission types, including air speeds and maneu- verability, which could affect wildlife hazard scores. During certain training procedures, mili- tary aircraft travel at high speeds and close to the ground, which likely increases the strike hazard compared to civil aircraft because of more time spent in bird rich altitudes [15]. Dam- age to aircraft is a function of kinetic energy which is the product of the mass of the bird in relation to the velocity of the aircraft [16]. However, birds with smaller body mass might still pose a substantial hazard to military, as compared to civil aircraft because of the increased air- craft speeds, particularly at lower altitudes, characteristic of military operations. In other words, whereas civil strikes involving smaller birds generally pose less hazard, collisions of small bird species with military aircraft can involve much higher airspeeds. Hence, a hazard difference between civil and military aviation may be more noticeable with the smaller rather than the larger birds. In addition, unlike civil aviation, military training operations can gener- ally adjust their flight schedule if hazards, including the risk of bird collisions, is predicted to be elevated [17]. Given these considerations, our purpose was to calculate avian RHS for the United States Navy (USN), including the United States Marine Corp, and the USAF. We predicted that air- craft type and body mass would influence RHS [11]. Because military airframes are designed for a variety of purposes and generally only conduct certain maneuvers (e.g. fighter airframes that accelerate rapidly and turn with high precision vs. cargo airframes designed to move heavy payloads), we calculated RHS separately for grouped military airframe type. Materials and methods We used wildlife strike records from databases maintained by the USN and the USAF. The USN wildlife strike database spanned 27 years (1990–2017) and contained 21,661 wildlife strike records. The USAF dataset spanned 23 years (1994–2017) and contained 104,129 wildlife strike records. Wildlife remains found on an aircraft after a strike event are required to be sent to the Feather Identification Lab at the Smithsonian Institution, where personnel identify wild- life based on feather characteristics and/or DNA [18]. Some species information for the USN database was gleaned from the Smithsonian Institution’s internal database by matching the event number. We filtered these databases to only include strikes that occurred within the U.S. where the species involved in the strike was identified. We did not filter strikes by altitude (i.e. < 152 m), which was done in similar studies with civil aviation to focus on the airport environment [11]. Military aircraft do not adhere to the 3˚ glide slope of ascent and decent of civil aircraft, from which the threshold of 152 m is based [19], and routinely conduct low-level flights. If multiple animals were involved in one strike event, they are entered as separate strikes, but with the same report number. To prevent duplication, only one strike per event was used (i.e. duplicate report numbers were removed). Duplicate report numbers were removed based on body size; the larger of the two species was retained. Less than 12% of all records involved more than one reported species, in which we kept the larger of the two species for the analysis. Only species with more than 20 strikes were used in our analyses [12, 14]. To reduce complexity and facili- tate management decisions, those species involved in fewer than 50 strikes were combined into species groups (S1 Table) based on phylogeny [20]. Species groups (n = 17) were com- posed of closely related species as per [20] and did not include any strikes in which guild was identified but not species. A total of 12 species with more than 20 but less than 50 strikes (noted in S1 Table) could not be grouped because they were not closely related to other species and left as is in the analyses A similar species grouping scheme is accepted by the FAA in the management of hazardous species at airports and this scheme allows us to compare to other studies [12, 14]. Introduction We pre- dicted that airframes with more sensitive components would experience higher RHS PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 2 / 16 Military avian hazard scores compared to robust airframes. Calculating hazard scores in this way can help prioritize spe- cies-specific wildlife management plans for military airfields across the U.S. serving different mission types. Based on previous work involving civil aircraft [6, 11], we predicted that body mass and migration flyway (Central, Mississippi, Pacific, Atlantic) would contribute to pre- dicting level of damage (see below) for military aircraft. Lastly, because of differing reporting rates, we predicted that probability of damage would differ between the two military branches. Furthermore, significant relationships between body mass and RHS could be used to calculate hazard scores for species that are not currently in our dataset (e.g. solve the equation for RHS based on avian body mass). PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Materials and methods Damage to aircraft from a wildlife strike in the military databases is recorded as a damage class (A/B/C/D/E/H, Table 1), based on monetary costs of repairs and human injuries rather than a categorical definition like in civil aviation. The A damage class is the most severe and H is the least [6, 8]. The USAF are required to report wildlife strikes under the Air Force Instruc- tion 91–202 Mishap Prevention Program [9] and the Air Force Instruction 91–204 Safety Investigations Reports [21]. The USN is required to report wildlife strikes under the Com- mander, Navy Installations Command Instruction 3750.1 [10]. The military databases were filtered by their damage class. None of the defined damage cat- egories are related to a strike in which no damage occurred, which is needed for the hazard ranking [6]. Therefore, strikes for which class severity was unknown in the USAF dataset were 3 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores Table 1. Definitions of United States Navy (USN) and United States Air Force (USAF) damage classes in U.S. dol- lars. Damage class ‘H’ was divided for the USN dataset based on internal discussions. Damage Class Associated monetary Cost A > $2,000,000 B $500,000 –$2,000,000 C $50,000 –$500,000 D $20,000 –$50,000 E < $50,000 H (damaging) > $55 H (non-damaging)  $55 https://doi.org/10.1371/journal.pone.0206599.t001 efinitions of United States Navy (USN) and United States Air Force (USAF) damage classes in U.S. dol- age class ‘H’ was divided for the USN dataset based on internal discussions. https://doi.org/10.1371/journal.pone.0206599.t001 considered to be non-damaging (pers. comm. D. Sullivan, Chief, USAF BASH Team). In the USN dataset, there were no records in which strike severity was unknown. After discussions with USN personnel, we separated Class H strikes in the USN dataset into damaging (> $55 of damage costs) or non-damaging ( $55 of damage costs) strikes. The $55 threshold is the aver- age cost of collecting wildlife remains from an aircraft after a strike occurs, and generally does not indicate damage to the aircraft. As these classifications differed from the civil database (minor vs. substantial damage) and were much broader, we used the median damage repair costs for civil aviation that had substantial damage to create a cutoff value between minor and substantial damage for the military dataset [6]. Materials and methods We then calculated separately the percentage of total strikes for each species group that resulted in damage and substantial damage [11, 14]. We were unable to use the effect-on-flight metric because these data only were recorded in the USAF and not the USN database. To increase sample size for our across-branch analysis, we combined the USN and USAF strike records, which operate similar aircraft types and are located across the U.S. We ranked species groups as per Dolbeer et al. [6], minus the effect-on-flight metric. This procedure involved determining the percentage of total strikes for each species group that resulted in damage and substantial damage. The RHS was calculated by summing those per- centages and scaling them to 100 by dividing the sum of percentages for that species by the maximum percentage for any species. Next, the species groups were ordered from most to least hazardous, including tied ranks. We then categorized airframe groups that were similar in size and flight patterns (S2 Table). Airframe groups were suggested by military aviation experts and included four groups: rotorcraft (e.g. helicopters), fighters, cargo, and stealth air- frames. Some airframes could be classified in multiple categories (i.e. F-35 is considered a stealth fighter). For our purposes we grouped airframe by mission type (quick maneuverabil- ity, moving payloads, or surveillance missions), which may differ from other airframe group- ings. We highlight these unique airframe groups in S2 Table. We calculated RHS for each airframe group from the combined USAF and USN dataset. We investigated differences in rel- ative hazard scores of species groups and airframes using contingency tables and the chi- square test for goodness of fit [22]. As previous research identified avian body mass as an important predictor for RHS, we examined the relationship between RHS and avian body mass using averaged avian body mass within species [23]. For species groups, we weighted averaged body masses by the respective number of strikes for each species in the group. The bird body masses were log-transformed to normalize the data. We regressed species RHS against log-transformed avian body masses for all aircraft and the four airframe groups separately via a quadratic function or linear regression (based on best model fit). These relationships do not have biological ties, but rather relate to the physics of the mass of the bird species and the rate and pattern of influence on the RHS. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Materials and methods 4 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores We assessed model fit by the coefficient of determination (r2 value) and p value (α = 0.05) [24]. To emphasize the differences in airframe and flight demands that likely contribute to var- iance in strikes between military and civil aircraft, we descriptively compared species RHS between military and civil aircraft [12]. Lastly, we converted damage class into two binary variables for damage (1 = any level of damage occurred, 0 = no damage occurred) and for substantial damage (1 = substantial dam- age occurred, 0 = no substantial damage occurred) and used a binary logistic regression to evaluate how log body mass, migration flyway, reporting military branch, and airframe influ- enced the probability damage [14]. All variables were considered fixed effects and were included based on a priori hypotheses. Log body mass was a continuous variable and the remainder were categorical variables. Migration flyway included four levels: Atlantic, Central, Mississippi, and Pacific. Reporting military branch included USAF or USN. We also included interaction variables of the predictors. Candidate models were evaluated by their Akaike’s Information Criterion (AIC) with the best model having the lowest AIC [25]. We also calcu- lated the McFadden’s r2 value as an indication of fit 1- log likelihood of the model over the log likelihood of the null model [26]. Some strikes involved more than one individual bird, and we could account for this random effect for the specific strike event by using a generalized linear mixed model approach with strike event as the random variable. However, with over 30,000 strikes, our models failed to converge. All database filtering and data analyses were conducted in R ver. 3.4.3. [27] and the ‘lme4’ and ‘arm’ packages [28,29]. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores Table 2. Relative hazard scores (RHS) for 108 species groups from most to least hazardous for military aircraft within the United States. Results From 1990–2017 an average of $20 million in damage and human injuries was attributed to wildlife strikes (n = 6,733) per annum for the USN in foreign and domestic operations. For the USAF in the U.S., from 1994–2017, an average of $38 million per annum in damage and human injuries was attributed to wildlife strikes (n = 104,129). After removing strikes that occurred in other countries, not identified to avian species, and involved a species struck more than 20 times, the combined dataset was reduced to 36,979 strikes. Of this 3,646 (10%) strike records were from the USN and 33,333 strike records (90%) came from the USAF. The median cost for civil aviation from 2000–2015 with substantial damage was $87,570. The maximum cost associated with Class D/E/H strikes fell below $87,570, so only Class A/B/ C strikes were considered as comprising substantial damage ($50,000 to  $2,000,000) in our military dataset. For both military branches there were 923 strikes with substantial damage (Class A/B/C). An additional 3,024 strikes reported minor damage (Class D/E/H), which brought the total of damaging strikes to 3,947. Strikes reported with no damage (damage class unknown) represented the majority of the strike records (n = 33,032). A total of 186 bird species was involved in 20 or more strikes with USN and USAF aircraft, and 3 species were struck more than 2,000 times: horned lark (Eremophila alpestris), mourning dove (Zenaida macroura), and barn swallow (Hirundo rustica). Birds were categorized into 108 groups. The percentage of strikes with some level of damage ranged from 1% (burrowing owl [Athene cunicularia]) to 74% (snow goose [Anser caerulescens]). There were 40 species or species groups involved in strikes with no substantial damage (Table 2). The species group involved in the highest percentage of strikes with substantial damage was the snow goose (42%). The top three species by their composite rank were snow goose, common loon (Gavia immer), and a tie between Canada goose (Branta canadensis) and black vulture (Coragyps atra- tus) (Table 2). Filtering the strike records to only fighter, cargo, stealth, and rotorcraft and to species or species groups that were struck over 20 times reduced the total number of strike records to 5 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Species % with damage Damage rank % with substantial damage Substantial damage rank Relative hazard score (RHS) Composite rank Snow goose (Anser caerulescens) 74 1 42 1 100 1 Common loon (Gavia immer) 70 2 30 2 86 2 Black vulture (Coragyps atratus) 58 3 25 4 72 3 Canada goose (Branta canadensis) 56 4 29 3 74 3 Turkey vulture (Cathartes aura) 48 5 21 5 60 5 Northern pintail (Anas acuta) 44 8 16 7 51 6 Mallard (Anas platyrhynchos) 45 6 13 10 50 7 Swainson’s hawk (Buteo swainsoni) 41 9 14 8 47 8 Double-crested cormorant (Phalacrocorax auritus) 44 7 13 11 48 9 Herring gull (Larus argentatus) 32 14 18 6 43 10 Red-tailed hawk (Buteo jamaicensis) 37 11 14 9 43 10 Bald eagle (Haliaeetus leucocephalus) 40 10 11 13 44 12 Other ducks 36 12 10 15 39 13 Pied-billed grebe (Podilymbus podiceps) 29 16 12 12 35 14 Great blue heron (Ardea herodias) 31 15 11 14 36 15 Osprey (Pandion haliaetus) 35 13 9 16 37 15  Other egrets 29 18 7 19 31 17  Other hawks 27 19 7 21 29 18 American coot (Fulica americana) 29 17 5 25 29 19 Ring-billed gull (Larus delawarensis) 19 25 8 18 23 20 Sprague’s pipit (Anthus spragueii) 17 28 8 17 22 21 Cattle egret (Bubulcus ibis) 18 26 6 22 21 22 White-winged dove (Zenaida asiatica) 20 23 5 26 21 23 Common grackle (Quiscalus quiscula) 18 27 6 24 21 24 Great horned owl (Bubo virginianus) 20 22 4 29 21 24  Other falcons 15 33 7 20 19 26 Mississippi kite (Ictinia mississippiensis) 23 20 3 35 22 27  Other gulls 16 30 4 28 18 28 American crow (Corvus brachyrhynchos) 13 37 6 23 16 29 Rock dove (Columba livia) 15 31 4 30 17 30 White-throated swift (Aeronautes saxatalis) 14 34 4 32 15 31 European starling (Sturnus vulgaris) 13 37 3 33 14 32 Yellow-billed Cuckoo (Coccyzus americanus) 15 32 2 38 15 32 Vaux’s swift (Chaetura vauxi) 12 40 4 31 14 34 Barn owl (Tyto alba) 12 41 3 34 12 35 Baltimore oriole (Icterus galbula) 10 48 2 36 11 36  Other blackbirds 9 58 5 27 12 37 Black-bellied plover (Pluvialis squatarola) 13 36 1 50 12 38 Scarlet tanager (Piranga olivacea) 14 35 1 52 13 39  Other doves 11 44 2 44 11 40 Purple martin (Progne subis) 11 42 1 48 11 41 American robin (Turdus migratorius) 10 49 2 43 10 42  Other plovers 11 46 2 46 11 42 (Continued) ores (RHS) for 108 species groups from most to least hazardous for military aircraft within the United States. Military avian hazard scores Table 2. (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 6 / 16 (Continued) Species % with damage Damage rank % with substantial damage Substantial damage rank Relative hazard score (RHS) Composite rank Yellow-bellied sapsucker (Sphyrapicus varius) 9 60 2 37 10 44 Mourning dove (Zenaida macroura) 9 55 2 45 9 45 Brown-headed cowbird (Molothrus ater) 10 47 1 55 10 46 Northern flicker (Colaptes auratus) 20 21 0 82 18 47 Ovenbird (Seiurus aurocapillus) 8 63 2 40 9 47  Other shorebirds 9 57 1 47 9 49  Other thrushes 11 43 1 62 10 50 Brown thrasher (Toxostoma rufum) 20 24 0 82 17 51 Cave swallow (Petrochelidon fulva) 11 45 1 61 10 51 Cardinals, grosbeaks, and allies 10 51 1 60 10 53 Red-winged blackbird (Agelaius phoeniceus) 7 70 2 41 8 53 Wood thrush (Hylocichla mustelina) 17 29 0 82 14 53 Great crested flycatcher (Myiarchus crinitus) 8 65 1 48 8 56  Meadowlarks 7 71 2 42 7 56 American kestrel (Falco sparverius) 7 68 1 51 7 58 Gray catbird (Dumetella carolinensis) 10 50 0 70 9 59 Ruby-crowned kinglet (Regulus calendula) 7 67 1 53 7 59 Violet-green swallow (Tachycineta thalassina) 12 39 0 82 10 61 Scissor-tailed flycatcher (Tyrannus forficatus) 9 56 1 68 8 62 Sora (Porzana carolina) 10 52 0 82 8 63 Blue-gray gnatcatcher (Polioptila caerulea) 7 69 1 66 7 64 Semipalmated sandpiper (Calidris pusilla) 10 53 0 82 8 64 Northern mockingbird (Mimus polyglottos) 9 54 0 82 8 66 Killdeer (Charadrius vociferous) 6 81 1 59 6 67 Orchard oriole (Icterus spurius) 9 58 0 82 8 67  Other flycatchers 8 61 0 82 7 69  Other terns 4 104 2 39 5 69  Other wood warblers 6 75 0 69 6 71 Short-eared owl (Asio flammeus) 8 62 0 82 7 71 Red-eyed vireo (Vireo olivaceus) 6 74 0 71 6 73 Barn swallow (Hirundo rustica) 6 79 1 67 6 74 Upland sandpiper (Bartramia longicauda) 8 64 0 82 7 74 House wren (Troglodytes aedon) 5 92 1 55 5 76 Lapland longspur (Calcarius lapponicus) 6 82 1 65 5 76 Dunlin (Calidris alpine) 8 66 0 82 7 78 Hermit thrush (Catharus guttatus) 6 76 0 75 6 79 Indigo bunting (Passerina cyanea) 5 88 1 63 5 79 Horned lark (Eremophila alpestris) 5 95 1 58 5 81 American goldfinch (Spinus tristis) 6 72 0 82 6 82 Chimney swift (Chaetura pelagica) 6 77 0 78 6 83 Ruby-throated hummingbird (Archilochus colubris) 6 73 0 82 6 83 American pipit (Anthus rubescens) 5 100 1 57 5 85 (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 7 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores ( ) Species % with damage Damage rank % with substantial damage Substantial damage rank Relative hazard score (RHS) Composite rank Cliff swallow (Petrochelidon pyrrhonota) 6 78 0 79 5 85 Western kingbird (Tyrannus verticalis) 4 105 1 54 4 87 Common nighthawk (Chordeiles minor) 5 96 1 64 5 88 Cedar waxwing (Bombycilla cedrorum) 5 84 0 77 5 89  Other wrens 6 80 0 82 5 90 Common snipe (Gallinago gallinago) 6 83 0 82 5 91  Other sparrows 5 94 0 72 5 92 Golden-crowned kinglet (Regulus satrapa) 5 85 0 82 5 93 Bank swallow (Riparia riparia) 5 86 0 82 4 94  Other vireos 5 87 0 82 4 95 Lesser nighthawk (Chordeiles acutipennis) 5 89 0 82 4 96 Least sandpiper (Calidris minutilla) 5 90 0 82 4 97 Yellow-rumped warbler (Dendroica coronate) 5 91 0 81 4 97 Common yellowthroat (Geothlypis trichas) 5 99 0 74 4 99 House finch (Haemorhous mexicanus) 5 92 0 82 4 100 Snow bunting (Plectrophenax nivalis) 5 97 0 82 4 101  Other longspurs 5 98 0 82 4 102 Savannah sparrow (Passerculus sandwichensis) 4 107 0 73 4 102 Dark-eyed junco (Junco hyemalis) 4 106 0 75 4 104 Tree swallow (Tachycineta bicolor) 5 101 0 80 4 104 Bobolink (Dolichonyx oryzivorus) 4 102 0 82 4 106 Wilson’s Snipe (Gallinago delicate) 4 103 0 82 4 107 Burrowing owl (Athene cunicularia) 1 108 0 82 1 108 31,082. There were 9,535 strike records for military fighter airframes (65 species struck over 20 times, S3 Table), 20,174 records for cargo airframes (98 species struck over 20 times, S4 Table), 674 strike records for stealth airframes (12 species struck over 20 times, S5 Table), and 699 strike records for rotorcraft (16 species struck over 20 times, S6 Table). There were 64 species groups that were similar for fighter and cargo airframes; however, only 6 species were repre- sented across all 4 aircraft groups and the civil dataset. RHS for the 6 species and species groups across airframe groups differed significantly (χ2 20 = 105.11 P < 0.001, Fig 1). Of the 6 species groups compared among airframe groups, all had higher RHS for the stealth than all civil airframes. There was a strong positive quadratic relationship between RHS and avian body mass for the species groups involved in bird strikes with all military aircraft (r2 = 0.76, Fig 2(D). The quadratic curve was steeper for the fighter airframe with a higher y-intercept, compared to the cargo trend (Fig 2). The best fit for rotorcraft and log avian body mass was a linear regression p g p See S1 Table for a list of species in each species group (i.e. Other ducks). Strike data are from separate databases maintained by the USN (1990–2017) and USAF (1994– 2017). https://doi.org/10.1371/journal.pone.0206599.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 8 / 16 p g p See S1 Table for a list of species in each species group (i.e. Other ducks). Strike data are from separate databases maintained by the USN (1990–2017) and USAF (1994– 2017). 31,082. There were 9,535 strike records for military fighter airframes (65 species struck over 20 times, S3 Table), 20,174 records for cargo airframes (98 species struck over 20 times, S4 Table), 674 strike records for stealth airframes (12 species struck over 20 times, S5 Table), and 699 strike records for rotorcraft (16 species struck over 20 times, S6 Table). There were 64 species groups that were similar for fighter and cargo airframes; however, only 6 species were repre- sented across all 4 aircraft groups and the civil dataset. RHS for the 6 species and species groups across airframe groups differed significantly (χ2 20 = 105.11 P < 0.001, Fig 1). Of the 6 species groups compared among airframe groups, all had higher RHS for the stealth than all civil airframes. There was a strong positive quadratic relationship between RHS and avian body mass for the species groups involved in bird strikes with all military aircraft (r2 = 0.76, Fig 2(D). The quadratic curve was steeper for the fighter airframe with a higher y-intercept, compared to the cargo trend (Fig 2). The best fit for rotorcraft and log avian body mass was a linear regression 8 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores Fig 1. Comparison of relative hazard scores for 6 avian species groups for 4 military airframe groups and all civil airframes for 6 species. Relative hazard scores are calculated from bird strikes within the U.S. See S2 Table for airframe group compositions. Civil relative hazard scores come from [11]. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 https://doi.org/10.1371/journal.pone.0206599.g002 https://doi.org/10.1371/journal.pone.0206599.g002 contributed to the probability of substantial damage. USAF aircraft were predicted to have a greater probability of a strike with substantial damage than USN aircraft (Table 4). Species are ordered from left to right by ascending averaged body mass. https://doi.org/10.1371/journal.pone.0206599.g001 Fig 1. Comparison of relative hazard scores for 6 avian species groups for 4 military airframe groups and all civil airframes for 6 species. Relative hazard scores are calculated from bird strikes within the U.S. See S2 Table for airframe group compositions. Civil relative hazard scores come from [11]. Species are ordered from left to right by ascending averaged body mass. https://doi.org/10.1371/journal.pone.0206599.g001 https://doi.org/10.1371/journal.pone.0206599.g001 (Fig 2(C), whereas the quadratic relationship was the best fit for fighter and cargo airframes (Fig 2(A) and 2(B), S7 Table). Stealth airframes did not exhibit a relationship with avian body mass regardless of equations used and is not included in Fig 2. The best logistic regression models predicting the probability of damage was the same for any damage and substantial damage and included all predictor variables and the airframe × avian body mass interaction (S8 Table). Probability of any damage and substantial damage increased with avian body mass. Within airframe type, fighters had the highest probability of damage or substantial damage compared to cargo airframes (Tables 3 & 4). Probability of dam- age or substantial damage was influenced by avian body mass, and this effect differed per air- frame. These probabilities differed per airframe type for bird species with the same body mass (Fig 3). Migration flyway, specifically the Mississippi flyway, had the lowest probability of sub- stantial damage compared to the Atlantic flyway (Table 4). Reporting military branch also PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 9 / 16 Military avian hazard scores Fig 2. Relationship between avian body mass and relative hazard score for avian groups with military cargo (a), fighter (b), rotorcraft (c) and all military airframes (d). Only strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the U.S. Navy (1990–2017) and U.S. Air Force (1994–2017). Equations and coefficient of determination (r2 values) and 95% confidence intervals are displayed. Fig 2. Relationship between avian body mass and relative hazard score for avian groups with military cargo (a), fighter (b), rotorcraft (c) and all military airframes (d). Only strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the U.S. Navy (1990–2017) and U.S. Air Force (1994–2017). Equations and coefficient of determination (r2 values) and 95% confidence intervals are displayed. Military avian hazard scores Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and th i f × i l b d i t ti Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and the airframe × avian log body mass interaction Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and the airframe × avian log body mass interaction. Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and the airframe × avian log body mass interaction. Discussion Ranking of wildlife hazards is an essential component of strike risk [12]. Although we did not estimate strike risk in this study, these RHS can help guide future research in developing air- frame-specific risk metrics for military airfields. We predicted that airframe and avian body mass, migration flyway, and reporting military branch would influence the relative hazard scores and the probability of damage. For the combined USAF and USN datasets, there was a strong quadratic trend between avian body mass and RHS. As avian body mass increased, so too did RHS, as observed in simi- lar studies [11]. This significant trend can be used to calculate RHS for species not included in PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 10 / 16 Reference categories include Atlantic flyway, cargo airframe, USAF branch, and the interaction term cargo× log mass. Reference categories include Atlantic flyway, cargo airframe, USAF branch, and the interaction term cargo× log mass. Confidence intervals Parameter Coefficient SE 2.5% 97.5% Intercept -5.28 0.09 -5.45 -5.11 Central 0.05 0.05 -0.05 0.14 Mississippi -0.06 0.05 -0.17 0.04 Pacific -0.09 0.06 -0.21 0.03 Fighter 1.45 0.13 1.20 1.70 Rotorcraft 0.59 0.36 -0.14 1.27 Stealth -0.01 0.39 -0.79 0.74 Log mass 0.70 0.02 0.66 0.73 USN 0.04 0.07 -0.10 0.18 Fighter × log mass -0.24 0.02 -0.29 -0.20 Rotorcraft × log mass -0.22 0.07 -0.35 -0.08 Stealth × log mass 0.00 0.08 -0.16 0.14 Reference categories include Atlantic flyway, cargo airframe, USAF branch, and the interaction term cargo × log mass. https://doi org/10 1371/journal pone 0206599 t003 our dataset (i.e. equations in Fig 2 can be solved for RHS with the given body mass of the bird species). We caution the use of the linear equation identified for rotorcraft for predicting RHS because of the low coefficient of determination (r2 value). The relationship between airframes and RHS, however, would be useful for the few species that are not included in the dataset because of the high coefficient of determination. g The stealth airframes had higher relative hazard scores compared to civil airframe groups. Interestingly, for 6 species struck by both military and civil aircraft, all had higher RHS for Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body mass interaction. Confidence intervals Parameter Coefficient SE 2.5% 97.5% Intercept -8.54 0.21 -8.96 -8.13 Central -0.18 0.09 -0.37 0.01 Mississippi -0.31 0.10 -0.51 -0.10 Pacific 0.19 0.11 -0.02 0.40 Fighter 1.94 0.29 1.37 2.50 Rotorcraft 1.42 1.02 -0.83 3.26 Stealth -1.47 1.05 -3.77 0.39 Log mass 0.98 0.03 0.91 1.04 USN -1.73 0.25 -2.25 -1.28 Fighter × log mass -0.26 0.05 -0.35 -0.17 Rotorcraft × log mass -0.32 0.18 -0.67 0.06 Stealth × log mass 0.32 0.16 0.03 0.66 Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). s://doi.org/10.1371/journal.pone.0206599.t004 McFadden’s r2 value is 0.23. Predictor variables include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body i t ti redicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the mation Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body mass interaction. Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log bod mass interaction. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 11 / 16 Military avian hazard scores Fig 3. Interaction plot of airframes, avian log body mass, and predicted probability of damage (a) and substantial damage (b). Only strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the United States Navy (1990–2017) and Air Force (1994–2017). https://doi.org/10.1371/journal.pone.0206599.g003 Fig 3. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Conclusion We found that avian hazards to military aviation differed from civil aviation. Specifically, spe- cies RHS were higher for military than civil aircraft. Furthermore, we found differences in RHS among airframe groups. As predicted, airframes that travel at greater speeds (fighters) experienced greater damage from birds when struck, especially at the lower end of the body mass scale. Although these results are based on strikes within and outside of the airfield envi- ronment, the calculated hazard scores can be used in conjunction with a frequency component to estimate risk. Military avian hazard scores high cruising altitudes [8, 15]. All airframes, except rotorcraft, exhibited an exponential rela- tionship between avian body mass and RHS. This relationship indicates that below a certain body mass, the RHS exhibits the opposite trend of increasing with decreasing body mass. We suggest that this trend is not biologically relevant, but a byproduct of the mathematical analysis such as from the numerous outliers. Furthermore, there are few RHS for birds with extremely small body masses. Interestingly, the trend between body mass and RHS for rotorcraft was not a strong relationship and the best fit was linear. Possible explanations for this result include differences in strike reporting rates, flight patterns, or airframe components. For example, strikes to wind screens, rotors, and tail rotors, components that are quite exposed, can result in frequent, substantial damage [30] We acknowledge that these RHS are based on a degree of frequency (species only included if struck 20 or more times) and thus influenced by differences in strike rates across species. Certain species are struck more frequently than others. Frequently struck birds use the airport for foraging and roosting activities [3] and generally are more maneuverable [31]. However, there is significant individual and species variability [32, 33] that influence avian responses to oncoming aircraft. Given this caveat, our results were similar to other studies in that birds associated with water were ranked as most hazardous [6, 8, 11, 14], as well as raptors [6, 8, 11]. Our results indicate that avian hazards to military aviation are airframe specific. Depending on the airfield, hazard scores can be calculated per airframe type. For example, if an airfield is primarily used for fighter missions/training, the fighter airframe RHS should be used, ensuring that species on the airfield would be prioritized based on the severity of the strike specific to that airframe. These results, coupled with data on species seasonal relative abundance on and near airfields [30, 34], will be important in directing airfield management [12]. Ultimately, the development of airframe-specific strike risk metrics will yield more accurate information on airframe vulnerability to bird strikes by mission type. Supporting information S1 Table. Bird species (n = 186) involved in more than 20 strikes with military aircraft grouped into 108 species groups. Species with fewer than 50 strikes were combined into spe- cies groups based on phylogeny. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz- ardous for fighter aircraft within the United States. (DOCX) Interaction plot of airframes, avian log body mass, and predicted probability of damage (a) and substantial damage (b). Only strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the United States Navy (1990–2017) and Air Force (1994–2017). Fig 3. Interaction plot of airframes, avian log body mass, and predicted probability of damage (a) and substantial damage (b). Only strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the United States Navy (1990–2017) and Air Force (1994–2017). https://doi.org/10.1371/journal.pone.0206599.g003 https://doi.org/10.1371/journal.pone.0206599.g003 https://doi.org/10.1371/journal.pone.0206599.g003 stealth than civil airframes. The mourning dove had a military RHS of 100 for stealth, but a score of 9 for civil aircraft [11]. We suspect that the materials composing stealth aircraft “skin” and specialized aircraft components have elevated sensitivity to strikes. For example, a strike in 2006 which involved a mourning dove (123 g) resulted in $129,787 damage to the wing of a stealth bomber. Mourning doves are capable of causing similar amounts of damage (> $130,000) for civil aircraft [7]; however, all of the reported mourning dove strikes from 2010–2015 involved the bird being ingesting into an engine, not external wing damage. Relative hazard scores for fighter airframes were generally higher than cargo and all civil airframes. Furthermore, our logistic regression models indicated that fighter airframes experi- enced a higher probability of damage with birds of a lower body mass compared to cargo air- frames. It is likely that higher speeds and lower flight altitudes of fighter airframes versus cargo aircraft contributed to higher RHS and probability of damage. Cargo airframes are likely simi- lar to civil aviation in terms of design and flight patterns, with long ascents and descents and PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 12 / 16 S2 Table. Airframe groups (n = 189) involved in bird strikes with military aircraft grouped into 8 airframe groups. (DOCX) Results from the binomial generalized linear model of factors that influence the probability of a bird strike to cause (a) any damage and the probability of a bird strike causing (b) substantial damage with military aircraft. (DOCX) Acknowledgments We thank the Commander, Navy Installations Command (CNIC) Airfield Operations Pro- gram for funding. We thank James E. Higgins, Dan Sullivan, Mike Begier, Jenny Washburn, Paul Block, Maguel Brooks, and Mark Schmidt for providing the data. We thank Jason Kougher, Eddie Earwood, and James Laughlin for their assistance and knowledge on the mili- tary strike databases and airframe groups. We thank Brian Washburn, Richard Dolbeer, and Thomas Seamans for reviewing earlier versions of this manuscript. Author Contributions Conceptualization: Bradley F. Blackwell, Travis L. DeVault. Conceptualization: Bradley F. Blackwell, Travis L. DeVault. Data curation: Morgan B. Pfeiffer, Travis L. DeVault. Methodology: Morgan B. Pfeiffer, Bradley F. Blackwell, Travis L. DeVault. Supervision: Travis L. DeVault. Visualization: Morgan B. Pfeiffer, Bradley F. Blackwell, Travis L. DeVault. Writing – original draft: Morgan B. Pfeiffer. Writing – original draft: Morgan B. Pfeiffer. Writing – review & editing: Morgan B. Pfeiffer, Bradley F. Blackwell, Travis L. DeVault. S2 Table. Airframe groups (n = 189) involved in bird strikes with military aircraft grouped into 8 airframe groups. (DOCX) S2 Table. Airframe groups (n = 189) involved in bird strikes with military aircraft grouped into 8 airframe groups. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz- ardous for fighter aircraft within the United States. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz- ardous for fighter aircraft within the United States. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz- ardous for fighter aircraft within the United States. (DOCX) 13 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores S4 Table. Relative hazard scores (RHS) for 65 species groups from most to least hazardous for cargo aircraft within the United States. (DOCX) S5 Table. Relative hazard scores (RHS) for 12 species groups from most to least hazardous for stealth aircraft within the United States. (DOCX) S6 Table. Relative hazard scores (RHS) for 16 species groups from most to least hazardous for stealth aircraft within the United States. (DOCX) S7 Table. Summary of quadratic and linear relationships with military airframe and avian log body mass. No significant relationship was found for stealth airframes and avian log body mass. Bold values indicate best fit by the coefficient of determination (r2 values). (DOCX) S8 Table. Results from the binomial generalized linear model of factors that influence the probability of a bird strike to cause (a) any damage and the probability of a bird strike causing (b) substantial damage with military aircraft. (DOCX) S4 Table. Relative hazard scores (RHS) for 65 species groups from most to least hazardous for cargo aircraft within the United States. (DOCX) S5 Table. Relative hazard scores (RHS) for 12 species groups from most to least hazardous for stealth aircraft within the United States. (DOCX) S6 Table. Relative hazard scores (RHS) for 16 species groups from most to least hazardous for stealth aircraft within the United States. (DOCX) S7 Table. Summary of quadratic and linear relationships with military airframe and avian log body mass. No significant relationship was found for stealth airframes and avian log body mass. Bold values indicate best fit by the coefficient of determination (r2 values). (DOCX) S8 Table. References 1. Conover MR. Resolving human-wildlife conflicts: the science of wildlife damage management. New York: CRC press; 2002. 14 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores 2. Seiler A, Helldin J. Mortality in wildlife due to transportation In: Davenport J, Davenport JL, editors. The ecology of transportation: managing mobility for the environment. Dordrecht: Springer; 2006. pp. 165– 89. 3. DeVault TL, Blackwell BF, Belant JL. Wildlife in airport environments: preventing animal–aircraft colli- sions through science-based management. Baltimore: The John Hopkins University Press; 2013. 4. Dolbeer RA, Eschenfelder P. Amplified bird-strike risks related to population increases of large birds in North America. International Bird Strike Committee Meeting 26; 2003. 5. Cleary EC, Wright SE, Dolbeer RA. Wildlife strikes to civil aircraft in the United States, 1990–1998. 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R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2017. 28. Bates D, Maechler M, Bolker B, Walker,S. lme4: Linear mixed-effects models using Eigen and S4 R package version 1.7 2014 29. Gelman, A, Su Y. arm: Data analysis using regression and multilevel/hierarchical models. R package version 1.8–6 2015 30. Federal Aviation Administration. Rotorcraft bird strike working group recommendations to the aviation rulemaking advisory committee (ARAC). 2017. 15 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599 November 1, 2018 Military avian hazard scores 31. Ferna´ndez-Juricic E, Brand J, Blackwell BF, Seamans TW, DeVault TL. Species with greater aerial maneuverability have higher frequency of collisions with aircraft: a compartive study. Front Ecol Evol 2018; 6: 17. 32. DeVault TL, Seamans TW, Blackwell BF, Lima SL, Ferna´ndez-Juricic E. Individual variation in avian avoidance behaviours in response to repeated, simulated vehicle approach. Can J Zool 2018; 96: 441– 446. 33. Efroymson RA, Suter GW II. Ecological risk assessment framework for low-altitude aircraft overflights: II. Estimating effects on wildlife. Risk Anal 2001; 21: 263–274. PMID: 11414536 34. Blackwell BF, Schmidt P, Martin JA. 2013. Avian survey methods for use at airports. Pages 153–166 in DeVault TL, Blackwell BF, and Belant JL, Editors. Wildlife in airport environments: preventing animal– aircraft collisions through science-based management. Baltimore: The John Hopkins University Press; 2013. 33. Efroymson RA, Suter GW II. Ecological risk assessment framework for low-altitude aircraft overflights: II. Estimating effects on wildlife. Risk Anal 2001; 21: 263–274. PMID: 11414536 References 16 / 16
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Sobre a Mulher Escrava no Rio Grande do Sul
Estudos ibero-americanos/Estudos Ibero-Americanos
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* Autora do livro RS: escravlsmo e abolição, Porto Alegre, Mercado Aberto, 1982. Professora do curso de Graduação e Pôs-Graduação de História da Universidade Federal do Rio Grande do Sul. 47 47 F.8tudos Ibero-Amerkanos. PUCRS, XVI(1,2):47-56, jul. e dez., 1990 Sobre a Mulher Escrava no Rio Grande do Sul Margaret M Bakos* Margaret M Bakos* A versão original desta comunicação foi elaborada a partir de um convite da Secretaria Municipal de Cultura, na pessoa do organizador do Curso, professor Mário Maestri, para falar sobre a mulher negra escrava no Rio Grande do Sul. O tema proposto instigou um questionamento inicial: em quais aspectos o escravismo seria diferente para o homem e para a mulher? As relações sociais escravistas coisificam os indivíduos, impedin- do-os de se movimentarem livremente, embotando e inibindo os seus sentimentos e instintos. Homens e mulheres, na escravidão, tomam-se seres de propriedade de outrem, com vontade sujeita à autoridade do dono e seu trabalho ou serviços são obtidos através da coerção. Nesta linha de raciocínio, toma-se difícil delinear e conseqüen- temente falar sobre a situação específica da mulher no cativeiro, pois a base estrutural da sua existência não está em ser fêmea. Está em ser escrava. Será impossível identificarmos, a nível estrutural, uma posição masculina ou feminina na escravidão? Toda diferença entre o papel do homem e da mulher no escravismo será sempre conjuntural e relacionada ou com a atividade que o negro exerce, ou com o momento hist6rico que vivencia. Homens e mulheres negros escravizados no Rio Grande do Sul, ao longo de dois séculos, F.8tudos Ibero-Amerkanos. PUCRS, XVI(1,2):47-56, jul. e dez., 1990 Estudos Ibero-Americanos, XVl( 1,2) - 1990 Estudos Ibero-Americanos, XVl( 1,2) - 1990 48 sofreram, capitularam e reagiram ao cativeiro através de ações, mais ou menos cooperativas ou agressivas, independentemente do seu sexo, relacionadas basicamente com as variáveis apontadas. A minha proposta nesta apresentação não é, portanto, estabelecer um inventário, no meu entender forçadíssimo, das diferenças entre homens e mulheres escravizados. A tendência, nesta perspectiva, é a de situar aos primeiros como heróis em potencial, em constante enfren- tamento com o jugo senhoril, caracterizando as segundas como vítimas duplas, da passividade inerente ao sexo e da violência dos senhores, conforme as retrataram inúmeras vezes os historiadores positivistas. Impõe-se uma nova visão, pois não é assim que vejo as mulheres escravas, genericamente falando. Acredito que entre os escravos encontramos gente assumida, sensível e combativa, mas também homens e mulheres entregues aos desmandos dos senhores, interna e profundamente auto- coisificados, independente do seu sexo. Ao tentar resgatar aspectos do engajamento feminino na luta contra a opressão, analiso três momentos definidos na relação escravista: I - No Trabalho A idéia desta abordagem originou-se a partir de um debate que se estabeleceu, ao final de uma palestra que proferi, em 1988, em Porto Alegre. Eu falara sobre a mulher escrava na capital do Estado e, para ilustrar o encontro, li alguns anúncios, publicados em jornais do século passado, que ou solicitavam ou ofereciam escravas negras para serviços domésticos. Muitos dos anúncios solicitavam que a serviçal fosse real- mente habilitada como cozinheira. Neste momento, uma das assistentes, estranhando o fato, espontaneamente, comentou que sempre pensara que as negras fossem "naturalmente" boas cozinheiras e competentes domés- ticas, de um modo geral. O questionamento que me ocorreu em torno desta intervenção foi sobre as origens históricas deste pensamento que, possivelmente, ainda se haja presente na cosmovisão de muitos gaúchos. O escravo negro começou a ser introduzido no Rio Grande do Sul nos finais do século XVIII, com vistas a atender as necessidades das charqueadas sulinas. Aos poucos começou-se a utilizar o escravo para as atividades de peonagem e agricultura. Nestes primeiros tempos, o que mais importava era possuir um escravo negro, fisicamente apto para estas Sobre a mulher escrava no ... 49 atividades. Foi com o desenvolvimento dos centros urbanos, a partir principalmente da primeira metade do século XIX, que a mulher escrava passou a ser uma peça cobiçada e fundamental para a sociedade gaúcha. Uma das poucas estatísticas que temos, e que diferencia a presença numérica dos escravos homens e mulheres, data de 1859. Ela arrola 25 cidades sedes de municípios, salientando-se como localidades que mais possuem escravos as seguintes: atividades. Foi com o desenvolvimento dos centros urbanos, a partir principalmente da primeira metade do século XIX, que a mulher escrava passou a ser uma peça cobiçada e fundamental para a sociedade gaúcha. Uma das poucas estatísticas que temos, e que diferencia a presença numérica dos escravos homens e mulheres, data de 1859. Ela arrola 25 cidades sedes de municípios, salientando-se como localidades que mais possuem escravos as seguintes: Localidade Porto Alegre Pelotas Jaguarão Rio Grande Cruz Alta Homens 4.556 3.095 2.929 2.503 2.315 Mulheres 3.861 1.693 2.127 1.866 1.704 Localidade Porto Alegre Pelotas Jaguarão Rio Grande Cruz Alta Vemos assim que nestas localidades urbanas havia uma presença de escravos negros, homens e mulheres, significativa, o que revela a necessidade de ambos para diferentes trabalhos. I - No Trabalho Uma análise do seu valor de compra, no mesmo ano (1859), nos permite resgatar que a diferença de preço também não era muito significativa: Preços de escravos negros no Rio Grande do Sul em 1859 Preços de escravos negros no Rio Grande do Sul em 1859 Para a agricultura Homens 1.250$000 Mulheres 1.150$000 Para serviços domésticos Homens 1.400$000 Mulheres 1.250$000 Para a agricultura Homens 1.250$000 Para serviços domésticos Homens 1.400$000 Para serviços domésticos Se as mulheres negras eram numericamente importantes e econo- micamente valorizadas, resta perguntar para que atividades eram des- tinadas. Talvez uma das melhores fontes para obter esta resposta seja justamente a análise dos anúncios de jornais. O O Diário de Porto Alegre, de 1827, é uma das mais antigas fontes para este tema e aponta cerca de 21 atividades para as quais o trabalho escravo era requisitado, em Porto Estudos Ibero-Americanos, XVI( 1,2) - 1990 50 Alegre. As mulheres eram procuradas para trabalharem como cos- tureiras, doceiras, lavadeiras, engomadeiras, cozinheiras, amas de leite, arrumadeiras, fiadeiras e refinadoras de açúcar. Com vistas a responder ao questionamento inicial, é importante chamar atenção para alguns ângulos sob os quais esta série de anúncios pode ser analisada: Alegre. As mulheres eram procuradas para trabalharem como cos- tureiras, doceiras, lavadeiras, engomadeiras, cozinheiras, amas de leite, arrumadeiras, fiadeiras e refinadoras de açúcar. Com vistas a responder ao questionamento inicial, é importante chamar atenção para alguns ângulos sob os quais esta série de anúncios pode ser analisada: a) A mulher escrava foi utilizada como mão-de-obra em dezenas de atividades designadas, para fins públicos, como domésticas, ligando-se a estas remotas origens uma série de preconceitos, que pode ser resu- mida no ditado popular machista e racista que diz ter a mulher negra um lugar garantido: "no palco, na cama e na cozinha, sem fazer con- curso público e com estabilidade assegurada". (O Tição, Ano 1, out. 80, p.3) b) O que chama a atenção nesta coletânea de anúncios, em número de 46, é que 13 relacionam-se com a atividade de fazer comida. Assim, não fica difícil entender por que foi tão profundamente internalizada, nas pessoas, a relação escrava negra \ cozinha. A ponto de alguém, no ano de 1988, ainda considerar que todas as negras eram em princípio boas cozinheiras. Entretanto, é justamente aí que se pode buscar os aspectos da resistênciavcoerçâo ao trabalho, que, em última análise, assinalam a semelhança entre homemvmulher, na escravidão. I - No Trabalho Explicando melhor, tanto faz, num regime de trabalho compulsório, se se é escravovescrava; a diferença entre um e outra não se faz por aí. Ser senhor'(senhora isto é o que importa. Os pólos da relação escravista, independente do seu sexo, dominam ou ao chicote ou apenas ao seu próprio corpo. Sob esta ótica repensei os dados e, em base às fontes que enfatizavam a preparação para o trabalho doméstico, que se exigia das escravas, tentei recuperar o processo de ensino'(aprendizagem que muitas negras devem ter passado entre o fogão e a despensa, desesperadas por não saberem o que fazer com panelas e víveres, que não conheciam, dos quais não gostavam e que eram obrigadas a cheirar, cozinhar, servir e comer. O chicote, a senzala e o terror foram certamente os mestres mais eficientes do período e os responsáveis pelo nossa preconceituosa idéia de que todas as negras são "naturalmente" boas cozinheiras. Até este momento, não se tem uma análise exaustiva da documentação que evidencie tais conclusões. Entretanto, isto não justifica que se aceite a História que forma este- reótipos, tomando como verdadeiras afirmações geradas pela tradi- ção oral e pela leitura simplista dos documentos, sem críticas e questionamentos. A cozinheira, a copeira e a babá permitiram que ai Sobre a mulher escrava no ... 51 senhora se alimentasse e que a sua família se perfumasse, se banhasse, namorasse nas janelas e saraus e procriasse novas sinhazinhas. É através da análise do sistema escravista que se evidencia a formação, a ferro e fogo, das habilidades "naturais" da mulher negra, cuja excelência somente será questionada quando a mulher imigrante empobrecida vier a disputar com a negra o trabalho doméstico, principalmente o urbano. Entretanto, até os dias de hoje, ainda são comuns os ditados populares, que a tradição oral preservou, e que nos fazem olhar com simpatia ou resistência seres humanos, com relação à sua capacidade do trabalho. Quem nunca ouviu: "Negra de canela fina é que é boa trabalhadeira!"? 11- Na resistência Considero como resistência toda reação de inconformidade do negro quanto à escravidão. Isto inclui desde tentativas de fugas, formação de quilombos, agressões contra senhores e feitores, assassinato dos próprios filhos, suicídios até o "corpo mole" inocente e preguiçoso, o "dengue" doce que alicia e comove, influenciando o humor dos circun- dantes. Esta abrangência se justifica pela impossibilidade de saber, atualmente, se o sorriso de submissão não teria sido tão falso quanto o famoso e decantado riso do palhaço de circo. Não há nenhum trabalho sistemático sobre fugas, quilombos e criminalidade escrava no Rio Grande do Sul que permita uma compa- ração estatística entre o número de mulheres e homens que fugiram. Este estudo seria difícil pelas inúmeras variáveis que teriam de ser levadas em conta. Resta, pois, exemplificar a resistência feminina, relendo fontes, até agora, utilizadas para generalizações em torno da resistência escrava. Um dos documentos mais antigos sobre a revolta dos escravos no Rio Grande do Sul refere-se a um episódio ocorrido na Real Feitoria do Linho cânhamo, em 2 de agosto de 1822. Nesta ocasião, houve uma denúncia de que havia pretos desta fazenda que costumavam roubar na vizinhança, sendo destacado um grupo de soldados para prendê-los, porém eles nada puderam fazer, pois os negros não se entregaram à prisão e, instigados por suas mulheres, que gritavam para que eles matassem os soldados, entraram nas suas moradias, armaram-se e atacaram a guarda que terminou voltando para os seus quartéis. (Feitoria Nacional do Linho Cânhamo, lata 300 - AHRGS.) ~ Estudos !bero-Americanos, XVI(1,2) -1990 Estudos !bero-Americanos, XVI(1,2) -1990 52 Neste episódio, resgata-se a negra que grita e instiga, que nada teme na luta contra o branco armado; mas não é preciso ficar apenas nesta inferência, podemos ver, além disso,a companheira de uma luta maior contra a escravidão, que se expressa quando ela incita uma atitude agressiva. Leitura nesta direção pode ser feita sobre o significado da participação feminina na história do quilombismo gaúcho. No quilombo do Negro Lucas, em Rio Grande, em 1833, par- ticipavam seis homens e quatro mulheres, que viviam em uma casa grande, onde preparavam sebo, graxa e charque para vender. Nos quilombos de Rio Pardo foram indiciadas sete mulheres que, juntamente com treze homens, viviam de roubos nas plantações das vizinhanças. 11- Na resistência Para viver em um quilombo era, primeiramente, no mínimo, necessário que o escravo enfrentasse a problemática de sua fuga, que era mais ou menos difícil, dependendo das atividades que ele exercia, do grau de vigilância que sofria e, naturalmente, da sua constituição física, que devia ser a melhor possível, pois que ele deveria percorrer longos percursos, alimentar-se esporadicamente, quando encontrasse o que comer, e sofrer de muita ansiedade com a ameaça permanente de enfrentamento aberto com autoridades constituídas - como o fami- gerado capitão de mato - até chegar a um refúgio. Essa foi também a saga da mulher quilombola, naturalmente no caso de ela ter ido espontaneamente para o quilombo. Em tal caso, há necessidade de esboçar um quadro mais realista de um ato de resistência? Até mesmo os representantes do sistema, com melhores vestimentas, montarias e esquemas de sobrevivência, amargavam na incumbência de perseguir os negros fugidos, como é sobejamente ilustrado na documentação oficial. Então, porque não valorizarmos ao máximo a ação quilombola como um alto de extrema coragem e valentia? Se eles não tinham o objetivo de transformar a sociedade, como afirma Gorender, pelo menos, a hos- tilizavam. Se buscavam recompor a sociedade tribal africana, segundo pensamento do mesmo autor, homens e mulheres partilhavam do mesmo desejo. Naturezas femininas e masculinas, sob a escravidão, interagiam com o medo, planejavam atingir o estado utópico, o nirvana de qualquer cativo, em qualquer época histórica: a liberdade! Há alguns, verdadeiramente não muitos, relatos de casos em que as escravas resistiram ao assédio de negros, seus companheiros de cativeiro, como o incidente que culminou no assassinato da esposa de um feitor Sobre a mulher escrava no ... 53 negro - em fazenda do Alegrete - porque ela não cedeu a um escravo que queria forçá-Ia "para fins libidinosos". (Relatório do Presidente da Província do Rio Grande do Sul João Capistrano Miranda Castro, 1848, Porto Alegre, Tipografia Porto Alegrense.) O ato que ficou registrado no processo crime aberto contra o assassino nos permite, agora, rever a vítima no seu comportamento corajoso, que a levou à morte, desmis- tificando a idéia romântica da mulher por índole submissa. Em uma pesquisa sobre os cárceres de Porto Alegre, na primeira metade do século XIX, encontrei mulheres negras aprisionadas ou pelo simples arbítrio de seus senhores ou por terem cometido crimes. (Bakos, 1982: 209-221.) Apenas na cadeia de Justiça, foram encontradas 13 escravas negras presas. 11- Na resistência Impossível saber quais os sofrimentos físicos e mentais, as torturas, que elas sofreram na passagem de um espaço para o outro, pois o cenário maior dos quadros continuava a ser o escravismo. Havia dorvchicote, fedorvsujeira, em todos os locais destinados aos negros. Nos autos dos processos, ficou registrado: um machado foi desviado, da lenha para a senhora, que na visão da escrava, foi umajuíza injusta dos seus atos. Um punhado de veneno para ratos foi parar nas baixelas senhoris. Tudo por mãos de negras, tudo para exteriorizar sentimentos de raiva, medo, ódio: pura emoção. Com esta leitura, não pretendo desculpar ou minorar um ato agressivo, muito menos justificar o emprego da violência nas relações humanas. Ao contrário, vejo a capacidade de agredir física ou mentalmente ao seu semelhante como a pior das características humanas. Por esta razão, por ser constitutiva e essencial na luta pela vida, a violência pode ser reduzida a um viés de análise histórica. Entender o porquê da violência, na visão de quem a pratica e a sofre, pode ser mais um meio para o historiador resgatar o sentido do jogo social pela vida. As respostas de Felisberta e Florinda são muito eloqüentes: "agredi, porque fui agredi da sem motivo", "agredi, mas não queria machucar, só queria assustar". Poderíamos analisar dezenas de processos como estes, mas não estão aí resumidos, apenas nestes dois, os princípios básicos do ser humano no seu relacionamento em sociedade? Para isto, não foi necessário que as negras saissem às ruas, fazendo parte de grupos armados, praticando violências planejadas. As cenas protagonizadas por Felisberta e Florinda aconteceram entre as paredes de casas, onde viviam pessoas ordeiras. Naturalmente ali se reproduziam as relações de trabalho escravistas, com muita crueza, pois na privacidade do espaço íntimo, as autoridades são mais presentes, despóticas, viciosas, impunes. Entretanto, no esquema rotineiro, uma 'coisa' saiu do seu lugar. Tanto faz se era uma 'coisa' masculina ou feminina. Interessa que ela saiu; com isso, mexeu com a ordem, mostrou que não era só 'coisa', era gente e igual a toda gente: homem ou mulher. Felisberta e Florinda, protagonistas da mudança, nossos objetos de análise, conseguiram, à época, com suas atitudes, saírem do espaço privado da casa. Se elas queriam sair, nunca saberemos. Mas o fato aí está para ser analisado: elas foram conduzidas a um fórum, foram interrogadas, ouvidas. Suas vozes ficaram registradas nos autos dos processos, chegaram até nós. 11- Na resistência A seguir, discrimino dois crimes de que escravas foram acusadas, no Rio Grande do Sul, procurando com isto apenas exemplificar o pen- samento de que as mulheres reagiram, como os homens, à escravidão, agredindo os seus senhores com as armas de que dispunham. Julgo importante apontar a existência de centenas de processos crimes envolvendo negras escravas ou livres, nos arquivos públicos do Rio Grande do Sul, os quais ainda não foram analisados ou o foram precariamente. Importantes referenciais sobre o significado do ato de agressão da cativa podem e devem ser inferidos das leituras destes processos, que se constituem, fora de dúvida, na mais rica fonte disponível para a compreensão das atitudes do escravo de ambos os sexos, resgatando para a atualidade elementos constitucionais das naturezas feminina e masculina. Dados do Escravo Ano Motivo Felisberta 1821 Castigada S. Gabriel sem motivo Florinda 1828 queria só P. Alegre ameaçar 1828 P. Alegre Florinda 54 Estudos Ibero-Americanos, XVI(1,2) -1990 Estudos Ibero-Americanos, XVI(1,2) -1990 As respostas de Felisberta e Florinda são muito eloqüentes: "agredi, porque fui agredi da sem motivo", "agredi, mas não queria machucar, só queria assustar". Poderíamos analisar dezenas de processos como estes, mas não estão aí resumidos, apenas nestes dois, os princípios básicos do ser humano no seu relacionamento em sociedade? Para isto, não foi necessário que as negras saissem às ruas, fazendo parte de grupos armados, praticando violências planejadas. As cenas protagonizadas por Felisberta e Florinda aconteceram entre as paredes de casas, onde viviam pessoas ordeiras. Naturalmente ali se reproduziam as relações de trabalho escravistas, com muita crueza, pois na privacidade do espaço íntimo, as autoridades são mais presentes, despóticas, viciosas, impunes. Entretanto, no esquema rotineiro, uma 'coisa' saiu do seu lugar. Tanto faz se era uma 'coisa' masculina ou feminina. Interessa que ela saiu; com isso, mexeu com a ordem, mostrou que não era só 'coisa', era gente e igual a toda gente: homem ou mulher. Felisberta e Florinda, protagonistas da mudança, nossos objetos de análise, conseguiram, à época, com suas atitudes, saírem do espaço privado da casa. Se elas queriam sair, nunca saberemos. Mas o fato aí está para ser analisado: elas foram conduzidas a um fórum, foram interrogadas, ouvidas. Suas vozes ficaram registradas nos autos dos processos, chegaram até nós. 11- Na resistência Impossível saber quais os sofrimentos físicos e mentais, as torturas, que elas sofreram na passagem de um espaço para o outro, pois o cenário maior dos quadros continuava a ser o escravismo. Havia dorvchicote, fedorvsujeira, em todos os locais destinados aos negros. Nos autos dos processos, ficou registrado: um machado foi desviado, da lenha para a senhora, que na visão da escrava, foi umajuíza injusta dos seus atos. Um punhado de veneno para ratos foi parar nas baixelas senhoris. Tudo por mãos de negras, tudo para exteriorizar sentimentos de raiva, medo, ódio: pura emoção. Com esta leitura, não pretendo desculpar ou minorar um ato agressivo, muito menos justificar o emprego da violência nas relações humanas. Ao contrário, vejo a capacidade de agredir física ou mentalmente ao seu semelhante como a pior das características humanas. Por esta razão, por ser constitutiva e essencial na luta pela vida, a violência pode ser reduzida a um viés de análise histórica. Entender o porquê da violência, na visão de quem a pratica e a sofre, pode ser mais um meio para o historiador resgatar o sentido do jogo social pela vida. 55 Sobre a mulher escrava no ... Ill- No engajamento social Uma outra instância em que a mulher escrava pode ser analisada é no seu engajamento na sociedade. Um bom exemplo é a sua participação na Confraria de Nossa Senhora do Rosário, fundada em Porto Alegre, em 1786, que tinha como principal objetivo a educação religiosa e a benemerência, mas que também funcionava como um local de agru- pamento dos negros e, muitas vezes, para conspirações ou ataques espontâneos à ordem constituída. Assim, embora institucionalizadas, as confrarias eram muitas vezes perseguidas e as suas procissões e festas proibidas e os seus membros visados pela polícia local. Mesmo assim, a presença da mulher negra livre ou escrava era uma constante, prevista, inclusive no Regimento da Confraria, mas da qual ela participava com todo o seu engajamento, conspirando também, como o homem ... Conclusão A submissão da mulher foi uma invenção de um mundo comandado por homens que também inventaram a escravidão. (Mott, 1988: 21) Mas, subjacente a estes garrotes ideológicos e concretos, a mulher, cons- titucionalmente, tinha forças e meios para resistir, com garra e oportunis- mo, tal como o homem. Por isto ela não merece ser registrada historicamente como dupla vítima do sistema. O que realmente lhe é devido é uma revalorização de seus atos, concedendo-lhes a devida dimensão e mérito. Isto apenas será possível se entendermos que a mulher escrava podia resistir ao jugo senhoril, de uma forma diferente que o homem. Se aceitarmos como resistência coisas do cotidiano, muito simples, como a indolência no trabalho, a dificuldade no aprendizado de novas tarefas, o tratamento "dengoso" que dispensava ao senhor e à sua família. Ao lado destas atitudes que a livravam dos excessos de trabalho e dos castigos, a escrava negra, como vimos, também agrediu a sociedade escravista: fugiu, furtou, matou, como todas no país. Foram protagonistas da História da escravidão negra no Brasil, vítimas, mas também agres- sores, todos os seres humanos vindos da África e os seus descendentes, independentemente do seu sexo: impossível fazer um recorte e falar sobre a mulher escrava especificamente, sem desenvolver uma argumentação machista! Estudos Ibero-Americanos, XVI(1,2) -1990 Estudos Ibero-Americanos, XVI(1,2) -1990 56 Bibliografia citada BAKOS, M. Os escravos negros nos cárceres de Porto Alegre: Sugestões de uma documentação do século XIX. In Revista de Estudos Ibero Americanos. PUCRS, 8(2):209-23, 1982. -. RS: Escravismo e Abolição. Porto Alegre, Mercado Aberto, 1982. CARDOSO, C. Escravo ou Camponês? São Paulo, Brasiliense, 1987. GIACOMINI, S. Mulher e Escrava. Rio de Janeiro, Vozes, 1988. GORENDER, J. Questionamento sobre a teoria econômica do escravismo colonial. In Estudos Econômicos. 13(1):7-41, 1983. MAESfRI, M.J. O Escravo no Rio Grande do Sul. Caldas do Sul, Escola Superior São Lourenço dos Brindes, 1984. MOTI, Mário Lúcio. Submissão e Resistência. São Paulo, Contexto, 1988. MOTIA, F. Crime e rebeldia escrava no Rio Grande do Sul (1820-1845). Trabalho de conclusão de bacharelado. UFRGS, jan. 1985.p.47. [Mimeo1 REIS, J.R. & SILVA, E. Negociação e Conflito. Rio de Janeiro, Cia. das Letras, 1989. SCHWARTZ, S. Mocambos, quilombos e palmares: a resistência escrava no Brasil Colonial. In Estudos Econômicos. 17(especial), 1987.
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Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides
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BioMed Central BioMed Central BioMed Central Research article Address: 1Department of Biotechnology, University of the Western Cape, Private Bag X17, Cape Town - Bellville 7535, South Africa, 2Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch 7701, South Africa and 3South African National Bioinformatics Institute, University of the Western Cape, Private Bag X17, Cape Town - Bellville 7535, South Africa mail: Stuart Meier - smeier@uwc.ac.za; René Bastian - rbastian@uwc.ac.za; Lara Donaldson - Lara@SCIENCE.uct.ac hane Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za Email: Stuart Meier - smeier@uwc.ac.za; René Bastian - rbastian@uwc.ac.za; Lara Donaldson - Lara@SCIENCE.uct.ac.za; Shane Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za * Corresponding author mail: Stuart Meier - smeier@uwc.ac.za; René Bastian - rbastian@uwc.ac.za; Lara Donaldson - Lara@SCIENCE.uct.ac hane Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za C di th e Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za responding author * Corresponding author Received: 10 September 2007 Accepted: 29 February 2008 Received: 10 September 2007 Accepted: 29 February 2008 Published: 29 February 2008 Published: 29 February 2008 BMC Plant Biology 2008, 8:24 doi:10.1186/1471-2229-8-24 This article is available from: http://www.biomedcentral.com/1471-2229/8/24 This article is available from: http://www.biomedcentral.com/1 © 2008 Meier 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. BMC Plant Biology Open Access Background tures respectively [15]. Further, irPNP rapidly and specifi- cally induced a transient elevation of cGMP levels in the conductive stele tissue of maize roots [6] and in stomatal guard cell protoplasts [7] and recently recombinant AtPNP-A was shown to stimulate protoplast swelling in a cGMP dependent manner [18]. IrPNPs also modulate ion fluxes across plant membranes [19] and recombinant AtPNP-A induced spatially dependent H+, K+ and Na+ fluxes in A. thaliana roots [20]. Endogenous levels of irPNP are increased in response to NaCl stress in whole- plants and in Arabidopsis suspension culture cells exposed to high salt or osmoticum [15]. Collectively, these studies indicate that PNP-like molecules may function as extracel- lular signalling molecules that directly affect water and solute transport in response to stress. Based on biochemi- cal and physiological data we propose mechanisms of action for AtPNP-A at the cellular level as summarised in Figure 1 (adapted from [21]). g Natriuretic Peptide (NP) systems have been identified in mammals, fish, amphibians, birds and reptiles. NPs and their receptors are commonly associated with organs involved in cardiac and osmoregulatory homeostasis. In amphibians, birds and fish, NPs have been shown to play a critical role in the regulation of blood fluid volume and composition [1]. The first indication that NPs function in plants came from radio-immuno assays on plant tissue extracts from Florida beauty [2] and it was shown that the rate of transpiration, solute flow and solute uptake in carnation and chrysan- themum was rapidly and significantly increased after exogenous application of synthetic human atrial NP (ANP) [3]. Subsequently it was demonstrated that rat ANP can induce stomatal opening in a concentration depend- ent manner [4] and this effect appears to be dependent on the intracellular second messenger cGMP (guanosine 3',5'-cyclic monophosphate) since it is inhibited by the guanylate cyclase inhibitor LY 83583, but can be induced by the synthetic cell permeant cGMP analogue 8-Br-cGMP [5-7]. Binding experiments of ANP to isolated leaf mem- branes provide evidence for specific receptor ligand inter- actions [8]. Despite an increasing body of physiological and biochem- ical data [21], the biological role of this systemically mobile peptide has remained elusive. In order to infer a Model of AtPNP-A action at the cellular level Figure 1 Model of AtPNP-A action at the cellular level. Background The model proposes that AtPNP-A can dock to receptor-like molecules that directly act as particulate guanylyl cyclases (pGCs) or indirectly activate soluble GCs (sGCs). GCs cata- lyse the reaction from GTP to cGMP. The latter acts as sec- ond messenger affecting cytosolic Ca2+ levels, modulating ion channels, activating phosphorylation through kinases and influences the transcriptome. Phosphodiesterases (PDEs) in turn metabolise cGMP to GMP (adapted from [21]). A plant NP (PNP) from A. thaliana (AtPNP-A) and several closely related sequences in different species have since been identified [9,10]. AtPNP-A, its most closely related sequence AtPNP-B, and orthologues in other higher plant species, share a family-45 glucosidase domain with the cell wall loosening expansins [11] and are related to expansins on the basis of this structural homology [9,12]. AtPNP-A (At2g18660) is a small protein of 126 amino acids in length (MW: 14016 kD; pI: 9.22) that is encoded by a gene with a single intron of 100 bp. The region most conserved between PNPs from different plant species has also been shown to be the key to its physiological activity [13]. Evidence for systemic mobility of PNPs comes from the structure and processing of the molecules [9]. The pro- tein contains an N-terminal 24 amino acid signal peptide (MW: 2249) that directs the molecule into the extracellu- lar space and PNPs that are recognized by anti-human atrial natriuretic polypeptide rabbit serum have been localised in situ in conductive tissue [14] and were isolated from xylem sap [15], and proteomics studies have identi- fied the AtPNP-A protein in the apoplastic space in A. thal- iana [16]. Model of AtPNP-A action at the cellular level Figure 1 Model of AtPNP-A action at the cellular level. The model proposes that AtPNP-A can dock to receptor-like molecules that directly act as particulate guanylyl cyclases (pGCs) or indirectly activate soluble GCs (sGCs). GCs cata- lyse the reaction from GTP to cGMP. The latter acts as sec- ond messenger affecting cytosolic Ca2+ levels, modulating ion channels, activating phosphorylation through kinases and influences the transcriptome. Phosphodiesterases (PDEs) in turn metabolise cGMP to GMP (adapted from [21]). Model of AtPNP-A action at the cellular level Figure 1 Model of AtPNP-A action at the cellular level. The model proposes that AtPNP-A can dock to receptor-like molecules that directly act as particulate guanylyl cyclases (pGCs) or indirectly activate soluble GCs (sGCs). GCs cata- lyse the reaction from GTP to cGMP. Abstract Background: Plant natriuretic peptides (PNPs) are a class of systemically mobile molecules distantly related to expansins. While several physiological responses to PNPs have been reported, their biological role has remained elusive. Here we use a combination of expression correlation analysis, meta-analysis of gene expression profiles in response to specific stimuli and in selected mutants, and promoter content analysis to infer the biological role of the Arabidopsis thaliana PNP, AtPNP-A. Results: A gene ontology analysis of AtPNP-A and the 25 most expression correlated genes revealed a significant over representation of genes annotated as part of the systemic acquired resistance (SAR) pathway. Transcription of these genes is strongly induced in response to salicylic acid (SA) and its functional synthetic analogue benzothiadiazole S-methylester (BTH), a number of biotic and abiotic stresses including many SA-mediated SAR-inducing conditions, as well as in the constitutive SAR expressing mutants cpr5 and mpk4 which have elevated SA levels. Furthermore, the expression of AtPNP-A was determined to be significantly correlated with the SAR annotated transcription factor, WRKY 70, and the promoters of AtPNP-A and the correlated genes contain an enrichment in the core WRKY binding W-box cis-elements. In constitutively expressing WRKY 70 lines the expression of AtPNP-A and the correlated genes, including the SAR marker genes, PR-2 and PR-5, were determined to be strongly induced. Conclusion: The co-expression analyses, both in wild type and mutants, provides compelling evidence that suggests AtPNP-A may function as a component of plant defence responses and SAR in particular. The presented evidence also suggests that the expression of AtPNP-A is controlled by WRKY transcription factors and WRKY 70 in particular. AtPNP-A shares many characteristics with PR proteins in that its transcription is strongly induced in response to pathogen challenges, it contains an N-terminal signalling peptide and is secreted into the extracellular space and along with PR-1, PR-2 and PR-5 proteins it has been isolated from the Arabidopsis apoplast. Based on these findings we suggest that AtPNP-A could be classified as a newly identified PR protein. Page 1 of 12 (page number not for citation purposes) (page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Background The latter acts as sec- ond messenger affecting cytosolic Ca2+ levels, modulating ion channels, activating phosphorylation through kinases and influences the transcriptome. Phosphodiesterases (PDEs) in turn metabolise cGMP to GMP (adapted from [21]). A number of physiological and biochemical studies have implicated AtPNP-A with a role in the regulation of ion and solute homeostasis. Immuno-reactive PNP (irPNP) extracts and recombinant AtPNP-A have been shown to induce swelling in leaf mesophyll protoplasts [17] and in protoplasts isolated from Arabidopsis cell suspension cul- Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 any bias in GO functional annotation terms in the corre- lated list (list 1) compared to the remainder of the A. thal- iana genome [see Additional file 1]. In the GO search category of biological process there is a significant (Family Wise Error Rate – FWER) adjusted p-value) enrichment in genes involved in biotic defence responses at a number of levels. The most notable bias being at level 8 with a signif- icant (adjusted p-value 0.0000038) enrichment in genes involved in SAR (Table 1). SAR is an inducible plant defense response against local pathogen infection that gives rise to a systemic long lasting resistance to a broad range of virulent pathogens [25]. The SAR response is characterised by the accumulation of endogenous salicylic acid (SA) in infected tissues and later in distal uninfected tissues with a subsequent induction of a select group of pathogenesis-related genes (PR genes) [26]. biological role for AtPNP-A, we made use of the large repositories of A. thaliana microarray data to study the expression profiles of AtPNP-A and the 25 most expres- sion correlated genes in response to various treatments as well as in mutants. We further analysed the promoters of these genes for known regulatory motifs. The results of our study predict a function for AtPNP-A in plant abiotic and biotic stress responses, and in particular in systemic acquired resistance (SAR). Furthermore, we demonstrate how computational analyses that link regulatory potential as encoded by promoter elements and expression data can provide novel insights into the function of a specific gene as well as groups of genes. Results and Discussion Expression Correlation and GO Analyses Page 3 of 12 (page number not for citation purposes) SAR: Systemic acquired resistance; RBS: Responsive to biotic stress Expression Correlation and GO Analyses In the first step of the analyses we extracted and ranked the 25 genes whose expressions are most tightly correlated with AtPNP-A (Table 1). The moderate correlation (r) val- ues of the listed genes (maximum r = 0.73) may reflect that the expression of AtPNP-A is subject to complex com- binatorial control via multiple promoter motifs with com- plex inputs from multiple, potentially antagonistic, signalling pathways. The enrichment in SAR annotated genes in our list is par- ticularly striking considering that in FatiGO the entire A. thaliana genome contains only 21 annotated SAR genes and four of these are present in our list of correlated genes. The four correlated SAR genes include NIMIN1 (At1g02450; r = 0.61) that is involved in the transcrip- tional regulation of PR genes [26], PR-1 (At2g14610; r = 0.63); PR-2 (At3g57260; r = 0.73) and PR-5 (At1g75040; 0.61) whose expression is commonly used as diagnostic markers of the SA dependent SAR response [27]. An In order to identify a functional role of AtPNP-A, the cor- related genes were analysed in FatiGO+ [22-24] to identify Table 1: List of genes that are expression correlated with AtPNP-A (At2g18660) Table 1: List of genes that are expression correlated with AtPNP-A (At2g18660) Locus r-value Annotation At3g57260 RBS, SAR 0.731 Pathogenesis-related protein 2 (PR-2) At5g10760 0.681 Aspartyl protease family protein At2g04450 RBS 0.676 Triphosphatase activity, stress response At5g52760 0.661 Heavy-metal-assoc. domain-containing At2g17040 0.659 No apical meristem (NAM) family protein At5g55450 RBS 0.647 Protease inhibitor/lipid transfer protein At1g21250 0.645 Wall-associated kinase 1 (WAK1) At4g23610 0.641 Hin1 – role in hypersensitive response At1g13470 0.634 Mitochondrial protein of unknown function At4g14365 0.634 Zinc finger (C3HC4-type RING) family At1g73800 0.630 Calmodulin binding protein At3g56710 0.629 SigA-binding protein, plastid sigma factor At4g04490 0.627 PK family, liposaccharide biosynthesis At2g14560 0.626 Protein of unknown function (DUF 567) At2g14610 RBS, SAR 0.626 Pathogenesis-related protein 1 (PR-1) At1g21520 0.626 Expressed protein At2g24850 0.626 Aminotransferase, resp. to wounding & JA At4g23150 0.625 Protein kinase family protein At3g60420 0.622 Phosphohistidine phosphatase activity At2g32680 0.620 Disease resistance, leucine rich-repeats At1g74440 0.614 Similar to YGL010w-like protein At1g02450 RBS, SAR 0.613 NPR1/NIM1-interacting prot. Expression Correlation and GO Analyses 1 (NIMIN1) At4g11890 0.606 Protein kinase family protein At1g75040 RBS, SAR 0.604 Pathogenesis-related protein 5 (PR-5) At1g08450 0.602 Calreticulin 3 precursor, Ca2+ binding SAR: Systemic acquired resistance; RBS: Responsive to biotic stress Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Expression profile of AtPNP-A and correlated genes in response to selected treatments Figure 2 Expression profile of AtPNP-A and correlated genes in response to selected treatments. The results presented illustrate the fold change (log2) in expression of AtPNP-A, WRKY 70 and WRKY 46 and the average fold change for the 25 correlated genes in response to abiotic stresses (A) and biotic and chemical treatments (B). (A) The treatments were: UV-B shoot 3 h (n = 2); O3 6 h (n = 3); Osmotic stress in the shoot after 3 h (n = 2); K+ starvation in the shoot after 7 days (n = 3); NaCl in the roots after 6 h (n = 2) and cold acclima- tion after 14 days (n = 3). (B) The treatments were: Erysiphe cichoracearum 3 days after inoculation (n = 4); Erysiphe orontii 3 days after inoculation (n = 2); Phytophthora infestans 1 day after inoculation (n = 3); BTH after 8 h (n = 3); SA after 3 h (n = 2) and cyclohexamide after 3 h (n = 2). Error bars repre- sent standard errors of the mean. extended correlation analysis revealed that an additional 11 SAR annotated genes, including NPR1 (or NIM1; At1g64280; r = 0.52), which is an essential key positive regulator of signal transduction leading to the SAR response and expression of PR proteins, are significantly correlated (9 positive, 2 negative; p < 0.01; bivariate nor- mal distribution) with the expression of AtPNP-A [see Additional file 2]. Other correlated genes in list 1 anno- tated to be involved in plant defence responses and response to biotic stimuli include a disease resistance fam- ily protein containing leucine rich-repeats (At2g32680), a stress responsive gene with triphosphatase activity (At2g04450) and a protease inhibitor (At5g55450). The GO analysis for the cellular component and molecular function category revealed no significant difference in bio- logically relevant labels between the two lists. Expression response t Figure 2 p p g p g Expression profile of AtPNP-A and correlated genes in response to selected treatments. The results presented illustrate the fold change (log2) in expression of AtPNP-A, WRKY 70 and WRKY 46 and the average fold change for the 25 correlated genes in response to abiotic stresses (A) and biotic and chemical treatments (B). (A) The treatments were: UV-B shoot 3 h (n = 2); O3 6 h (n = 3); Osmotic stress in the shoot after 3 h (n = 2); K+ starvation in the shoot after 7 days (n = 3); NaCl in the roots after 6 h (n = 2) and cold acclima- tion after 14 days (n = 3). (B) The treatments were: Erysiphe cichoracearum 3 days after inoculation (n = 4); Erysiphe orontii 3 days after inoculation (n = 2); Phytophthora infestans 1 day after inoculation (n = 3); BTH after 8 h (n = 3); SA after 3 h (n = 2) and cyclohexamide after 3 h (n = 2). Error bars repre- sent standard errors of the mean. The strong up-regulation of AtPNP-A and correlated genes by SA and benzothiadiazole S-methylester (BTH), a syn- thetic functional SA analogue [28], is a key indicator that these genes are involved in plant defence and specifically SAR since SA has been shown to be essential and sufficient to induce the SAR response in plants [29] (Figure 2B). In addition to AtPNP-A, all of the 25 correlated genes were significantly (ANOVA p-value < 0.05) up-regulated by more than two fold after 8 h and 24 h treatments with 60 µM BTH (Supplementary Table 3 in [28]) further linking these genes to the SAR defense pathway. Expression of AtPNP-A is also significantly correlated with the isochoris- mate synthase-1 (ICS-1) gene (At1g74710; r = 0.50) that is critical for SA biosynthesis [29]. expression and the correlated genes in response to these pathogens is in accord with the literature informing that SA-dependent defenses generally act against biotrophs in contrast to jamonic acid- and ethylene-dependent responses that counteract necrotrophs [30]. The large increases in gene expression induced by ozone and UV-B (+6.86log2 and +8.09log2 respectively for AtPNP- A) is consistent with these genes being part of the SAR response since both these treatments have previously been shown to stimulate SA production and induce the expression of PR genes [31-34]. Expression Correlation and GO Analyses The results of the Swiss-Prot keyword search also identi- fied a significant enrichment in genes annotated as PR proteins (adjusted p value = 0.002), involved in signalling (adjusted p value = 0.026) and associated with the apo- plast (adjusted p value = 0.040) in list 1. It was noted that along with PR-1, PR-2 and PR-5, AtPNP-A is one of six genes in list 1 annotated as having signalling function. Microarray Expression Profiles The over representation of genes involved in defence responses, and specifically SAR is consistent with the observation that AtPNP-A and the correlated genes are most highly expressed in microarray experiments where defence responses are elicited. The treatments that induce up-regulation of AtPNP-A and the correlated genes more than two-fold include SA and other SAR inducing condi- tions as well as a number of abiotic stresses (Figure 2). Expression response t Figure 2 The expression of Ca2+ sensing molecules is rap- idly induced in response to biotic and abiotic stresses [51] and functions to decode Ca2+signatures and/or relay sig- nals to downstream targets, including kinases, which fur- ther amplify the Ca2+ signal by inducing downstream phosphorylation cascades [38]. The presence of three kinases (At4g04490; r = 0.63, At4g23150; r = 0.63 and At4g11890; r = 0.61) in amongst the correlated genes (Table 1) is entirely consistent with such a signaling cas- cade. Moreover, the expression correlation of three stress responsive mitogen-activated protein kinase (MAP kinase) genes MAPKK (At4g26070; r = 0.59), MPK 11 (At1g01560; r = 0.58) and MAPKK (At4g29810; r = 0.57) to AtPNP-A also ties in with the proposed cascades [see Additional file 2]. Activation of MAPKs has indeed been reported after exposure to pathogens [52] as well as a number of abiotic stresses [53]. K+ is the key inorganic ion required in high quantities by plants while Na+ on the other hand is toxic at high concen- trations [38]. Na+ is able to compete with K+ ions for uptake and binding sites thus maintaining the correct Na+/K+ ratio in plants is of the utmost importance [39]. Decreases in K+ might cause the plant to take up more Na+ in order to maintain adequate osmotic pressure [40]. Therefore either the increase in cytosolic Na+ or a decrease in osmotic pressure as a consequence of K+ starvation or a combination of both may cause AtPNP-A induction. Elevated expression of AtPNP-A and correlated genes, par- ticularly defence genes and SAR annotated genes, by abi- otic osmotic stresses as well as defence eliciting treatments may well reflect that both types of challenges lead to com- mon homeostatic disturbances which in turn transcrip- tionally activate a set of common response genes. This concept is supported by several studies that recognise a role of SA in abiotic stresses such as drought, salinity and temperature [41,42] and the accumulation of PR proteins is in fact a common plant response to both abiotic and biotic stresses further highlighting the overlap in biotic and abiotic defence mechanisms [43]. While transcriptional responses to some stresses, includ- ing the osmotic, salt, UV-B and some of the biotic treat- ments, were measured over multiple time points, the data presented here are generally the earliest time point that induced the largest increase in AtPNP-A expression. Expression response t Figure 2 The biotic stresses that induced the largest increase in expression of AtPNP-A include infection with the bio- trophic pathogens Phytophthora infestans, Erysiphe cichora- cearum and Erysiphe orontii that depend on living host tissue for survival (Figure 2B). Activation of AtPNP-A Page 4 of 12 (page number not for citation purposes) Page 4 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 http://www.biomedcentral.com/1471-2229/8/24 The expression of AtPNP-A and the correlated genes is also strongly modulated by a number of abiotic stresses including K+ starvation, osmotic, and NaCl stress as well as cold acclimation (Figure 2A). A common element of abiotic stresses is that they decrease water potential [35]. It is noteworthy that the induction of AtPNP-A in response to ion and osmotic stresses is tissue specific with the response to high Na+ being specific to root tissue with lit- tle change observed in shoots while both low K+ and high osmolarity induced elevated transcription in shoots only [36]. This is of interest since the AtPNP-A protein has been shown to affect water movement in shoots [37] and pro- toplasts [17] as well as ion fluxes in roots [20]. It is thus tempting to speculate that AtPNP-A may have a role in maintaining plant water and ion homeostasis under stress conditions. cell membranes is also correlated with that of AtPNP-A (r = 0.60) [see Additional file 2] since ion conductance in these channels is regulated by cGMP as well as Ca2+ and calmodulin. These channels have also been implicated in regulating SA-dependent biotic defense responses [46]. AtPNP-A expression is also correlated with a number of Ca2+ sensing/binding proteins including, the above men- tioned CNGC20, calreticulin 3 (At1g08450; r = 0.60), two calmodulin-binding proteins (At1g73800; r = 0.63 and At1g73805; r = 0.588) with family members involved in the induction of plant defense responses (NCBI sequence viewer, pfam07887) and a Ca2+-binding EF hand domain containing protein (At3g47480; r = 0.59). One of the ear- liest responses to biotic and abiotic stresses is an increase in cytosolic free Ca2+[47] that in turn plays a role in acti- vating the oxidative burst after elicitor treatment [48,49] and is also linked to signaling SA-induced PR gene expres- sion [50]. Page 5 of 12 (page number not for citation purposes) Common Motifs in cis and Transcription Factors f p The common expression profiles of APNP-A and the 25 correlated genes in response to both biotic and abiotic stresses suggests that these genes are under common regu- latory control and are thus likely to share common cis-ele- ments in their promoter regions. To reveal aspects of common transcriptional activation we analyzed promoter regions of these genes 1 kb upstream of the predicted tran- scription start site (TSS) for the presence of known plant cis-elements. in the A. thaliana genome suggesting that they are impor- tant regulatory elements in these expression correlated genes. In plants, W-box cis-elements are known to bind WRKY TFs [60] indicating that these TFs may be important in reg- ulating the expression of the correlated genes. This is com- pletely consistent with our expression analysis results since the WRKY family of TFs [60] have well established roles in regulating disease responses in plants [61]. In addition, they have also been documented to mediate abi- otic plant responses to freezing [62], wounding [63], oxi- dative stress [64], drought, salinity, cold, and heat [65- 67]. In our study, expression of AtPNP-A is moderately correlated with the expression of WRKY 70 (At3g56400; r = 0.60) and WRKY 46 (At2g46400; r = 0.56) [see Addi- tional file 2]. When viewing the expression profiles of WRKY 70 and WRKY 46 genes it is apparent that the vari- ous treatments which induced large increases in the expression of the correlated genes in Table 1 also induced marked changes in the expression of the WRKY genes (Fig- ure 2). This links the expression of AtPNP-A and the WRKY genes to common biological responses and raises The analysis in POBO [58] indicated that the invariant core TTGAC W-box motif was present in 25/26 of our cor- related genes a total number of 78 times at an average of 2.99 copies/promoter compared to the average of 2.24 across all A. thaliana promoters (t-test p-value >0.0001) (Figure 3 and [see Additional file 3]). The analysis in Ath- ena [59] identified that the extended and more stringent TTGAC(A/T) W-box motif was present in 22/26 genes a total of 54 times at an average of 2.08 copies/promoter (p- value = 0.0037; data not shown). Expression response t Figure 2 The expression of AtPNP-A in some cases showed induction at earlier time points than considered in this study, however, in all cases the expression of AtPNP-A generally increased over time and thus high transcript levels were sustained for the duration of the stress, e.g. five days for E. orontii and 24 h for osmotic, salt and UV-B treatments (data not shown). The UV-B experiment can be distinguished from the other experiments in that the stress was not main- tained for the duration of the experiment. Rather, plants were irradiated for 15 minutes before being transferred back to the standard phytochamber conditions until har- vest. The expression of AtPNP-A in shoots was elevated at 30 minutes (1.84log2; data not shown), peaked at 3 h (8.09log2, > 250 fold) (Figure 2A) and remained elevated The generation of reactive oxygen species and changes in ion fluxes have been identified as early responses to both abiotic and biotic stresses, including an influx of H+ and Ca2+ and an efflux of K+ and Cl- [35]. AtPNP-A has been shown to modulate H+, Na+ and K+ fluxes [20] thus further implicating AtPNP-A in plant stress responses as do stud- ies which indicate that AtPNP-A signals via the intracellu- lar second messenger cGMP [5,6,18] since cGMP has been shown to be an important signaling molecule in pathogen [44] and osmotic stress responses [45] in plants. It seems particularly relevant that the expression of a gene encod- ing a cyclic nucleotide-gated channel (CNGC20; At3g17700), which has been shown to be involved in the transport of Ca2+ and K+, and in some cases Na+, across Page 5 of 12 (page number not for citation purposes) Page 5 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Frequency of occurrence of the W-box (TTGAC) core motif in artificial clusters generated in POBO for A. thaliana back- ground promoters compared to the promoters of AtPNP-A and the 25 most positively expression correlated genes Figure 3 Frequency of occurrence of the W-box (TTGAC) core motif in artificial clusters generated in POBO for A. thaliana background promoters compared to the promoters of AtPNP-A and the 25 most posi- tively expression correlated genes. Expression response t Figure 2 The 1 kb upstream promoter sequences of the 26 expression correlated genes were analysed in POBO (see methods and [see Additional file 3]) to determine the frequency of occurrence of the TTGAC W-box core motif. The analysis determined that compared to the A. thaliana background (2.24 copies/pro- moter), there was a significant (t-test: p-value > 0.0001) enrichment in the frequency of the TTGAC motif in our dataset (2.99 copies/promoter). (6.46log2; data not shown) at 24 h after irradiation. This documents that AtPNP-A expression remains very high and sustained after the stress has been removed and thus may indicate that the initial damage inflicted, and not the actual presence of the stress itself, is the driving force for the maintained transcriptional activation. The increase in expression of AtPNP-A (4.49log2) in response to the protein synthesis inhibitor cycloheximide (CHX) implies that transcription of AtPNP-A can occur independently of de novo protein synthesis and that con- curs with the definition of immediate early response genes [54] that have been proposed to play important roles in the early regulation of defence responses [55]. It has been postulated that CHX induces gene expression via dual mechanisms; by preventing synthesis or activation of a short-lived transcriptional repressor or by removing spe- cific labile transcript degrading enzymes [56]. There is evi- dence that the induced expression of genes encoding secreted proteins, such as AtPNP-A does not require de novo protein synthesis [57]. The ability to rapidly induce expression of AtPNP-A independently of de novo protein synthesis thus implies both an important and early role for this gene in response to specific elicitors. Frequency in artificial ground pro and the 25 Figure 3 q y ( ) g g p p p p y p g g Frequency of occurrence of the W-box (TTGAC) core motif in artificial clusters generated in POBO for A. thaliana background promoters compared to the promoters of AtPNP-A and the 25 most posi- tively expression correlated genes. The 1 kb upstream promoter sequences of the 26 expression correlated genes were analysed in POBO (see methods and [see Additional file 3]) to determine the frequency of occurrence of the TTGAC W-box core motif. The analysis determined that compared to the A. thaliana background (2.24 copies/pro- moter), there was a significant (t-test: p-value > 0.0001) enrichment in the frequency of the TTGAC motif in our dataset (2.99 copies/promoter). q y ( ) g g p p p p y p g g Frequency of occurrence of the W-box (TTGAC) core motif in artificial clusters generated in POBO for A. thaliana background promoters compared to the promoters of AtPNP-A and the 25 most posi- tively expression correlated genes. The 1 kb upstream promoter sequences of the 26 expression correlated genes were analysed in POBO (see methods and [see Additional file 3]) to determine the frequency of occurrence of the TTGAC W-box core motif. The analysis determined that compared to the A. thaliana background (2.24 copies/pro- moter), there was a significant (t-test: p-value > 0.0001) enrichment in the frequency of the TTGAC motif in our dataset (2.99 copies/promoter). Page 6 of 12 (page number not for citation purposes) Expression in selected Figure 4 The expression correlation between AtPNP-A and the dis- cussed WRKY genes and the overrepresentation of W- boxes in the correlated genes prompted a manual analysis of the promoter of AtPNP-A which revealed the presence of four copies of the core TTGAC W-box motif and two occurrences of more stringent TTGAC(C/T) motif clus- tered in close proximity (starting at -738 and -775) rela- tive to the predicated TSS. The result of the manual inspection coincided with the results returned form Ath- ena and POBO. Similar frequencies of these motifs were observed in a study of 26 SAR regulated genes (termed PR- 1 regulon genes) in which only W-boxes were present in the promoters of all 26 genes at an average of 4.3 copies of the core and 2.1 copies of the more stringent W-box ele- ments within 1100 bp upstream of the predicated TSS [60]. These values represent a significant enrichment in W-boxes since these authors determined that the statisti- cal expectation for a randomly distributed pentamer (TTGAC) was 2.1 copies and for the hexamer (TTGAC(C/ T)) 1.1 copies per 1100 bp of promoter [60]. Expression profile of AtPNP-A and selected corre- lated genes in selected A. thaliana mutants. The expression profiles of AtPNP-A and the correlated genes were examined in a number of SA/SAR related mutants. The expression of AtPNP-A and selected genes is greatly elevated in WRKY 70 over-expresser lines and in mutants with ele- vated SA levels such as cpr5 and mpk4. Conversely, in the SA deficient mutant nahG, expression of the selected genes is markedly reduced. Error bars represent standard errors of the mean. Expression profile of AtPNP-A and selected corre- lated genes in selected A. thaliana mutants. The expression profiles of AtPNP-A and the correlated genes were examined in a number of SA/SAR related mutants. The expression of AtPNP-A and selected genes is greatly elevated in WRKY 70 over-expresser lines and in mutants with ele- vated SA levels such as cpr5 and mpk4. Conversely, in the SA deficient mutant nahG, expression of the selected genes is markedly reduced. Error bars represent standard errors of the mean. WRKY 70 is a SAR annotated TF in FatiGO and has been shown to be an essential factor in plant defense responses necessary for the induction of PR gene expression in A. thaliana [28,69]. Expression in selected Figure 4 In a microarray study, a constitutive over- expressor of WRKY 70 was shown to induce constitutive expression of SA induced PR genes and five of our corre- lated genes, including PR-2 and PR-5, which are widely considered SAR marker genes, correspond to genes in this study that were up-regulated > 2.5 fold compared to con- trols [69]. While the 8K Affymetrix chip used for this study did not contain an AtPNP-A representative sequence, in an unpublished experiment using the 24K chip, AtPNP-A was up-regulated over 50 fold in over-expressing WRKY 70 lines and was amongst the top 20 genes that are up-reg- ulated in this study (Figure 4; Personal communication: Gunter Brader, Faculty of Biosciences, University of Hel- sinki). Additionally, a strong induction in expression of the 25 correlated genes is also observed in this experiment providing further evidence that indicates that WRKY 70 may positively regulate the expression of AtPNP-A and the expression correlated genes. In summary, the presented evidence is entirely consistent with transcription of AtPNP-A and the correlated genes being positively regulated by WRKY TFs. The promoter of AtPNP-A and the correlated genes contain an enrichment of the core W-box motif and expression of AtPNP-A is cor- related with two WRKY genes in response to various SAR eliciting and biotic and abiotic stresses. The correlation of WRKY 46 in response to ion and osmotic abiotc stresses was both treatment and tissue specific. In the light of these facts we suggest that the expression of AtPNP-A may be closely regulated by WRKY TFs in response to SAR-induc- ing and abiotic stresses. While transcription of WRKY 70 and WRKY 46 is gener- ally strongly induced in response to SAR eliciting treat- ments, only WRKY 46 is consistently co-expressed with AtPNP-A in response to the abiotic (ion and osmotic) stresses (Figure 2A and 2B). As previously described for AtPNP-A and the correlated genes, the induced expression of WRKY 46 is specific to shoots in response to K+ starva- tion and osmotic stress and to roots in response to NaCl stress. Common Motifs in cis and Transcription Factors Although in Athena this p-value does not qualify the W-box motifs to be enriched in our correlated genes according to the stringent enrich- ment threshold of <10-4 (Bonferroni correction), it does show that a very high percentage of our genes contain multiple copies of the stringent W-box. Both these pro- moter analysis methods indicate that multiple copies of the W-box elements are present in a high percentage of our correlated genes with the core TTGAC motifs being significantly enriched compared to expected frequencies Page 6 of 12 (page number not for citation purposes) Page 6 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Expression profile of AtPNP-A and selected correlated genes in selected A. thaliana mutants Figure 4 Expression profile of AtPNP-A and selected corre- lated genes in selected A. thaliana mutants. The expression profiles of AtPNP-A and the correlated genes were examined in a number of SA/SAR related mutants. The expression of AtPNP-A and selected genes is greatly elevated in WRKY 70 over-expresser lines and in mutants with ele- vated SA levels such as cpr5 and mpk4. Conversely, in the SA deficient mutant nahG, expression of the selected genes is markedly reduced. Error bars represent standard errors of the mean. the possibility that WRKY 70 and WRKY 46 may positively regulate AtPNP-A transcription. AtPNP-A as PR Protein Based on the above results we suggest that AtPNP-A could be classified as a PR protein since it possesses many of the criteria that define this class of proteins. The name "patho- genesis related protein" is a collective term that encom- passes all proteins that are present at almost undetectable levels in healthy tissue but are induced at the protein level following pathogen infection. The classification of these proteins is based on their pathogen inducible expression rather than defined functional roles in defence [43]. This point is brought into focus when considering PR-1, which is the quintessential marker of the SAR response yet its biological role is largely unknown [80]. Although AtPNP- A is yet to be proven to be induced at the protein level in response to pathogens, elevated protein levels have been shown as a result of abiotic stresses [15]. In addition, tran- scription of AtPNP-A is low under control conditions but strongly induced in response to biotic and abiotic stresses and the protein has been identified and isolated from the A. thaliana apoplast together PR-1, PR-2 and PR-5 proteins [16]. AtPNP-A has other features characteristic of PR pro- teins including an N-terminal signal peptide [43] that directs the molecule into the extracellular space. Further, induction of AtPNP-A at the transcript level appears to occur independent of de novo protein synthesis character- istic of genes encoding secreted proteins [28]. The evolu- tionary history of AtPNP-A suggests that PNPs, like the related expansins, derived from ancestral family-45 endoglucanases that have lost their hydrolytic activity and have sub-functionalized into extracellular, systemically mobile signalling molecules [9]. Conversely, the nahG mutant that is defective in SA pro- duction and signalling, is the only experiment presented in this study that documents a large reduction in the expression of AtPNP-A (-4.6log2) and the correlated genes (Figure 4). This experiment was performed in senescing leaves [72] to identify SA-dependent global gene expres- sion patterns during developmental senescence since SA has previously been shown to be required for expression of some senescence-induced genes [73]. In this study, and in the ATGE developmental series of A. thaliana microar- ray experiments [74] transcript levels of AtPNP-A were ele- vated approximately 2.8 fold in senescing leaves compared to adult green leaves (data not shown) indicat- ing that AtPNP-A is a senescence enhanced gene. Insights from AtPNP-A Expression in Mutants In the cpr5 (constitutive expresser of pathogenesis related genes) and mpk4 mutants that have elevated levels of SA and display constitutive expression of PR genes [70,71], the expression of AtPNP-A and the correlated genes was markedly elevated (Figure 4). It is of particular interest that the four listed mutants that displayed the largest increase in the expression of AtPNP-A in Genevestigator The link between AtPNP-A, SA signalling and the WRKY TFs is also supported by expression profiles of AtPNP-A and the correlated genes in mutants including a WRKY 70 over-expresser and various SAR related mutants present in the mutant surveyor in Genevestigator [68]. Page 7 of 12 (page number not for citation purposes) Page 7 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 http://www.biomedcentral.com/1471-2229/8/24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 are all cpr5 mutants, being cpr5/scv1, cpr5/npr1, cpr5, cpr5/ npr1/svi1 (range +7.55 to +6.21log2) (data not shown). AtPNP-A as PR Protein Further- more, since transcript levels of AtPNP-A were reduced beyond detection limits in senescing leaves in the nahG mutant this induction appears to be SA-dependent. This pattern is completely consistent with other results since premature senescence, including leaf yellowing and necrosis can be induced by biotic and aboitic stresses that stimulate SA production, including ozone [75] and UV-B [76] which also induce large increases in expression of AtPNP-A. Thus, there is evidence documenting induction of AtPNP-A expression in SA-mediated natural develop- mental and stress activated processes which both culmi- nate in cell death indicating that AtPNP-A may be involved in these processes. The mutant analyses further enforce that the transcriptional regulation of AtPNP-A and the correlated genes is largely controlled by SA. Future directions In order to determine the physiological role of AtPNP-A in A. thaliana a T-DNA insertion mutant, that is available from SALK, could be used. Phenotyping this mutant in response to SA-inducing abiotic and biotic stresses as well as during developmental senescence, will help character- ise specific physiological processes in which AtPNP-A is involved. If such a mutant demonstrated a compromised SAR response, it would greatly strengthen the claim that AtPNP-A is indeed involved in the SAR response pathway. Additionally, it will be interesting to look at the expres- sion of the correlated genes in an AtPNP-A mutant in response to SAR inducing conditions since this may ena- ble us to determine a role for AtPNP-A in the context of a SAR response pathway. The role of a TGA TF in PR gene expression Additional evidence for co-regulation of AtPNP-A with SAR annotated genes is provided by the observation that expression of another SAR annotated TF, the TGA3 bZIP TF (At1g22070), is correlated with that of AtPNP-A (r = 0.49). Upon induction of SAR, NIMIN1 and NPR1 form a ternary complex with TGA factors in the nucleus which enhances their binding to the positive regulatory as-1 (activator sequence 1) or as-1-like (TGACG) cis-elements that are present in the promoter of several plant genes acti- vated during defense, including A. thaliana PR-1 [77-79]. A manual inspection of the AtPNP-A promoter identified two occurrences of the TGACG motif in close proximity to the TSS (start at -94 and +24). The correlation in expres- sion between AtPNP-A, NPR1, NIMIN1 and TGA3 (Table 1, and [see Additional file 2]) along with the identifica- tion of TGA3 cis-elements in the promoter of AtPNP-A is strong evidence that these two factors contribute to the regulation of AtPNP-A expression. Page 8 of 12 (page number not for citation purposes) Promoter analysis The web-based Athena [59] and POBO [58]) applications were used to analyse the promoters (-1 kb upstream of the predicted TSS) of AtPNP- and the 25 top correlated genes. In POBO [58], the 1 kb promoter sequences were uploaded and the analysis was run against A. thaliana background (clean) searching for the TTGAC W-box core motif using the default settings (number of sequences to pick-out = 50, number of samples to generate = 1000, sequence length = 1000 bps). A two-tailed p-value was cal- culated in the linked online GraphPad web-site using the generated t-value and degrees of freedom to determine the statistical differences between input sequences and back- ground. Identification of Correlated Genes Identification of Correlated Genes We downloaded (01/07/2005) A. thaliana gene expres- sion levels for 1877 experiments from the NASCArrays database [81], using the bulk data download option. Perl scripts were used to calculate non-parametric correlation coefficients (Spearman's rho) between the expression of AtPNP-A (At2g18660) and each of the approximately 22,000 genes represented on the Affymetrix array that was used to generate this data set. We ranked genes according to the correlation coefficient and reported genes that were most positively correlated with At2g18660. The p-values were calculated using the bivariate normal distribution, with p representing the probability of observing an equal or larger positive or negative correlation by chance. Functional Classification and expression analysis of Correlated Genes To characterise the correlated genes the web-based 'FatiGO+' program [22] was used to search for differential distributions of gene ontology (GO) and biological terms within this list. The search was conducted using AtPNP-A (At2g18660) and the 25 most positively correlated genes in Table 1 (list 1 = 26 genes). This list was compared to a reference gene list that contained the remaining genes in the entire A. thaliana genome (list 2 = 26147 genes). Sta- tistical significance was determined using the Family Wise Error Rate (FWER) to calculate the adjusted p-value. In Athena, the analysis was performed with the visualisa- tion tool using the 26 correlated genes with settings of 1000 bp upstream and do not cut off at adjacent genes. The statistical significance of over-represented TF binding sites is automatically calculated using a hypergeometric probability model to calculate the p-value. A Bonferroni correction was automatically used in Athena to account for multiple hypotheses testing (up to 105 different TF binding sites) and determined that the p-value threshold for significant enrichment was < 10-4. The expression profiles of AtPNP-A and the positively cor- related genes (Table 1) were initially examined using Affymetrix public microarray data in the gene response viewer tool (GRV) in Genevestigator [68]). The analysis was performed using the ATH1: 22K array chip type and included all of the available 2507 chip sources. For better temporal and spatial response resolution we obtained normalised microarray data from the following sites: http://www.biomedcentral.com/1471-2229/8/24 http://www.biomedcentral.com/1471-2229/8/24 NASCArrays, Ozone-26 (reference ID); P. infestans-123; UV-B stress-144; Potassium starvation-105; BHT-392. TAIR (ATGenExpress): Salicylic acid-ME00364; E.orontii- ME00354; Salt stress-ME00328; Osmotic stress-ME00327; Cold acclimation-ME00369; Cyclohexamide-ME00361. GEO (NCBI): E. cichoracearum-GSE431. and abiotic stimuli and in mutant studies. The expression of AtPNP-A is correlated with the ICS-1 gene that is involved in SA biosynthesis and NPR1 and NIMIN1, which are key positive regulators of the SAR response, and two annotated SAR TFs TGA 3 and WRKY 70. Addition- ally, like the PR genes, the promoter of AtPNP-A contains as-1 and W-box cis-elements that correspond to binding sites for the TGA 3 and WRKY TFs. Further, over expressing WRKY 70 lines have been shown to cause a greater than 50-fold increase in the expression of AtPNP-A which is consistent with this TF being a positive regulator of AtPNP-A transcription. The induced expression of AtPNP- A by SAR elicitors and its secretion into the apoplast is similar to that of PR proteins and strongly implicates a role for AtPNP-A in plant SAR defence responses which may involve the modification of cellular ion and water homeostasis during stress responses. In order to further reveal the relationship of AtPNP-A expression with that of key genes involved in the SAR response, the mutant surveyor in Genevestigator was used to compare gene expression in different types of defence related A. thaliana mutants. The genes investigated in this study included AtPNP-A, the 25 correlated genes, and WRKY 70 and WRKY 46. Normalised array data from the mutant experiments were obtained from: TAIR-ME00373 for cpr5/npr1 mutants; NASCarray-52 for the nahG mutant and array express (EBI) for the mpk4 mutant (E-MEXP- 173). For the WRKY 70 over expresser, data was obtained through personal communication with Gunter Brader, Faculty of Biosciences, University of Helsinki. Conclusion AtPNP-A is an annotated "signal" molecule that is secreted into the apoplastic space and has been implicated with a role in the control of ion and solute movements in plants (Figure 1). The expression of AtPNP-A is signifi- cantly correlated with that of genes involved in the SAR defence response pathway in response to various biotic Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 Additional file 3 Promoter analysis. AtPNP-A (At2g18660) and the expression correlated genes were analysed in POBO and Athena for the presence of W-boxes. Click here for file p p 18. Wang Y, Gehring C, Cahill D, Irving H: Plant natriuretic peptide active site determination and effects on cGMP and cell vol- ume regulation. Funct Plant Biol 2007, 34:645-653. [http://www.biomedcentral.com/content/supplementary/1471- 2229-8-24-S3.doc] 19. Pharmawati M, Shabala SN, Newman IA, Gehring CA: Natriuretic peptides and cGMP modulate K+, Na+, and H+ fluxes in Zea mays roots. Mol Cell Biol Res Commun 1999, 2:53-57. 20. Ludidi N, Morse M, Sayed M, Wherrett T, Shabala S, Gehring C: A recombinant plant natriuretic peptide causes rapid and spa- tially differentiated K+, Na+ and H+ flux changes in Arabi- dopsis thaliana roots. Plant Cell Physiol 2004, 45:1093-1098. http://www.biomedcentral.com/1471-2229/8/24 http://www.biomedcentral.com/1471-2229/8/24 Authors' contributions 9. Ludidi NN, Heazlewood JL, Seoighe C, Irving HR, Gehring CA: Expansin-like molecules: novel functions derived from com- mon domains. J Mol Evol 2002, 54:587-594. The project was conceived by CG and VB. StM, RB, LD and ShM have extracted and interpreted the data. The manu- script was written by StM and CG. All authors read and approved the final manuscript. J 10. Nembaware V, Seoighe C, Sayed M, Gehring C: A plant natriuretic peptide-like gene in the bacterial pathogen Xanthomonas axonopodis may induce hyper-hydration in the plant host: a hypothesis of molecular mimicry. Bmc Evol Biol 2004, 4:10. 11. McQueen-Mason S, Cosgrove DJ: Expansin mode of action on cell walls: analysis of wall hydrolysis, stress relaxation, and binding. Plant Physiol 1995, 107:87-100. Abbreviations NP – natriuretic peptide; PNP – plant natriuretic peptide; SAR – systemic acquired resistance; cGMP – guanosine 3',5'-cyclic monophosphate; TF – transcription factor; TSS – translation start site. Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) BMC Plant Biology 2008, 8:24 Additional file 2 15. Rafudeen S, Gxaba G, Makgoke G, Bradley G, Pironcheva G, Raitt L, Irving H, Gehring C: A role for plant natriuretic peptide immuno-analogues in NaCl- and drought-stress responses. Physiol Plantarum 2003, 119:554-562. Extended list of genes expression correlated with AtPNP-A. An extended list containing all genes that are expression correlated with AtPNP-A (At2g18660) and a list of all annotated SAR genes in Arabidopsis thal- iana. y 16. Boudart G, Jamet E, Rossignol M, Lafitte C, Borderies G, Jauneau A, Esquerre-Tugaye MT, Pont-Lezica R: Cell wall proteins in apo- plastic fluids of Arabidopsis thaliana rosettes: Identification by mass spectrometry and bioinformatics. Proteomics 2004, 5:212-221. [http://www.biomedcentral.com/content/supplementary/1471- 2229-8-24-S2.xls] 17. Maryani MM, Bradley G, Cahill DM, Gehring CA: Natriuretic pep- tides and immunoreactants modify osmoticum-dependent volume changes in Solanum tuberosum L. mesophyll cell protoplasts. Plant Sci 2001, 161:443-452. Acknowledgements p y 21. Gehring CA, Irving HR: Natriuretic peptides - a class of heterol- ogous molecules in plants. Int J Biochem Cell Biol 2003, 35:1318-1322. This project was supported by the South African National Research Foun- dation and an Ernest Oppenheimer Memorial Trust fellowship to C.G. 22. Al-Shahrour F, Diaz-Uriarte R, Dopazo J: FatiGO: a web tool for finding significant associations of gene ontology terms with groups of genes. Bioinf 2004, 20:578-580. References 1. Takei Y: Does the natriuretic peptide system exist through- out the animal and plant kingdom? Comp Biochem Physiol B: Bio- chem Mol Biol 2001, 129:559-573. 1. Takei Y: Does the natriuretic peptide system exist through- out the animal and plant kingdom? Comp Biochem Physiol B: Bio- chem Mol Biol 2001, 129:559-573. 1. Takei Y: Does the natriuretic peptide system exist through- out the animal and plant kingdom? Comp Biochem Physiol B: Bio- chem Mol Biol 2001, 129:559-573. 23. Al-Shahrour F, Minguez P, Vaquerizas JM, Conde L, Dopazo J: BABE- LOMICS: a suite of web tools for functional annotation and analysis of groups of genes in high-throughput experiments. Nucleic Acids Res 2005, 33(Web Server issue):W460-464. , 2. Vesely DL, Giordano AT: Atrial natriuretic peptide hormonal system in plants Biochem Biophys Res Commun 1991 179:695 700 2. Vesely DL, Giordano AT: Atrial natriuretic peptide hormonal system in plants Biochem Biophys Res Commun 1991 179:695 700 y p p system in plants. Biochem Biophys Res Commun 1991, 179:695-700. 3. Vesely DL, Gower WR, Giordano AT: Atrial natriuretic peptides are present throughout the plant kingdom and enhance sol- ute flow in plants. Am J Physiol 1993, 265:E465-E477. system in plants. Biochem Biophys Res Commun 1991, 179:695-700. 3. Vesely DL, Gower WR, Giordano AT: Atrial natriuretic peptides are present throughout the plant kingdom and enhance sol- ute flow in plants. Am J Physiol 1993, 265:E465-E477. 24. Al-Shahrour F, Minguez P, Tarraga J, Medina I, Alloza E, Montaner D, Dopazo J: FatiGO +: a functional profiling tool for genomic data. Integration of functional annotation, regulatory motifs and interaction data with microarray experiments. Nucleic Acids Res 2007, 35(Web Server issue):W91-96. p J y 4. Gehring CA, Khalid KM, Toop T, Donald JA: Rat natriuretic pep- tide binds specifically to plant membranes and induces sto- matal opening. Biochem Biophys Res Commun 1996, 228:739-744. 4. Gehring CA, Khalid KM, Toop T, Donald JA: Rat natriuretic pep- tide binds specifically to plant membranes and induces sto- matal opening. Biochem Biophys Res Commun 1996, 228:739-744. 25. Durrant WE, Dong X: Systemic acquired resistance. Annu Rev Phytopathol 2004, 42:185-209. p g p y 5. Pharmawati M, Billington T, Gehring CA: Stomatal guard cell responses to kinetin and natriuretic peptides are cGMP- dependent. Cell Mol Life Sci 1998, 54:272-276. p g p y 5. Pharmawati M, Billington T, Gehring CA: Stomatal guard cell responses to kinetin and natriuretic peptides are cGMP- dependent. Additional file 1 p 13. Morse M, Pironcheva G, Gehring C: AtPNP-A is a systemically mobile natriuretic peptide immunoanalogue with a role in Arabidopsis thaliana cell volume regulation. Febs Lett 2004, 556:99-103. 14. Maryani MM, Morse MV, Bradley G, Irving HR, Cahill DM, Gehring CA: In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tis- sue and stomata. J Exp Bot 2003, 54:1553-1564. Additional material g y 12. Kende H, Bradford K, Brummell D, Cho HT, Cosgrove D, Fleming A, Gehring C, Lee Y, McQueen-Mason S, Rose J, Voesenek LA: Nomen- clature for members of the expansin superfamily of genes and proteins. Plant Mol Biol 2004, 55:311-314. Additional file 1 GO analysis of AtPNP-A expression correlated genes. List of significantly enriched GO terms associated with AtPNP-A (At2g18660) expression correlated genes in FatiGO+. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2229-8-24-S1.pdf] Additional file 2 Extended list of genes expression correlated with AtPNP-A. An extended list containing all genes that are expression correlated with AtPNP-A (At2g18660) and a list of all annotated SAR genes in Arabidopsis thal- iana. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2229-8-24-S2.xls] Additional file 3 Promoter analysis. AtPNP-A (At2g18660) and the expression correlated genes were analysed in POBO and Athena for the presence of W-boxes. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2229-8-24-S3.doc] Additional file 1 GO analysis of AtPNP-A expression correlated genes. List of significantly enriched GO terms associated with AtPNP-A (At2g18660) expression correlated genes in FatiGO+. 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Craigon DJ, James N, Okyere J, Higgins J, Jotham J, May S: NASCAr- rays: a repository for microarray data generated by NASC's transcriptomics service. Nucleic Acids Res 2004, 32(Database issue):D575-577. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 12 of 12 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge
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Experiences of an earthquake during pregnancy, antenatal mental health and infants’ birthweight in Bhaktapur District, Nepal, 2015: a population-based cohort study
BMC pregnancy and childbirth
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Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 https://doi.org/10.1186/s12884-020-03086-5 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 https://doi.org/10.1186/s12884-020-03086-5 (2020) 20:414 Open Access Experiences of an earthquake during pregnancy, antenatal mental health and infants’ birthweight in Bhaktapur District, Nepal, 2015: a population-based cohort study Experiences of an earthquake during pregnancy, antenatal mental health and infants’ birthweight in Bhaktapur District, Nepal, 2015: a population-based cohort study Goma Kumari Khatri1* , Thach Duc Tran1, Sushil Baral2 and Jane Fisher1 Goma Kumari Khatri1* , Thach Duc Tran1, Sushil Baral2 and Jane Fisher1 Abstract Background: Infant birthweight is an important public health indicator that is a proxy of maternal and baby’s health. Earthquakes can cause acute distress, but can also contribute to chronic stress through long-term disruptions to social, economic and domestic circumstances. The aims of this study were to examine the direct effect of earthquake experiences on the birthweight of infants of women who experienced the 2015 Nepal Earthquakes during pregnancy and whether mental health mediated this relationship. Methods: This is a prospective, population-based cohort study. Pregnant women in Bhaktapur District, Nepal who had experienced the 2015 earthquakes after conception were recruited. Baseline data were collected in structured individual interviews. Follow up was a short telephone interview two weeks after the baby’s birth. Infant birthweight recorded on the hospital-issued birth certificate. The direct effect of earthquake experiences on birthweight and the indirect effect via antenatal common mental disorders (CMDs) were tested using Sobel tests simultaneously. Results: In total 497/498 women contributed baseline data and 469 (94.4%) women also provided the birth weight of their infants. In total 6% of infants had low birth weight (< 2.5 kg). The earthquake experiences did not have a significant direct effect on birthweight (p = 0.116). However, the effect of earthquake experiences had a significant indirect effect on infant birthweight via CMDs. When 15 covariates were controlled, women who had moderate to severe earthquake experiences had an increase of 1.58 scores of antenatal CMD symptoms that resulted in their babies being 20.50 g lighter than those who had low earthquake experiences (p = 0.026). Conclusions: The findings from this study indicate that the relationship is mediated by the mental health of women during pregnancy. Data demonstrate that the mental health of women who are pregnant should be prioritised in post-disaster management not only because of the burden experienced by women but also because of the risk for the growth and development of their babies. Keywords: Birthweight, Earthquake experiences, Antenatal, Common mental disorders * Correspondence: shikak2003@gmail.com; goma.khatri@monash.edu 1School of Public Health and Preventive Medicine, Monash University, Level 4, 553 St Kilda Rd, Melbourne, Victoria 3004, Australia Full list of author information is available at the end of the article Background from ‘the United States’ and ‘non-United States coun- tries’ and found that relative risk of LBW was signifi- cantly higher among women who had ‘antenatal depression’ compared to those who had not. The study also reported that LBW was significantly higher in ‘non- United States’ countries compared to those in ‘the United States’ [6]. g Infant birthweight is an important public health indica- tor. It reflects maternal health as well as a predictor of the health and development of the baby [1]. Worldwide, it is estimated that low birthweight (LBW), usually de- fined as being born weighing less than 2500 g, contrib- utes to up to 80% of all neonatal deaths [2]. It is estimated that about 96.5% of infants born with LBW are living in resource-constrained low-income countries [2]. Finding from a national survey in Nepal revealed that LBW of infants based on the reported birth weight by mothers who had a live birth in the five years prior to the survey was 12.3%. About 5 and 12% of the mothers reported that their babies were small and very small at birth respectively [1]. Babies with LBW who survive, are at higher risk of compromised growth and development in their subsequent lives [3]. There have been seven studies that reported that women’s experiences of an earthquake might increase the risk of giving birth to LBW of their infants. Among them, six [15–20] were from middle-and high-income countries and only one [21] from a low-income country. All were audits of birth records or secondary analyses of existing data. None investigated directly, the relation- ships among women’s experiences of the earthquake while pregnant, their mental health and its relationship with the birthweight of their infants. Multiple causative factors are associated with LBW, but maternal health is a well-established determinant. While poor maternal nutrition, and chronic or acute stresses during pregnancy independently increase the likelihood of LBW, these may interact with each other in ways that amplify the risk of LBW [2, 4]. These causative factors are highly prevalent in low-income settings [2, 5, 6]. There is to date no study examining the possible path- ways of the effect of earthquakes on birth outcomes, but this is of central importance for effective post-disaster re- sponses. Antenatal mental health that can be compro- mised by experiencing a natural disaster and might play a mediating role in this relationship. Study design A prospective, population-based study of a cohort of women who were pregnant and had experienced the 2015 Earthquakes since conceiving. It included a base- line survey in late pregnancy and a follow-up interview conducted two weeks after birth. g y g Earthquakes cause acute distress, but can also contrib- ute to chronic stress through long-term disruptions to so- cial, economic and domestic circumstances [8–10]. Our study [11] was conducted after the 2015 Nepal earth- quakes and reported that 21.9% of participants had Edinburgh Postnatal Depression Scale (EPDS) scores> 12, and another 17.1% scored 10–12 indicating a high preva- lence of clinically-significant common mental disorders (CMDs) symptoms, which include anxiety, depressive, ad- justment, and somatoform disorders [12]. This is a higher prevalence than those reported in a systematic review and meta-analysis (about 16%) from low and lower-middle- income countries in general circumstances [5]. Another study by Kane et al. [13] was conducted in the same earth- quake context among the general population. The study reported that 34.3% experienced depressive symptoms, 33.8% anxiety and 5.2% had PTSD symptoms. The study found that women were more likely to experience depres- sion, anxiety and PTSD then men [13]. Study setting Data were collected in Bhaktapur District from October 2015 to April 2016. Bhaktapur is one of the 14 of 77 dis- tricts in Nepal that were severely affected by the April 2015 Earthquakes which were measured at 7.8 on the Richter Scale [22]. Bhaktapur has six sub-administrative divisions that include rural and urban communities. It also includes a UNESCO-designated World Heritage Site of major cultural, historical, and religious significance. Conceptual framework Conceptual framework in (Fig. 1) shows the proposed direction of associations among study variables. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 12 Page 2 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Background The aims of this study were to examine the direct effect of earthquake experi- ences on the birthweight of infants of women who experi- enced the 2015 Nepal Earthquakes during pregnancy and whether mental health mediated this relationship. In Nepal, Sharma et al. [7] conducted a case-control study in a tertiary hospital to identify determinants of LBW. They found that women who had a history of premature birth, had not been able to consume nutritious food during pregnancy, and were of younger (< 20years) age had a significantly higher risk of giving birth to a LBW infant [7]. We are not aware of any study in Nepal that has examined and reported on associations between pregnancy mental health and infant birthweight. Procedure Participants were recruited from all Bhaktapur sub- administrative divisions. Potential participants were identified in three ways: (i) together, the district public health office, district public health nurses, community health centre staff and female community health volun- teers (FCHVs) created a list of eligible pregnant women (ii) The Women and Children Development district offi- cers identified eligible women who had not been listed (iii) formal and informal local community leaders, eld- erly women and pregnant women were asked to identify potential participants in their villages. With the address given in the list of eligible women, we visited their home to inform them about the study and invite to participate in the study. All women were invited to participate in the study if they met the following criteria: (1) to be aged at least 15 years, (2) living in Bhaktapur District, and (3) pregnant during the 2015 Earthquakes. Pregnant women were excluded if they: (1) had a multifetal pregnancy, or (2) had a cognitive dis- ability, or (3) were local but outside Bhaktapur at the time of the major 2015 earthquakes, or (4) moved (migrated) to Bhaktapur to live after the major earthquakes. Those who contributed baseline data were invited again to participate in the follow-up interview. Women who had a stillbirth and those who did not measure the weight of their babies at birth were excluded from analyses on birthweight. There were two waves of data collection: one during pregnancy and the other two weeks after women had given birth. Participants The sample size was calculated to test the indirect effect of earthquake experiences on birthweight via antenatal CMD symptoms with a study power of 80% and an alpha error of In general circumstances, antenatal CMDs appear to increase the risk of LBW [6, 14]. Grote et al. [6] con- ducted a systematic review of eleven studies reported Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 3 of 12 Fig. 1 Conceptual framework. Source: Khatri, GK (2019) [23]. Note. Arrow signs show the postulated direction of association among variables. Plus (+) sign indicates positive and minus (−) sign indicates a negative association between variables Fig. 1 Conceptual framework. Source: Khatri, GK (2019) [23]. Note. Arrow signs show the postulated direction of association among variables. Plus (+) sign indicates positive and minus (−) sign indicates a negative association between variables 0.05 (5% level of significance). In total 480 women were re- quired to identify Cohen’s effect ≥0.14 of earthquake expe- riences on CMD symptoms and ≥0.26 of CMD symptoms on birthweight, allowing for attrition of 20% [24]. Public involvement Members of the local community were included in di- verse ways. Local bilingual health experts contributed to the translation of the interview schedules to Nepali, cul- tural verification and back translation to English. Twelve pregnant women are meeting inclusion criteria apart from living in Bhaktapur pilot-tested the schedules for acceptability and comprehensibility. Health workers, community leaders, elderly women, female community health volunteers, and staff of the Bhaktapur District Women’s Development Office identified eligible preg- nant women. HERD International, a local research or- ganisation, partnered in conducting this study. Potential participants were informed about the study by giving an explanatory statement. The statement was read by a researcher or a family member to those who could not read it. Participants were assured that their privacy and confidentiality would be maintained strictly. Women who were willing to participate gave consent by signing the form or providing a thumbprint. When a participant gave consent, she was invited to complete an interview in person for the first wave of data collection (baseline). All interviews were conducted in Nepali in private rooms at local health centres or, rarely, in a private space at their homes. Six trained and female interviewers who had experi- ence in data collection for other research projects man- aged by HERD International conducted the interviews following training from and with supervision from bilin- gual co-investigator (GKK). Primary data sources The primary data source was a structured interview schedule, which included standardised tools and study- specific questions (Table 1, Questionnaires). Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 4 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Table 1 Primary data sources and coding Interview time Variables Primary data sources and coding birth outcome Follow up Birthweight In Nepal, a birth certificate is prov facility. It records the date and tim In Nepal, a birth certificate is provided to each mother who has given birth in a medical facility. It records the date and time of the birth and the baby’s birthweight. Women were asked to report the birth weight of their babies from the birth certificate. Women who gave birth at home and did not have a birth certificate but where a skilled person had measured birth weight using a standard scale were also asked to report the weight. Those who did not measure baby birth weight were not able to be included in analyses. The weight in grams was used as a continuous variable to examine an association among covariates. Birth weight was also categorized into two groups: ≥2.5 kg deemed to be normal and < 2.5 kg as low birth weight. Mediating variable Baseline Symptoms of Common Mental Disorders (CMDs) during pregnancy The Edinburgh Postnatal Depression Scale-Nepali version (EPDS-N) comprises 10 items, scored from 0 to 3 with a total score of 0 to 30. This version has been formally validated against Diagnostic and Statistical Manual (DSM) IV diagnostic criteria among women who had recently given birth in Nepal: Sensitivity 68.43%; Specificity 93.80%; Positive Pre- dictive Value 65% and Negative Predictive Value 94.64 [31]. The EPDS total score was used as a continuous variable to examine a mediation effect. We also reported propor- tions of the scores: < 9, 10 to 12 and ≥13 to enable comparison between groups. Independent variables Baseline Earthquake experiences Study-specific questions adapted from previous studies [9, 20] collecting information about 17 items: where they are living now (1 item); property damage (3 items); impact on daily life including basic needs (6 items); being injured/trapped (2 items); witnessing injury/people trapped/death (4 items), intensity of fear experienced during the earthquake (1 item). Details of these items have reported elsewhere [11]. Each item was scored 0 (not experienced) to 1 (experienced) and summed to create a total score. Data management and statistical analysis Data were collected on paper forms and double-entered using Epidata version 3.1 computer software [25] at HERD Nepal. Data analyses were performed using Stata version 14 [26]. Descriptive analyses were used to compare partic- ipants’ characteristics in the group who provided complete data (which could be included in the final analyses) with those of participants who provided incomplete data (which could not be included) and between groups with babies of different birthweights. Fisher exact test for cat- egorical and Mann-Whitney test for continuous variables were used to test socio-demographic differences between the groups were and were not included in the analyses. In order to examine the interaction effect between the exposure (earthquake experiences) and mediator (depres- sive symptoms) on birthweight and the confounding effect of depressive symptoms on the effect of earthquake expe- riences on birthweight, we conducted multivariate regres- sion analyses stratified by having depressive symptoms or not. The criterion for not having clinically significant de- pressive symptoms was an EPDS score ≤12. The criterion for having clinically significant depressive symptoms was an EPDS score ≥13. These criteria were based on a formal validation of the EPDS among women who had recently given birth in Nepal [31]. The multivariate regression pre- dicting birthweight included all covariates that had been included in the mediation analysis. The direct effect of earthquake experiences on birth- weight and the indirect effect via EPDS total score were tested using simultaneous Sobel tests [27, 28]. According to Pek and Hoyle [28], we assumed that women’s mental health problems (EPDS total score) mediated the effect of earthquake experiences on birthweights. In this analysis, birth weight (in grams) was the primary outcome, the ex- posure variable was earthquake experiences, and EPDS total score was a mediating variable [28]. This analysis controlled for 15 covariates that were selected based on the results of bivariable analyses and previous studies. Primary data sources BMC Pregnancy and Childbirth (2020) 20:414 Table 1 Primary data sources and coding (Continued) Interview time Variables Primary data sources and coding Violence Against Women Questionnaire, including lifetime experiences of controlling be- haviour (7 items), emotional (4 items), physical (6 items), and sexual (3 items) violence by the current intimate partner [43]. Any lifetime experiences of intimate partner violence calculated combining any of four sub-types of violence. Baseline Practical and emotional support specific to the earthquakes Study-specific questions adapted from previous studies [10, 20]. Participants were asked whether they received any support including shelter, food, clothes, informal emotional and professional psychological support and rescue from the hazardous place during/ after earthquakes by any individual or any governmental and non-governmental organization. Violence Against Women Questionnaire, including lifetime experiences of controlling be- haviour (7 items), emotional (4 items), physical (6 items), and sexual (3 items) violence by the current intimate partner [43]. Any lifetime experiences of intimate partner violence calculated combining any of four sub-types of violence. Study-specific questions adapted from previous studies [10, 20]. Participants were asked whether they received any support including shelter, food, clothes, informal emotional and professional psychological support and rescue from the hazardous place during/ after earthquakes by any individual or any governmental and non-governmental organization. The second interview (follow-up) was a very short tele- phone interview two weeks after childbirth to ask about the date, place and mode of birth, and the baby’s birth- weight. All these interviews were conducted by GKK. The second model predicted EPDS scores from earth- quake experiences and other covariates. The final model predicted birthweight from all variables including earth- quake experiences, EPDS scores and other covariates. Fi- nally, the test results were summarized including the estimations and significant tests of the direct effect of the earthquake experiences on birthweight; the indirect effect of the earthquake experiences on birthweight via antenatal CMD symptoms, and the total effect of the earthquake experiences on birthweight. The second model predicted EPDS scores from earth- quake experiences and other covariates. The final model predicted birthweight from all variables including earth- quake experiences, EPDS scores and other covariates. Fi- nally, the test results were summarized including the estimations and significant tests of the direct effect of the earthquake experiences on birthweight; the indirect effect of the earthquake experiences on birthweight via antenatal CMD symptoms, and the total effect of the earthquake experiences on birthweight. Results Sample In total 497/498 eligible pregnant women were recruited and contributed baseline data. All gave consent to be followed up, and 492 women participated in the follow- up interview. Altogether 469 (94.4%) were able to pro- vide the baby’s weight at birth and were included in the analyses (Fig. 2). Bivariate analyses were conducted using the chi-square test or regression to select potential covariates that should be included in the final model of the mediation analysis. Covariates were selected on the basis that there were sig- nificant differences at p ≤0.1. However, some covariates such as household wealth, intimate partner violence and education levels were included in the final model even though they were not significant because they are estab- lished risk factors for low birthweight [29, 30]. Primary data sources The score ranged from 0 to 17. The total score was grouped in tertle, and low tertile was labelled as low, and middle and upper tertile labelled as middle/high earthquake experiences. Covariates Baseline and follow up Length of gestation at birth Date of the baby’s birth and the date of the last menstrual period were recorded to calculate the length of gestation at birth. At baseline, we asked participants about their date of last menstrual period. In the follow-up interview, date of baby birth was recorded. The last date of menstru- ation was subtracted from date of baby birth and divided by seven to calculate the total weeks as the length of gestation at birth. We used weeks as a continuous variable to examine an association with other variables. Study-specific questions adapted from our prior research [39] to ascertain women’s age, highest educational level, employment, consume chewing tobacco/smoking, and alcohol, household economics, family structure, and husband’s education, employment, consumption of chewing tobacco/smoking, and alcohol. Household economics was calculated based on household characteristics and durable assets using the World Bank method [39, 40]. Woman’s weight was measured using a portable digital weighing scale. Height was measured against the smooth but hard wall. Height and weight were used to calculate body mass index using international formula (weight/height2). It was used as a continuous variable to examine its associations with other covariates. Study-specific questions adapted from our prior research [39] about gravidity, parity, prior spontaneous/induced abortions, pregnancy intention, taking iron tablets during current pregnancy, foetal health, sex of index foetus and a number of children. Single yes/no question was used to assess if the participant had been diagnosed with or treated for any psychiatric illness within last year. The Relationship Assessment Scale (RAS) comprises 7 items scored from 1 to 5 and total scored from 7 to 35. Mean inter-item correlation of the scale is 0.49; test-retest reliability (among undergraduate students in USA) is 0.85 and high correlation with Dyadic Adjust- ment Scale [41] (0.86 to .88). Examples of items are: ‘How well does your partner meet your needs? In general, how satisfied are you with your relationship? How much do you love your partner?’ Higher scores indicate a better quality of the relationship [42]. Items from the World Health Organization Multi-Country Study on Women’s Health and Page 5 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Khatri et al. Birth weight Birthweight ranged from 1800 g to 5000 g. Mean birth- weight was 3045.3 ± 447.8 g. About 6% (5.8%) women re- ported birthweight less than 2500 g. Most women reported a rounded birthweight: 13.65% reported 3000 g; 11.51% reported 3500 g; 9.81% reported 2500 g; 8.10% reported 2700 g; 6.18% reported 2800 g and about 5% in each weight category reported 2900 g, 3000 g, 3100 g and 3200 g. The multivariate regression analyses predicting birth- weight stratified by having or not having clinically sig- nificant depressive symptoms revealed that moderate to high experiences of the earthquake had no significant ef- fect on birthweight in the absence of depressive symp- toms (adjusted coefficient = 52.26, p = 0.29) or in the presence of depressive symptoms (adjusted coefficient = 171.94, p = 0.10). Participants’ characteristics A comparison of the characteristics of participants whose data could and could not be included in the final analyses is presented in Table 2. Compared to the group who could be included, the group who were not in- cluded had significantly younger age, lower body mass index, no formal or primary education, more women consumed chewing tobacco/smoking, and lower In the Sobel test, three different multiple linear regres- sion models were performed simultaneously. The first model predicted birthweight from earthquake experi- ences and other covariates but without EPDS scores. Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 6 of 12 Fig. 2 Flow diagram of participants able to provide data about their baby’s birth weight. Note. n = number Fig. 2 Flow diagram of participants able to provide data about their baby’s birth weight. Note. n = number that earthquake experiences did not have a significant direct effect on birth weight. Compared to low experi- ences of the earthquakes, women who had middle or high earthquake experiences had higher EPDS scores. Women who had higher EPDS scores had babies with lower birthweights. household wealth. In addition, they were more likely to have a no-formal educated partner, experienced emo- tional and physical/sexual violence perpetrated by an in- timate partner, had an inconvenient/unwanted pregnancy, lower ANC check-ups and iron tablet intake. No women reported a history of psychiatric illness. Antenatal CMDs symptoms significantly mediated the effect of earthquake experiences on baby birthweight (Fig. 3). This means that while controlling for all covari- ates, women who had moderate to high earthquake ex- periences had an increase of 1.58 points in the EPDS scores that resulted in a 20.50 g reduction in their in- fants’ birth weight compared to those who had low earthquake experiences. Association among earthquake experiences, antenatal CMDs symptoms and birth weight The main findings of the Sobel test analysis are pre- sented in Fig. 3, and more detailed findings are pre- sented in Tables S1, S2, S3 and S4. The results showed Page 7 of 12 Khatri et al. Association among earthquake experiences, antenatal CMDs symptoms and birth weight BMC Pregnancy and Childbirth (2020) 20:414 Page 8 of 12 Table 2 Characteristics of participants at the baseline interview (Continued) Table 2 Characteristics of participants at the baseline interview (Continued) Characteristics Included (n = 469) Not included (n = 28) p-value Girl 207 (44.1) 9 (39.1) Number of children, n % 0.6 None 246 (52.5) 13 (46.4) One or more 223 (47.5) 15 (53.6) Length of gestation at baby birth (weeks), mean (SD) 39.9 (1.5) 39.1 (2.5) 0.2 Earthquake experiences, mean (SD) 3.3 (1.3) 3.6 (1.3) 0.2 Having practical/emotional social support, n % 348 (74.2) 18 (64.2) 0.3 EPDS, mean (SD) 8.0 (5.1) 10.6 (6.7) 0.06 EPDS total score categories, n % 0.2 < 9 score 289 (61.6) 14 (50.0) 10 to 12 score 81 (17.3) 4 (14.3) ≥13 score 99 (21.1) 10 (35.7) Note. ‘Included’ means data included for final analysis; ‘not included’ means data not included for final analysis (loss to follow up) n = number; % = percentage; SD = standard deviation; EPDS = Edinburgh Postnatal Depression Scale Note. ‘Included’ means data included for final analysis; ‘not included’ means data not included for final analysis (loss to follow up) n = number; % = percentage; SD = standard deviation; EPDS = Edinburgh Postnatal Depression Scale 20.50 g less compared to women who had low earthquake experiences via antenatal CMD symptoms. Independent of other covariates, women who had higher CMD symp- toms gave birth to babies with significantly lower birth- weight compared to women with lower CMD symptoms. Among the covariates, body mass index of women during advanced pregnancy and length of gestational age at birth were also associated consistently and independ- ently with birthweight (Table S1, S3). There is no study examining the direct and indirect ef- fect of earthquake experiences on birth weight simultan- eously to compare our findings. However, the findings of our study are in line with the findings of audits of hos- pital records that babies born after earthquakes had lower birthweight compared to those who born before the earthquakes [16, 18–21] and indicate that experien- cing an earthquake while pregnant can adversely affect Association among earthquake experiences, antenatal CMDs symptoms and birth weight BMC Pregnancy and Childbirth (2020) 20:414 Table 2 Characteristics of participants at the baseline interview Characteristics Included (n = 469) Not included (n = 28) p-value Socio-demographic characteristics Age (years), mean (SD) 26.5 (4.8) 24.9 (5.2) 0.05 Body mass index during late pregnancy, mean (SD) 27.1 (3.8) 25.4 (3.8) 0.01 Education, n % < 0.001 No formal/ primary education 86 (18.3) 17 (60.7) Secondary and above education 383 (81.7) 11 (39.3) Having income-generating work, n % 152 (32.4) 8 (28.6) 0.8 Consume chewing tobacco/smoking, n % 12 (2.6) 7 (25.0) < 0.001 Alcohol consumption, n % 118 (25.2) 7 (25.0) 1.0 Family structure, n % 0.3 Joint/extended family 234 (49.9) 11 (39.3) Nuclear family 235 (50.1) 17 (60.7) Education of partners, n % < 0.001 No formal/ primary education 51 (10.9) 13 (46.4) secondary and above education 418 (89.1) 15 (53.6) Partners with income-generating work, n % 437 (93.2) 27 (96.4) 1.0 Consume chewing tobacco/smoking by partner, n % 157 (33.5) 20 (71.4) < 0.001 Alcohol consumption by partner, n % 239 (51.0) 19 (67.9) 0.1 Household wealth, mean (SD) .09 (2.03) −1.53 (1.66) < 0.001 Intimate partner relationship Quality of relationship with an intimate partner, n % 0.002 Least optimal (< 28 RAS score) 105 (22.4) 14 (50.0) Optimal (> 28 RAS score) 364 (77.6) 14 (50.0) Any lifetime experience of intimate partner controlling behaviour, n % 163 (34.8) 15 (50.0) 0.1 Any lifetime experience of intimate partner emotional violence, n % 191 (40.7) 17 (60.7) 0.05 Any lifetime experience of intimate partner physical and or sexual violence, n % 109 (23.2) 16 (57.1) < 0.001 Any lifetime experiences of any intimate partner violence, n % 270 (57.6) 20 (71.4) 0.2 Reproductive characteristics History of pregnancy, n % Nulliparous 211 (45.0) 9 (32.1) 0.2 Two or more pregnancies 258 (55.0) 19 (67.9) History of miscarriage/abortion, n % 85 (18.1) 7 (25.0) 0.3 Taking iron tablet during current pregnancy, n % 449 (95.7) 20 (71.4) < 0.001 The good foetal health, n % 447 (95.3) 24 (85.7) 0.05 Unwelcome/inconvenient pregnancy, n % 61 (13.0) 10 (35.7) 0.003 Attended antenatal check-up for current pregnancy 466 (99.4) 25 (89.3) 0.003 Number of the antenatal visit for current pregnancy < 0.001 none 3 (0.6) 3 (10.7) once 8 (1.7) 2 (7.1) twice 15 (3.2) 5 (17.9) Three times 33 (7.0) 5 (17.9) Four or more times 410 (87.4) 13 (46.4) Sex of index foetus 0.7 Boy 262 (55.9) 14 (60.9) Khatri et al. Discussion One possible explanation for this is that earthquake experiences may increase stress in preg- nant women that negatively affect the growth and devel- opment of their foetus rather than there being a direct effect of earthquake experiences on the foetus [6, 32]. About 6% of women reported that their babies had LBW (< 2500 g), but a further 10% of women reported a rounded Fig. 2500 g. It is common practice for rounding up to occur and so it is likely that almost 16% of babies who had been exposed in utero to the earthquake were of LBW. In contrast to the national scenario, only two partici- pants had a low body mass index (< 18.5 kg/m2) in this study. Most of the study participants reported that they had received advice about nutrition and diet (96.2%) and took iron tablets (94.4%) during the current pregnancy. This could be related to the unique circumstance of the earthquake that this cohort of women may receive spe- cial attention to protect their foetus. When we used body mass index as a continuous variable to predict birthweight, it showed that with the increment of body mass index, birthweight of baby increased significantly and independently. Our study reported a higher prevalence of LBW than those of other studies conducted in an earthquake con- text. For instance, Tan et al. [19] audited birthweight of babies from hospital records before and after the 2008 Wenchuan earthquake in China. They found that 5.01% of babies born after the earthquake had LBW (≤2500 g) which was significantly higher than those were born be- fore the earthquake (3.72%). China is a middle-income country where the rate of LBW is much lower than in Nepal in general circumstances too [2]. Women in China may receive better care and services in post- earthquake management than those of Nepali women that may affect the difference of baby birthweight. y In contrast to a previous study [29], our study did not find household wealth to be a risk factor. The household wealth of this sample was relatively homogeneous, and there may have been insufficient variance to detect dif- ferences in birthweight between households with higher and lower wealth. A household wealth of women who were ‘included’ and were ‘not included’ in final analyses was significantly different. Discussion To our knowledge, this is the world’s first study in a low- income country to examine the relationship between preg- nant women’s experiences of an earthquake and the birth- weight of their infants and whether antenatal CMD symptoms mediate the effect of earthquake experiences on baby birthweight. Women who had moderate or high earthquake experiences gave birth to babies who weighed Fig. 3 Key findings of the Sobel test of the direct effect of earthquake experiences on birthweight and the indirect effect mediated by EPDS total score. Note. Single-headed solid arrows show the direction of association among variables. EPDS = Edinburgh Postnatal Depression Scale; ß = linear regression coefficient Fig. 3 Key findings of the Sobel test of the direct effect of earthquake experiences on birthweight and the indirect effect mediated by EPDS total score. Note. Single-headed solid arrows show the direction of association among variables. EPDS = Edinburgh Postnatal Depression Scale; ß = linear regression coefficient Fig. 3 Key findings of the Sobel test of the direct effect of earthquake experiences on birthweight and the indirect effect mediated by EPDS total score. Note. Single-headed solid arrows show the direction of association among variables. EPDS = Edinburgh Postnatal Depression Scale; ß = linear regression coefficient Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 9 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 This practice applies to pregnant women too. Even though there are different nutritional policies and pro- grams to combat nutritional problems [1], there is no policy or practise to ensure specific food security for pregnant women in post-earthquake management in Nepal [33]. It was reported that nutritious food was dis- tributed to pregnant women immediately after the earth- quake, but the report did not specify what nutritious food and what number of women benefited. The report did not provide details about who benefited from the supply: all women or only women in urban or in rural areas and how long the supplementation was sustained for [34, 35]. birth outcomes. Further, our study findings are in line with previous investigations of the relationship between antenatal CMDs and baby birthweight in general cir- cumstances, which have found a significant negative as- sociation [6, 14]. Discussion This suggests that ‘not in- cluded’ women were even more likely than those who were included to have a baby with LBW and that our findings were more likely to have been under- than over-estimates of the impact of the earthquake on birthweight. In Nepal context, our study reported a higher preva- lence of LBW than that reported in the recent Nepal Demographic and Health Survey (NDHS) 2016 (12% baby had LBW < 2500 g) [1]. Our study site was in Bhak- tapur district, which was relatively well resourced, with more accessible health services than in other districts in Nepal. The earthquake created a unique circumstance. There were anecdotal records from hospital staff that women heard informally that earthquakes might have a negative impact on foetal health and growth and ‘many women’ sought termination of their pregnancies. Women who elected to continue their pregnancies were reported to be cautious about foetal wellbeing, and they presented for antenatal check-ups (Table 2) at a much higher rate than the national average [1]. Despite this healthcare, LBW was still very high. Our study reported a small number of women (4.1%) had a preterm birth (< 37 weeks). This could be because we missed the group at high risk of preterm birth or who sought an abortion before we approached them since the data collected six months after the major earthquake. Because of the small number of preterm birth, length of gestation at birth was used as a continu- ous variable to predict birthweight. Consistent with pre- vious studies [2, 7, 36], our data revealed that the higher the length of gestation at birth, the higher the weight of babies. This study confirms previous findings [2, 4] that ma- ternal body mass index is associated significantly with baby birthweight. While malnourishment among women in reproductive age in Nepal is high, women rarely con- sume additional nutritious food during pregnancy [1, 7]. According to NDHS 2016, 17.3% of women of repro- ductive age had a low body mass index (< 18.5 kg/m2), an indicator of undernutrition [1]. In Nepal, women, generally feed other family members first and then they consume any remaining food at the end of the meal. Implications and conclusions The study has brought to attention the public health im- portance of the experiences of earthquakes for women who are pregnant not only for their own health but also because of the impact on their baby’s birthweight. These findings indicate that antenatal CMDs among women should be a focus of post-disaster interventions not only because of the burden [5] imposed on women’s partici- pation but also because of the risk for the growth and development of their foetuses [3, 6]. In Nepal, menstruation is considered unclean, and it is culturally prohibited for women to attend everyday reli- gious observations or sacred ceremonies or to go into the home kitchen or to prepare food when they are menstruating. These are publicly observable circum- stances and readily linked to specific ceremonies or events, and so it is easy for women in Nepal to recall the first day of the last menstrual period. It is generally be- lieved that they rarely recall the date inaccurately. Nevertheless, we acknowledge that the date of the last menstrual period was self-reported. Although we think this is unlikely, it might not have been recalled precisely and have led to an inaccurate estimation of the length of gestation at birth. In our study, a very few women had a low body mass index and preterm birth, which are well-established risk factors of LBW. Despite that our study reported a high prevalence of LBW. This indicates that women’s mental health problems were a key factor for this high preva- lence of LBW. While most participants reported that they received antenatal check-ups, they had a high prevalence of symptoms of antenatal CMDs [11], none of which were detected or treated. Maternal and child health is a na- tional priority in Nepal, and the country has made pro- gress to reduce maternal and child morbidity and mortality in recent decades. However, rates are still very high compared to other countries [1]. In Nepal, there is no specific programs and intervention for the mental health of women in the perinatal period, but this could assist in the reduction of maternal and child morbidity and mortality. These data clearly indicate that specialised consideration of perinatal mental health is a grave need in the post-disaster context in Nepal. We acknowledge that mediation analysis is a sophisti- cated statistical technique that requires assumptions. Strengths The study was a population-based prospective study. Population-based studies permit the findings to be gen- eralised to equivalent settings. We were able to do a Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 10 of 12 Page 10 of 12 Page 10 of 12 validated against a gold standard comparator for use among pregnant women [28]. postpartum follow-up as a prospective study. Prospective investigations are able to ascertain causal relationships including, in this study, between earthquake experiences, pregnant women’s mental health and birthweight. This is a robust design for mediation analyses [28]. The study was adequately powered to detect the direct and indirect effects of the earthquake on birthweight. The study was powered to detect within-group vari- ation of earthquake experiences on birthweight, but as the earthquake affected the entire country, it was not possible to recruit a comparison group of women who had no experience of the earthquakes. Data were col- lected about six months after the earthquake. Therefore, the sample did not include women who had already given birth, including those who had premature births. We were told that some women had sought abortions immediately after the earthquakes because they feared that there might have been adverse effects on the foetus. This could lead to underestimates of the impact of the earthquake on infant birthweight. Nevertheless, the study included all eligible women who met inclusion cri- teria during the study period. The strengths of this study outweigh the limitations and can be generalised with some confidence. There were very high recruitment and retention frac- tions. Data were collected in individual interviews which enabled women with low literacy to participate. We con- trolled for other known risk and protective factors for baby birthweight and antenatal CMD symptoms to con- trol for potential confounding effects. Limitations We acknowledge the limitation of the study that baby birthweight was based on the birth certificates provided by health institutions rather than being measured by the research team. Most birthweights were recorded on birth certificates as figures that had been rounded up; it is possible that the percentage of LBW was underesti- mated. For this reason, we used birthweight as a con- tinuous measure in outcome analyses. The earthquake experience measurement scale was adapted from one used in previous studies and was carefully pilot tested, but it was not standardised or locally validated. Acknowledgements We are indebted to the research participants who contributed their time and trusted us with their intimate experiences despite the major disruption of the earthquakes. We are very grateful to the six HERD health researchers for their courage and dedication to collecting high-quality data despite the ad- verse circumstances in Nepal at the time of data collection. We would like to acknowledge the important contributions of the Female Community Health Volunteers (FCHV), the health staff of Bhaktapur district, the officers of the Women and Children Development Office Bhaktapur, and members of the community for their valuable support during data collection. We would like to acknowledge the organizations that supported the authors by providing scholarship and fellowships. 6. Grote NKP, Bridge JAP, Gavin ARP, Melville JLMD, Iyengar SP, Katon WJMD. A meta-analysis of depression during pregnancy and the risk of preterm birth, low birth weight, and intrauterine growth restriction. Arch Gen Psychiatry. 2010;67(10):1012. 6. Grote NKP, Bridge JAP, Gavin ARP, Melville JLMD, Iyengar SP, Katon WJMD. A meta-analysis of depression during pregnancy and the risk of preterm birth, low birth weight, and intrauterine growth restriction. Arch Gen Psychiatry. 2010;67(10):1012. 7. Sharma SR, Giri S, Timalsina U, Bhandari SS, Basyal B, Wagle K, Shrestha L: Low birth weight at term and its determinants in a tertiary hospital of Nepal: A case-control study. PLoS One 2015, 10(4). 8. Anwar J, Mpofu E, Matthews LR, Brock KE. Risk factors of posttraumatic stress disorder after an earthquake disaster. J Nerv Ment Dis. 2013;201(12): 1045–52. 9. Qu Z, Tian D, Zhang Q, Wang X, He H, Zhang X, Huang L, Xu F. The impact of the catastrophic earthquake in China's Sichuan province on the mental health of pregnant women. J Affect Disord. 2012;136(1–2):117–23. Funding GKK is supported by an Australia Award PhD scholarship, TT is supported by a National Health and Medical Research Council Early Career Fellowship, JF is supported by Finkel Professorial Fellowship which receives funding from the Finkel Family Foundation. The funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The authors received no separate funding for this project. Abbreviations 4. Wadhwa PD, Entringer S, Buss C, Lu MC. The contribution of maternal stress to preterm birth: issues and considerations. Clin Perinatol. 2011;38(3):351–84. CMD: Common mental disorder; EPDS: The Edinburgh Postnatal Depression Scale; LBW: Low birthweight CMD: Common mental disorder; EPDS: The Edinburgh Postnatal Depression Scale; LBW: Low birthweight 5. Fisher J, de Mello MC, Patel V, Rahman A, Tran T, Holton S, Holmes W. Prevalence and determinants of common perinatal mental disorders in women in low-and lower-middle-income countries: a systematic review. Bull World Health Organ. 2012;90(2):139–49. Implications and conclusions The design we used in this study, a prospective cohort, can help us to be confident that pregnancy mental health is a mediator rather than a confounder or an effect-modifier. Nevertheless, the associations between exposure, mediator and the outcome can be confound- ing or effect modification. Secondly, unmeasured vari- ables including ‘post-traumatic growth’ may have a counter effect on mental health problems that may influ- ence the mediation effect of mental health problems on birthweight. Also, there is the possibility of measurement errors of earthquake experiences and pregnancy mental health because the scales were not locally formally Even though it is challenging to conduct ethical, com- prehensive and culturally-sensitive studies after a natural disaster, it is essential to generate evidence to inform ef- fective post-disaster interventions. These data Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 11 of 12 Page 11 of 12 demonstrate that it is feasible to generate robust evidence in this situation. for data collection and assisted with data management. All authors (GKK, TT, SB and JF) read and approved the manuscript. for data collection and assisted with data management. All authors (GKK, TT, SB and JF) read and approved the manuscript. In conclusion, this study has addressed an important knowledge gap. These findings provide evidence about the importance of women’s antenatal mental health in post-disaster management which is currently missing from the World Health Organization’s guideline “Key Steps for Maternal and Newborn Health Care in Hu- manitarian Crisis” [37]. The data inform enhancements to existing maternal and child health programs in Nepal and will strengthen the country’s capacity to meet the Sustainable Development Goals [38]. Author details 1 f Author details 1School of Public Health and Preventive Medicine, Monash University, Level 4, 553 St Kilda Rd, Melbourne, Victoria 3004, Australia. 2HERD International, Kathmandu 44600, Nepal. Additional file 2 Supplementary Table 2 (S2) Model 2 in Sobel test analysis. Multiple linear regression model predicting the symptoms of CMDs from earthquake experiences and other covariates Additional file 2 Supplementary Table 2 (S2) Model 2 in Sobel test analysis. Multiple linear regression model predicting the symptoms of CMDs from earthquake experiences and other covariates Received: 10 July 2018 Accepted: 2 July 2020 Ethics approval and consent to participate Ethics approvals for this study were obtained from Monash University Human Research Ethics Committee (CF15/2776–2015001130) on 19th August 2015 and Nepal Health Research Council (219/2015) on 4th October 2015. Written informed consent was obtained from all participants. References O Additional file 4 Supplementary Table 4 (S4) Key results of mediation analysis. The Sobel test of the effect of earthquake experiences on birthweight mediated by symptoms of CMDs Additional file 4 Supplementary Table 4 (S4) Key results of mediation analysis. The Sobel test of the effect of earthquake experiences on birthweight mediated by symptoms of CMDs 1. MOH, ERA N, International I: Nepal demographic health and population survey 2016. In. Nepal; 2017. 2. UNICEF, WHO: Low birth weight: country, regional and global estimates. 2004. Additional file 5. Questionnaires. English version of the set of Questionnaires that used to collect primary data Additional file 5. Questionnaires. English version of the set of Questionnaires that used to collect primary data 3. Walker SP, Wachs TD, Meeks Gardner J, Lozoff B, Wasserman GA, Pollitt Carter JA. Child development: risk factors for adverse outcomes in developing countries. Lancet. 2007;369(9556):145–57. Consent for publication Not applicable. Consent for publication Not applicable. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12884-020-03086-5. Received: 10 July 2018 Accepted: 2 July 2020 Additional file 3 Supplementary Table 3 (S3) Model 3 in Sobel test analysis. Multiple linear regression model predicting birthweight from the earthquake experiences, the symptoms of CMDs and other covariates Additional file 3 Supplementary Table 3 (S3) Model 3 in Sobel test analysis. Multiple linear regression model predicting birthweight from the earthquake experiences, the symptoms of CMDs and other covariates Availability of data and material The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. 10. Ren J, Jiang X, Yao J, Li X, Liu X, Pang M, Chiang CLV: Depression, social support, and coping styles among pregnant women after the Lushan earthquake in Ya'an, China. PLoS ONE, ArtID e0135809 10 2015, 10(8):1–19. Competing interests None declared. Additional file 1 Supplementary Table 1 (S1) Model 1 in Sobel test analysis. Multiple linear regression model predicting birthweight from earthquake experiences and other covariates (not including the symptoms of CMDs, the mediator) Additional file 1 Supplementary Table 1 (S1) Model 1 in Sobel test analysis. Multiple linear regression model predicting birthweight from earthquake experiences and other covariates (not including the symptoms of CMDs, the mediator) 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. 19. Tan CE, Li HJ, Zhang XG, Zhang H, Han PY, An Q, Ding WJ, Wang MQ. The impact of the Wenchuan earthquake on birth outcomes. PLoS One. 2009;4(12):e8200. 20. Chang HL, Chang TC, Lin TY, Kuo SS. Psychiatric morbidity and pregnancy outcome in a disaster area of Taiwan 921 earthquake. Psychiatry Clin Neurosci. 2002;56(2):139–44. 21. Harville EW, Do M. 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The EpiData Association: Odense, Denmark; 2005. 26. StataCorp: Stata: Release 14 Statistical Software. In., vol. Special edition, 14 edn. College Station, TX: StataCorp LP; 2015. 26. StataCorp: Stata: Release 14 Statistical Software. In., vol. Special edition, 14 edn. College Station, TX: StataCorp LP; 2015. 27. Hayes AF. Beyond baron and Kenny: statistical mediation analysis in the new millennium. Commun Monogr. 2009;76(4):408–20. 27. Hayes AF. Beyond baron and Kenny: statistical mediation analysis in the new millennium. Commun Monogr. 2009;76(4):408–20. g 28. Pek J, Hoyle RH. On the (in) validity of tests of simple mediation: threats and solutions. Soc Personal Psychol Compass. 2016;10(3):150–63. g 28. Pek J, Hoyle RH. On the (in) validity of tests of simple mediation: threats and solutions. Soc Personal Psychol Compass. 2016;10(3):150–63. and solutions. Soc Personal Psychol Compass. 2016;10(3):150–63 29. Blumenshine P, Egerter S, Barclay CJ, Cubbin C, Braveman PA. Socioeconomic disparities in adverse birth outcomes: a systematic review: a systematic review. Am J Prev Med. 2010;39(3):263–72. 30. Murphy CC, Schei B, Myhr TL, Du Mont J. Abuse: a risk factor for low birth weight? Authors’ contributions GKK JF d TT Prevalence of intimate partner violence: findings from the WHO multi-country study on women's health and domestic violence. Lancet. 2006;368(9543):1260–69. 17. Kim B, Carruthers CK, Harris MC: Maternal stress and birth outcomes: Evidence from the 1994 Northridge earthquake. J Econ Behav Organ 2017, 140:354–373. 18. Oyarzo C, Bertoglia P, Avendaño R, Bacigalupo F, Escudero A, Acurio J, Escudero C. Adverse perinatal outcomes after the February 27th 2010 Chilean earthquake. J Matern Fetal Neonatal Med. 2012;25(10):1868–73. Authors’ contributions GKK JF d TT GKK, JF and TT conceptualized and designed the study and selected the methods. 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J Marriage Fam. 1976;38(1):15–28. 15. Torche F. The effect of maternal stress on birth outcomes: exploiting a natural experiment. Demography. 2011;48(4):1473–91. 42. Hendrick SS, Dicke A, Hendrick C. The relationship assessment scale. J Soc Pers Relatsh. 1998;15(1):137–42. 16. Bahmanjanbeh F, Kohan S, Yarmohammadian MH, Haghshenas A. Evaluation of reproductive health indicators in women affected by East Azarbaijan earthquake on august 2012. Iran J Nurs Midwifery Res. 2016;21(5):504–9. 43. Garcia-Moreno C, Jansen HA, Ellsberg M, Heise L, Watts CH. Publisher’s Note A systematic review and meta-analysis. CMAJ Canadian Medical Association Journal. 2001;164(11):1567–72. 31. Nepal M, Sharma V, Koirala B, Khalid A, Shrestha P. Validation of the Nepalese version of Edinburgh postnatal depression scale in tertiary health care facilities in Nepal. Nepalese J Psychiatry. 1999;1(1):46–50. 31. Nepal M, Sharma V, Koirala B, Khalid A, Shrestha P. Validation of the Nepalese version of Edinburgh postnatal depression scale in tertiary health care facilities in Nepal. Nepalese J Psychiatry. 1999;1(1):46–50. 32. Ren JH, Chiang CLV, Jiang XL, Luo BR, Liu XH, Pang MC. Mental disorders of pregnant and postpartum women after earthquakes: a systematic review. Disaster Med Public Health Preparedness. 2014;8(4):315–25. 32. Ren JH, Chiang CLV, Jiang XL, Luo BR, Liu XH, Pang MC. Mental disorders of pregnant and postpartum women after earthquakes: a systematic review. Disaster Med Public Health Preparedness. 2014;8(4):315–25. 33. DOHS: Annual report Department of Health Services 207 34. GoN: Response of the government of Nepal to the list of issues and questions in relation to the sixth periodic report of Nepal adopted by the committee on the convention on the elimination of all forms of discrimination against women In. Nepal; 2018. 34. 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  Citation: Kumar, R.; Srivastava, A.; Lakhani, R. Industrial Wastes- Cum-Strength Enhancing Additives Incorporated Lightweight Aggregate Concrete (LWAC) for Energy Efficient Building: A Comprehensive Review. Sustainability 2022, 14, 331. https:// doi.org/10.3390/su14010331 Academic Editors: Elena Michelini, Daniele Ferretti and Syed Minhaj Saleem Kazmi Keywords: lightweight aggregate concrete; industrial waste; thermal insulation; energy-efficient building; sustainability Industrial Wastes-Cum-Strength Enhancing Additives Incorporated Lightweight Aggregate Concrete (LWAC) for Energy Efficient Building: A Comprehensive Review Rajesh Kumar Rajesh Kumar * , Abhishek Srivastava and Rajni Lakhani Rajesh Kumar * , Abhishek Srivastava and Rajni Lakhani Organic Building Materials (OBM) Group, CSIR-Central Building Research Institute, Roorkee 247667, Uttarakhand, India; ashish27.srivastav@gmail.com (A.S.); rlakhani@cbri.res.in (R.L.) * Correspondence: rajeshkumar@cbri.res.in or rk2896315@gmail.com Abstract: Lightweight aggregate concrete (LWAC) exhibits the advantages of thermal insulation, reduces energy consumption building costs, improves building efficiency and easy construction. Furthermore, the utilization of industrial wastes in concrete is advantageous in terms of environ- mental sustainability. In order to explore this, several researchers investigated the idea of integrating industrial wastes in LWAC. However, the lack of knowledge regarding the performance of industrial waste-based lightweight aggregate concrete hinders the adaptation of this concept and application of LWAC in the construction sector. Therefore, this paper summarizes the research in relation to the sustainable LWACs containing oil palm shell (OPS), lightweight expanded clay aggregate (LECA), vermiculite, perlite, pumice and sintered fly ash as lightweight aggregate, along with industrial wastes and strength-enhancing additives (viz. fibers, polymers, etc.). Firstly, desirable physical, chemical, morphological and mineralogical characterization of different lightweight aggregates are presented, and then a comprehensive overview on fresh, hardened, durability and thermal prop- erties of LWAC incorporating industrial wastes are discussed in comparison with normal weight concrete. The review also highlights the current challenges and suggests the research gaps for further development of eco-friendly LWAC. It is concluded that vermiculite, perlite, pumice, OPS, sintered fly ash and LECA with some suitable industrial waste materials have the potential to be used in the construction sector. Moreover, LWAC with industrial waste has 50–65% lower carbon emission (kg CO2 eq/m3) in the environment. The scientific contribution of this paper provides insights into different LWACs and the knowledge base for future research and paradigm shift of using LWACs as more common alternative building materials. sustainability sustainability sustainability Figure 2 Location-wise research trend Figure 2. Location-wise research trend. Figure 2. Location-wise research trend. Figure 2 Location-wise research trend Figure 2. Location-wise research trend. Figure 2. Location-wise research trend. The use of lightweight aggregates (LWA) in concrete has the following advantages: The use of lightweight aggregates (LWA) in concrete has the following advantages: The use of lightweight aggregates (LWA) in concrete has the following advantages: The use of lightweight aggregates (LWA) in concrete has the following advantages: The use of lightweight aggregates (LWA) in concrete has the following advantages The use of lightweight aggregates (LWA) in concrete has the following advantages: The use of lightweight aggregates (LWA) in concrete has the followin 1 Less dead load structure stability as well as economic viability [2]; The use of lightweight aggregates (LWA) in concrete has the follow 1 Less dead load structure stability as well as economic viability [2]; 1. Less dead load, structure stability as well as economic viability [2]; 1. Less dead load, structure stability as well as 2 Cost reduction [3]; 1. Less dead load, structure stability as well as 2 Cost reduction [3]; 2. Cost reduction [3]; . Cost reduction [3]; Relati ely lo the al o du ti ity [4 6] 2. Cost reduction [3]; 3 Relatively low thermal conductivity [4 6]; 3. Relatively low thermal conductivity [4–6]; 3. Relatively low thermal conductivity [4–6]; 4 H li ti i t d t hi h i b ildi t [7 8] 3. Relatively low thermal conductivity [4–6]; 4 Has application in prestressed concrete high rise buildings etc [7 8 4. Has application in prestressed concrete, high rise buildings, etc. [7,8] 4. Has application in prestressed concrete, high rise buildings, etc. [7,8]; 5 I th k bilit if tt d i t i t [9] 4. Has application in prestressed concrete, high rise buildings, etc. [7,8] 5 Improves the workability if pre wetted prior to use in concrete [9] 5. Improves the workability if pre-wetted prior to use in concrete [9]. Has application in prestressed concrete, high rise buildings, etc. [7,8]; I th k bilit if tt d i t i t [9] Has application in prestressed concrete, high rise buildings, etc. [7,8]; Improves the workability if pre wetted prior to use in concrete [9] Improves the workability if pre-wetted prior to use in concrete [9]. 5. Improves the workability if pre-wetted prior to use in concrete [9]. Figure 2 Location-wise research trend Figure 2. Location-wise research trend. Figure 2. Location-wise research trend. However, lower absolute mechanical strength, higher water absorption, porosity, etc., are some of the negative impacts. LWAC is an alternative solution to NWC, especially when lightweight energy-efficient solutions are needed. Thus, to overcome the problem of natural stone deterioration and for making energy-efficient buildings, different light- weight concretes are used. Chart 1 shows the classification of LWAC. It can be ascertained from the flow chart that LWAC can be used for load-bearing and non-load-bearing pur- poses. Different LWAs commonly used in making LWAC are vermiculite, OPS, perlite, LECA, pumice, scoria, tuff, cinder, lytag, etc. (Koksal et al. [1]). LWAs are a type of coarse/fine aggregates used for the manufacturing of LWC products, and these products are used in different structural work (Alengaram et al. [2]). LWAs have a cellular type structure and were used for producing different types of masonry blocks, wall panels, cladding and LWC. When they are used as fine aggregates, they function similar to active 5. Improves the workability if pre wetted prior to use in concrete [9]. However, lower absolute mechanical strength, higher water absorption, porosity, etc., are some of the negative impacts. LWAC is an alternative solution to NWC, especially when lightweight energy-efficient solutions are needed. Thus, to overcome the problem of natural stone deterioration and for making energy-efficient buildings, different light- weight concretes are used. Chart 1 shows the classification of LWAC. It can be ascertained from the flow chart that LWAC can be used for load-bearing and non-load-bearing pur- poses. Different LWAs commonly used in making LWAC are vermiculite, OPS, perlite, LECA, pumice, scoria, tuff, cinder, lytag, etc. (Koksal et al. [1]). LWAs are a type of coarse/fine aggregates used for the manufacturing of LWC products, and these products are used in different structural work (Alengaram et al. [2]). LWAs have a cellular type structure and were used for producing different types of masonry blocks, wall panels, cladding and LWC. When they are used as fine aggregates, they function similar to active However, lower absolute mechanical strength, higher water absorption, porosity, etc., are some of the negative impacts. LWAC is an alternative solution to NWC, especially when lightweight energy-efficient solutions are needed. Thus, to overcome the problem of natural stone deterioration and for making energy-efficient buildings, different lightweight concretes are used. Chart 1 shows the classification of LWAC. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. In the 21st century, energy-efficient buildings are one of the important issues, which include both techno-economic and sustainable environmental factors [1]. Energy-efficient buildings are designed to use energy as little as possible. Many developing countries are leading towards the construction of green buildings using cost-effective, durable concrete. The huge demand for concrete in the infrastructural development using normal-weight aggregates (NWAs) has reduced the natural stone deposits, which causes irreplaceable damage to our environment. As a result, the priority of searching for sustainable materials has been enhanced worldwide. LWAC is one such alternative to normal weight aggregate concrete (NWC) with various physical, mechanical, social and economic advantages [2]. Figures 1 and 2 show the current trend of research (year-wise and location-wise) in the area of lightweight aggregate concrete (Source: https://www.scopus.com/; accessed on 12 September 2021). The trend shows that day by day, the practical use of LWACs is increasing and thus requires the current status of the research conducted to date. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sustainability Sustainability 2022, 14, 331. https://doi.org/10.3390/su14010331 2 of 21 creas- ncreas- Sustainability 2022, 14, 331 Figure 1. Year-wise research trend. Figure 2 Location wise research trend Figure 1. Year-wise research trend. Figure 1. Year-wise research trend. Figure 2 Location-wise research trend Figure 2. Location-wise research trend. Figure 1. Year-wise research trend. Figure 1. Year-wise research trend. Figure 1. Year-wise research trend. h d 2. Types of LWAs In general, LWAs can be divided into two categories. Chart 1. Classification of lightweight concretes. Chart 1. Classification of lightweight concretes. The cellular structure within the particles is formed at high temperatures, generally at 1100 ◦C or higher. Due to the cellular structure of particular aggregate particles, the aggregates are light in weight, and the specific gravity, as well as the unit weight of LWAs, is lower than that of NWAs. The maximum size grading designations of LWAs generally available are 19 mm, 13 mm or 10 mm. LWAs have more water absorption (up to 5 to 20%) and allow limited access for fresh cement paste into the open pores of the LWAs. Due to their porous structure, LWAs require to be wet for 24 h before adding it into the mix. In the case of LWC, the bond between the aggregate and the matrix is stronger than the normal concrete. LWC provides a reduction in dead loads and improves the thermal and fire properties of buildings. It is necessary to limit the slump to improve the workability, durability and avoid segregation. In LWC, excessive mixing should be avoided because it tends to crack the LWA particles (ACI 213 [10]). p The paper presents the wide classification of different kind of LWAs that are used to develop LWACs and discuss their physio-chemical and morphological characterization. Additionally, fresh, hardened, functional and durable properties of sustainable LWAC are presented along with the effect of different SCMs and fibers to improve the performance of LWACs. Furthermore, ongoing research activities at CSIR-CBRI on the effect of marble slurry and Class-F fly ash on LECA incorporated LWAC is also discussed. Figure 2 Location-wise research trend Figure 2. Location-wise research trend. Figure 2. Location-wise research trend. It can be ascertained from the flow chart that LWAC can be used for load-bearing and non-load-bearing purposes. Different LWAs commonly used in making LWAC are vermiculite, OPS, perlite, LECA, pumice, scoria, tuff, cinder, lytag, etc. (Koksal et al. [1]). LWAs are a type of coarse/fine aggregates used for the manufacturing of LWC products, and these products are used in different structural work (Alengaram et al. [2]). LWAs have a cellular type structure and were used for producing different types of masonry blocks, wall panels, cladding and LWC. When they are used as fine aggregates, they function similar to active pozzolanic materials. It can be produced from the naturally available raw materials such as expanded clay, shale, slate, etc., as well as from SCMs such as FA and slags, etc. 3 of 21 marble Sustainability 2022, 14, 331 Chart 1. Classification of lightweight concretes. Chart 1. Classification of lightweight concretes. Chart 1. Classification of lightweight concretes. Chart 1. Classification of lightweight concretes. 2.1. Natural Aggregate Natural LWAs are the materials that are available as natural resources. These are naturally ready to use with mechanical treatment, i.e., crushing and sieving. Mostly, natural LWAs are of volcanic origin, e.g., pumice and scoria [11,12]. Thus, they are only found in a few areas of the world. Pumice is formed when the molten lava from the explosive eruption of a volcano cools [13]. Quick cooling freezes the material existing at the molten Sustainability 2022, 14, 331 4 of 21 state, which does not have zero probability of a crystallization process. Scoria is darker in color than pumice but has all the properties same as that of pumice. Volcanic tuffs are a kind of volcanic rock, which develops pores through rapid cooling and hardening of lava. The pore may vary from 10% to 60%. These tuffs are classified as rhyolite, dacite, andesite, etc. Similarly, volcanic slag is also derived from lava, which is less vitreous and more crystalline slag-like materials. These all-natural LWAs are used to produce LWC with a density range from 1860 to 1988 kg/m3 [14–16]. 2.2. Manufactured/Artificial Aggregate These LWAs are classified as brick rubble, cinder, sintered cinder, blast-furnace slag, LECA, etc. These types of aggregates are produced by thermal treatment of either naturally occurring materials such as clay, shale, vermiculite, perlite and slate, etc., or industrial byproducts/waste materials such as FA, municipal solid waste (MSW), waste of dredging, blast furnace slag, etc. Cinder LWAs are produced as coal burnt residues in the industrial boilers. The residue is melted and sintered to form cinders. Sintered fly ash is developed at a temperature of 1100–1300 ◦C [17]. They have a hard, coarse red shell and fine pore structures, which are commercially available in a size range of 7 to 30 mm. Similarly, foamed blast-furnace slag aggregates are produced by sintering slag at a temperature of 1400–1600 ◦C. The size range lies between less than 3 mm and 20 mm, with a varying bulk density of 300 to 700 kg/m3. LECA is produced using clay or shale after heating at the point of incipient vitrification (at a temperature of about 1200 ◦C) [18]. After exposure, clay/shale expands or bloats to seven times their original volume and forms a cellular structure that remains stable even after cooling. In the market, different kind of LECA is available by different names such as hydite, rocklite, lytag, aglite, keramzit, etc. In a similar manner, expanded perlite is developed after exposing perlite at a temperature of 900–1100 ◦C, which leads to an increase in volume by 15–20 times [19]. Vermiculite is a type of mica with high magnesium content, which is formed at a temperature of 900 ◦C [20]. 3. Different LWAs and Their Use to Develop Green Building Materials During the extracting process of oil from the oil palm tree, OPS are generated, which is a waste product. In order to protect the environment, action was taken to utilize OPS as LWAs. The dumped oil palm kernel shells left at the factory yard are shown in Figure 3. Figure 4 shows that OPS aggregate has an irregular flaky, angular shape. Sustainability 2022, 14, x FOR PEER REVIEW 6 of 22 Table 2. Chemical Composition of LWAs. Table 2. Chemical Composition of LWAs. Table 2. Chemical Composition of LWAs. Type of Aggregate References Chemical Components (%) Main Minor SiO2 Al2O3 Fe2O3 CaO MgO SO3 K2O Na2O LOI OPS Shafigh et al. [14]; Foong et al. [38] 18.47–21.32 4.27–6.20 2.06–3.62 64.09–65.41 2.08–2.43 4.25–5.50 0.28–0.73 0.21–0.25 1.41–1.80 LECA Al-Bahar and Bogahawatta [39]; Sajedi and Shafigh [40]; Masoud et al. [41] 53.3–66.05 15.05–19.78 6.2–9.52 1.05–2.98 0.78–3.67 0.23–0.25 2.55–4.1 0.17–1.54 1.37–15.11 Vermiculite Koksal et al. [1]; Abidi et al. [42]; Sayadi et al. [43] 36.9–46 10–17.7 5.50–11.2 1–3.5 16–35 0.02–0.10 1–6 0.13–0.2 7.5–9.2 Perlite Turkmen and Kantarci [44] 71–76 9.91–16 0.40–1.57 0.5–2.19 0.01–0.28 0.04–0.10 4–5 2.9–4 1.48–2.0 Pumice Demirel and Kelestemur [45]; Binici et al. [35]; Onoue et al. [46] 41.41–63.4 12.97–21.9 1.26–11.41 1.8–13.73 0.3–15 0.44–0.50 1.73–5.40 1.80–5.20 1.60–7.32 Sintered Fly ash Kayali [47]; Kockal and Ozturan [15]; Guneyisi et al. [36] 56.2–64.60 19.58–28.5 4.0–7.23 0.54–4.24 0.66–4.64 0.30–0.69 0.01–5.95 0.32–2.06 0.49–5.10 3. Different LWAs and Their Use to Develop Green Building Materials There are different types of LWAs that are used in the construction of buildings [21]. Some vitally used LWAs are vermiculite, OPS, perlite, LECA, pumice and sintered fly ash. Tables 1 and 2 summarize the physical and chemical characterization results obtained by several researchers. Table 1. Physical properties of LWAs. Type of Aggregate References Specific Gravity Water Absorption for 24 h (%) Fineness Modulus (FM) Bulk Density (Compacted) (kg/m3) OPS Mannan and Ganapathy [22]; Teo et al. [4]; Shafigh et al. [14]; Sobuz et al. [23]; Eziefula et al. [21] 1.17–1.30 33.0–19.6 5.64–6.24 572–656 LECA Maghsoudi et al. [5]; Real et al. [24]; Bogas and Cunha [11]; Shafigh et al. [25] 0.51–1.18 16.42–26.5 15.8–5.96 273–667 Vermiculite Schackow et al. [26]; Divya et al. [27]; Arun et al. [28] 1.10–3.0 2.65 2.46 - Perlite Demirbog and Gul [29]; Karakoc and Demirboga [19]; Oktay et al. [30]; Zulkifeli and Saman [31] 0.12–0.42 82.5 - 200 Pumice Hossain [32]; Sari and Pasamehmetoglu [33]; Gunduz and Ugur [34]; Binici et al. [35]; Kockal and Ozturan [15] 1.57–3.10 - - 870 Sintered Fly ash Guneyisi et al. [36]; Gomathi and Sivakumar [37] 2.10–2.25 0.14 6.24 - Table 1. Physical properties of LWAs. 5 of 21 Sustainability 2022, 14, 331 Table 2. Chemical Composition of LWAs. Type of Aggregate References Chemical Components (%) Main Minor SiO2 Al2O3 Fe2O3 CaO MgO SO3 K2O Na2O LOI OPS Shafigh et al. [14]; Foong et al. [38] 18.47–21.32 4.27–6.20 2.06–3.62 64.09–65.41 2.08–2.43 4.25–5.50 0.28–0.73 0.21–0.25 1.41–1.80 LECA Al-Bahar and Bogahawatta [39]; Sajedi and Shafigh [40]; Masoud et al. [41] 53.3–66.05 15.05–19.78 6.2–9.52 1.05–2.98 0.78–3.67 0.23–0.25 2.55–4.1 0.17–1.54 1.37–15.11 Vermiculite Koksal et al. [1]; Abidi et al. [42]; Sayadi et al. [43] 36.9–46 10–17.7 5.50–11.2 1–3.5 16–35 0.02–0.10 1–6 0.13–0.2 7.5–9.2 Perlite Turkmen and Kantarci [44] 71–76 9.91–16 0.40–1.57 0.5–2.19 0.01–0.28 0.04–0.10 4–5 2.9–4 1.48–2.0 Pumice Demirel and Kelestemur [45]; Binici et al. [35]; Onoue et al. [46] 41.41–63.4 12.97–21.9 1.26–11.41 1.8–13.73 0.3–15 0.44–0.50 1.73–5.40 1.80–5.20 1.60–7.32 Sintered Fly ash Kayali [47]; Kockal and Ozturan [15]; Guneyisi et al. [36] 56.2–64.60 19.58–28.5 4.0–7.23 0.54–4.24 0.66–4.64 0.30–0.69 0.01–5.95 0.32–2.06 0.49–5.10 3.1. OPS Concrete (OPSC) OPS are considered as agricultural waste and lighter in weight, which is found in abundance all over the world. Malaysia is one of the largest oil palm producers in the world, which produces 4 MT of OPS annually [2]. 3.1. OPS Concrete (OPSC) OPS are considered as agricultural waste and lighter in weight, which is found in abundance all over the world. Malaysia is one of the largest oil palm producers in the world, which produces 4 MT of OPS annually [2]. During the extracting process of oil from the oil palm tree, OPS are generated, which is a waste product. In order to protect the environment, action was taken to utilize OPS as LWAs. The dumped oil palm kernel shells left at the factory yard are shown in Figure 3. Figure 4 shows that OPS aggregate has an irregular flaky, angular shape. REVIEW 6 of 22 Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). 6 of 21 m et al Sustainability 2022, 14, 331 Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). The concrete with compressive strength of more than 25 MPa was achieved using OPS as a lightweight coarse aggregate. The construction of low-cost buildings can be per- formed using OPS concrete. Mannan and Ganapathy [22] compared the different proper- ties of OPS concrete with control concrete. It was found that the 28-day compressive strength, flexural strength and splitting tensile strength of OPS concrete were between 20 and 24 MPa, 2.75 and 4.00 MPa and 1.78 and 2.41 MPa, respectively. The 28-day elasticity modulus of OPS concrete was 0.70–0.76 × 104 MPa. OPS concrete showed 14% higher dry- ing shrinkage than control concrete at 90 days, and it also had higher water absorption. Mannan and Ganapathy [48] studied the different properties of concrete using OPS as a coarse aggregate and fly ash (FA). 3.1. OPS Concrete (OPSC) It was found that the OPS concrete containing FA showed lower air content compared to control concrete without FA. The density of the fresh concrete of OPS concrete lies between 1910 and 1958 kg/m3. The 28-day compressive strength of samples was in the range of 20.1–24.2 MPa, which satisfies the requirement of structural LWC. Olanipekun et al. [3] studied the strength characteristics as well as the cost analysis of concrete made by using OPS and crushed granular coconut shells. The level of replacement for LWA with coarse aggregate was in the range of 25 to 100%. The results show that by increasing fractions of OPS, the uniaxial compressive strength of LWA concrete was decreased. A cost reduction between 30% and 42% could be achieved using OPS as a coarse aggregate in concrete production. The concrete with compressive strength of more than 25 MPa was achieved using OPS as a lightweight coarse aggregate. The construction of low-cost buildings can be performed using OPS concrete. Mannan and Ganapathy [22] compared the different properties of OPS concrete with control concrete. It was found that the 28-day compressive strength, flexural strength and splitting tensile strength of OPS concrete were between 20 and 24 MPa, 2.75 and 4.00 MPa and 1.78 and 2.41 MPa, respectively. The 28-day elasticity modulus of OPS concrete was 0.70–0.76 × 104 MPa. OPS concrete showed 14% higher drying shrinkage than control concrete at 90 days, and it also had higher water absorption. Mannan and Ganapathy [48] studied the different properties of concrete using OPS as a coarse aggregate and fly ash (FA). It was found that the OPS concrete containing FA showed lower air content compared to control concrete without FA. The density of the fresh concrete of OPS concrete lies between 1910 and 1958 kg/m3. The 28-day compressive strength of samples was in the range of 20.1–24.2 MPa, which satisfies the requirement of structural LWC. Olanipekun et al. [3] studied the strength characteristics as well as the cost analysis of concrete made by using OPS and crushed granular coconut shells. The level of replacement for LWA with coarse aggregate was in the range of 25 to 100%. The results show that by increasing fractions of OPS, the uniaxial compressive strength of LWA concrete was decreased. A cost reduction between 30% and 42% could be achieved using OPS as a coarse aggregate in concrete production. 3.1. OPS Concrete (OPSC) g gg g p p Teo et al. [4] investigated the properties of OPS-based LWAC and found that the 28-day air-dry density and compressive strength were 1960 kg/m3 and 28.12 MPa, respectively. The 28-day tensile strength, flexural strength and elastic modulus were found as 2.02 MPa, 4.97 MPa and 5.31 GPa, respectively. The curing condition affects the durability of OPS concrete in a significant manner. OPS concrete in water curing gave better durability performance than normal weight concrete. Shafigh et al. [14] studied the different properties such as air content, cube compressive strength, density and water absorption of OPS-based high strength lightweight concrete (HSLC). Five types of curing conditions with different OPS concrete mixtures were taken, and then their compressive strength was determined. The effect of partial replacement of limestone powder (as a filler material) by the fine aggregate was determined. It was observed that the workability of the developed concrete was decreased by increasing limestone powder content. Water absorption of high-strength OPS concrete varies from low to high range, which is a good sign of strong concretes. Alengaram et al. [2] investigated that OPSC inherited high strength to density ratio than that of NWC. It was also shown that OPSC could be used to produce medium to high strength concrete. Liu et al. [49] studied the physico-mechanical and thermal properties of OPS foamed geopolymer concrete (OPSFGC). OPSFGC mixtures were prepared to have densities of 1300, Sustainability 2022, 14, 331 7 of 21 7 of 21 1500 and 1700 kg/m3 in which an artificial foaming agent was used for casting. The control OPS non-foamed geopolymer concrete (OPSNFGC) was cast for comparison studies. It was found that the 28-day compressive strengths of OPSFGC of all three densities were lower than the OPSNFGC. As the density decreases, the 28-day splitting tensile strength and modulus of rupture also decreases. The thermal conductivity of the mix with a density of 1300 and 1500 kg/m3 was 0.47 and 0.50 W/mK, respectively. Sobuz et al. [23] studied the characteristics of concrete produced using OPS fractions ranging from 0 to 50% by replacing conventional coarse aggregate. The compressive strength was reduced with the increasing percentage of OPS aggregates. The structural requirement of LWC was satisfactory. Mo et al. [50] described the effect of steel fiber on the hardness features such as flexural toughness, compressive toughness and fracture parameters of steel fiber OPS concrete (SFOPSC). 3.1. OPS Concrete (OPSC) The outcome of adding steel fiber in the range of 0.5 to 1.0% in SFOPSC on the tensile strength, compressive toughness and flexural toughness was improved by 1.41 times, 6 times and 16 times, respectively. Mo et al. [51] made a comparison between lightweight OPS concrete and NWC of 25, 35 and 45 MPa strength grades. It was concluded that young modulus, indirect tensile strength and drying shrinkage of OPS concrete was lower than that of NWC, while the bond strength of OPS concrete was 80% higher. g g Mo et al. [52] studied the durability properties of OPS concrete in which cement and natural sand were replaced with ground granulated blast furnace slag (GGBS) and manufactured sand, respectively. GGBS was also found to be of great use in improving the long-term compressive strength gain as well as bringing down the strength reduction in OPS concrete when exposed to heat. The use of GGBS as a partial cement substitution in the OPS concrete resulted in an increase in compressive strength gain compared to OPS concrete without GGBS. Eziefula et al. [21] performed comparative studies of different mechanical properties of OPSC with periwinkle shell concrete. The mechanical strength and bulk density of periwinkle shells and OPS-based concrete qualify for the requirements of LWACs. However, the strength characteristics of OPS concrete were inferior to that of the periwinkle shell-based concrete. Khankhaje et al. [53] investigated different properties of OPSC and cockleshell concrete (CSC). It was found that CSC possesses better properties compared to OPSC. It was due to the high water absorption capacity of OPS. Alengaram et al. [54] studied microscopic analysis of OPS using scanning electron microscope as shown in Figure 5, which shows micro pores (size: 16–24 µm) on the convex surface of OPS surface, which causes high water absorption. REVIEW 8 of 22 Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011), 2021, Elsevier). Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011), 2021, Elsevier). Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011), 2021, Elsevier) Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011), 2021, Elsevier). Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011) 2021 Elsevier) Figure 5. Micro-pores on outer surface [54]. 3.1. OPS Concrete (OPSC) (Reprinted with permission from Alengaram et al. (2011), 2021, Elsevier). 3 2 LECA C t 3.2. LECA Concrete 3.2. LECA Concrete LECA aggregates are manufactured from low-lime plastic clay. For aggregate prep- aration clay is firstly heated and then dried After drying sintering is performed in spe LECA aggregates are manufactured from low-lime plastic clay. For aggregate prepara- tion, clay is firstly heated and then dried. After drying, sintering is performed in specific Sustainability 2022, 14, 331 8 of 21 kinds of rotary kilns at a temperature of 1100–1300 ◦C. In this process, the gas, i.e., released after the heating process, becomes entrapped inside the pellets during cooling. LECA aggregates have rounded irregular shapes [55] and incorporate many multi-separated interconnected voids of different sizes, which make them lightweight. The morphology of LECA is shown in Figure 6. LECA is mostly dark brown or reddish or brown-red or gray in colors. These aggregates are also available in yellow or black color due to variation in chemical composition and process of manufacturing. It has a pH value of 7 and is thus inert in nature. It can be used as a replacement for both coarse and fine aggregates. cific kinds of rotary kilns at a temperature of 1100–1300 °C. In this process, the gas, i.e., released after the heating process, becomes entrapped inside the pellets during cooling. LECA aggregates have rounded irregular shapes [55] and incorporate many multi-sepa- rated interconnected voids of different sizes, which make them lightweight. The morphol- ogy of LECA is shown in Figure 6. LECA is mostly dark brown or reddish or brown-red or gray in colors. These aggregates are also available in yellow or black color due to vari- ation in chemical composition and process of manufacturing. It has a pH value of 7 and is thus inert in nature. It can be used as a replacement for both coarse and fine aggregates. Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with per- mission from Nkansah et al. (2012), 2021, Elsevier). Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with permis- sion from Nkansah et al. (2012), 2021, Elsevier). Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with per- mission from Nkansah et al. (2012), 2021, Elsevier). Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with permis- sion from Nkansah et al. (2012), 2021, Elsevier). Zach et al. 3 2 LECA C t 3.2. LECA Concrete [5] studied the possible use of the non-stationary hot-wire method for de- termining the thermal conductivity of the LECA concrete coefficient. It was found that the thermal conductivity of LECA concrete ranged from 0.14 to 0.16 W/m.K. Maghsoudi et al. [57] developed self-compacting lightweight concrete (SCLC) using LECA. Various mix designs of SCLC were cast to obtain the final standard SCLC, with a compressive strength of 20.8–28.5 MPa at 28 days. Zohrabi et al. [58] investigated the mechanical properties of LWC containing LECA with metakaolin, polypropylene (PP) and steel fibers. The PP fi- bers have more effect on the energy absorption capability of LWC rather than on strength Zach et al. [5] studied the possible use of the non-stationary hot-wire method for determining the thermal conductivity of the LECA concrete coefficient. It was found that the thermal conductivity of LECA concrete ranged from 0.14 to 0.16 W/m.K. Maghsoudi et al. [57] developed self-compacting lightweight concrete (SCLC) using LECA. Various mix designs of SCLC were cast to obtain the final standard SCLC, with a compressive strength of 20.8–28.5 MPa at 28 days. Zohrabi et al. [58] investigated the mechanical properties of LWC containing LECA with metakaolin, polypropylene (PP) and steel fibers. The PP fibers have more effect on the energy absorption capability of LWC rather than on strength properties. The addition of 1% steel fibers increased the compressive strength and flexural strength by 36.3% and 52.6%, respectively. Grabois et al. [6] investigated the fresh and hardened properties of steel fibers incorporated SCLC. The 28-day compressive strength was found above 30 MPa for a density of 1700–1900 kg/m3. Concretes with coarse and fine LWAs depicted higher drying shrinkage than those containing only coarse LWAs. The results showed that the thermal conductivity decreased by 60% by using coarse and fine LECA aggregates. gg g Bogas and Cunha [11] investigated the physico-mechanical behavior of non-structural lightweight concrete (NSLWC), which was developed by volcanic scoria. The different properties of various NSLWC fill solutions were analyzed. It was found that the concrete incorporated with coarse LECA aggregate, with a diameter of 4–8 mm showing thermal conductivity of 0.23 W/mK, while the tensile and compressive strength at 28 days were found as 0.5 and 5.3 MPa, respectively. The 28-day modulus of elasticity was 4.9 GPa with capillary absorption of 0.501 × 10−3 mm/min0.5. Vijayalakshmi and Ramanagopal [59] reviewed the utilization of LECA to produce structural LWC. 3.3. Vermiculite Concrete Vermiculite LWA is formed due to weathering action or hydrothermal changes in biotite or phlogopite. It is basically a hydrous phyllosilicate or hydrated magnesium aluminum silicate mineral, which shows volumetric expansion after the heating process and thus causes exfoliation. After exfoliation, vermiculite forms elongated concertina particles, which have specific characteristics such as incombustible, lightweight, compressible, inert and highly absorbent. It has an appearance similar to mica and is available in various parts of the world such as India, the USA, Australia, Russia, South Africa, Bulgaria, etc. In construction practices, these aggregates impart good thermal resistance at a cheaper cost. Sundhakumar [62] investigated the microstructure of expanded vermiculite (EV) and inferred that the lower thermal conductivity of EV-containing cementitious products is caused by a large number of air voids presented in it. The SEM micrograph of vermiculite is shown in Figure 7. REVIEW 10 of 22 Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Lorenzon et al. [63] investigated the properties of cement–vermiculite mortar (CVM), which showed similar physical characteristics to that of the wood in terms of thermal per- formance. CVM absorbed more amount of water and lost water rapidly compared to the pinewood manufactured box. As a result, it was obtained that the exfoliated vermiculite mortars can be used as boxes for honeybees instead of pinewood. Al-Jabri et al. [64] uti- lized vermiculite and polystyrene beads by replacing OPC cement and developed con- crete blocks. It was found that polystyrene incorporated LWC blocks provide lower ther- mal conductivity as compared to vermiculite and conventional concrete. Schackow et al. Lorenzon et al. [63] investigated the properties of cement–vermiculite mortar (CVM), which showed similar physical characteristics to that of the wood in terms of thermal performance. CVM absorbed more amount of water and lost water rapidly compared to the pinewood manufactured box. As a result, it was obtained that the exfoliated vermiculite mortars can be used as boxes for honeybees instead of pinewood. Al-Jabri et al. [64] utilized vermiculite and polystyrene beads by replacing OPC cement and developed concrete blocks. 3 2 LECA C t 3.2. LECA Concrete Further, different fresh and mechanical properties of the developed concrete using LECA aggregates were compared with NWCs. Shafigh et al. [25] compared the engineering characteristics of NWCs with concrete containing two types of LWAs, namely, LECA concrete and oil-palm-boiler-clinker (OPBC) concrete. Natural coarse aggregates were replaced with these LWAs, and then fresh and hardened properties were explored. It was found that the 28-day compressive strength Sustainability 2022, 14, 331 9 of 21 OPBC concrete was about 27% higher than the LECA concrete. While the modulus of elasticity of the OPBC and LECA concretes was 59% and 37% of that of NWC, respectively. OPBC concrete was about 27% higher than the LECA concrete. While the modulus of elasticity of the OPBC and LECA concretes was 59% and 37% of that of NWC, respectively. y p y Heiza et al. [60] analyzed the effect of varying reinforcement ratios on reinforced two-way slabs keeping the dimensions constant. LECA was used to produce a structural LWC with low density and high self-compacting features. LECA was suggested to be pre-wetted since it provides higher strength. Reddy et al. [61] studied the flexural strength of conventional concrete with partial to complete substitution of coarse aggregate by LECA. Thus, the overall use of LECA fine and coarse aggregates can reduce the demand for natural aggregates while designing concrete structures. The LECA concrete gives low density compared to conventional concrete and provides better insulation against heat and sound, while the cube’s compressive strength was reduced continuously with the increase in percent substitution of natural aggregate by LECA. 3.3. Vermiculite Concrete It was found that polystyrene incorporated LWC blocks provide lower thermal conductivity as compared to vermiculite and conventional concrete. Schackow et al. [26] Sustainability 2022, 14, 331 10 of 21 10 of 21 developed the LWC containing vermiculite and expanded polystyrene (EPS) along with a water-reducing super plasticizer and air-entraining agent (AEA). After developing LWC, different mechanical and thermal properties were compared. At a lower incorporation percentage, both AEA and LWAs impart higher compressive strength for LWC. The density observed for EPS LWC and vermiculite LWC ranged from 1.070 g/cm3 to 1.250 g/cm3 and 1.130 g/cm3 to 1.290 g/cm3, respectively. Vermiculite LWC had less thermal conductivity. Koksal et al. [1] compared the physico-mechanical, thermal and micro structural properties of EV and silica fume (SF) incorporated cement-based mortars. At elevated temperatures, the strength and durability attributes were improved using SF in vermiculite containing lightweight mortars. The thermal conductivity of developed mortars depicted a decrement up to 0.257 W/mK, which showed an increment of 58.2% in overall thermal performance behavior. Abidi et al. [42] utilized vermiculite, perlite and cement to produce novel lightweight composite material for building construction, and then various thermo- mechanical properties were studied. It was found that thermal conductivity was decreased with an increase in porosity rate. When the matrix was reinforced with 5 to 25 by wt% of vermiculite, the thermal conductivity of the composite decreased from 0.50 to 0.45 W/mK. Gunasekaran et al. [65] studied the properties of mortar by replacing natural sand with vermiculite in the range of 5 to 30% by wt. of sand. It was concluded that the use of vermiculite mortar is quite economical and provides better compressive strength. Divya et al. [27] studied the different parameters such as compressive strength, flexural strength and split tensile strength after using vermiculite aggregates as partial substitution with 40 to 60% by weight of fine aggregate. The optimum strength was observed to be at a 50% substitution ratio. Sairam and Sailaja [66] replaced different percentages of cement and fine aggregate with vermiculite and FA mineral admixtures. Then, different mechanical properties of the M35 grade of LWC were investigated, and it was found that strength decreased with an increasing amount of vermiculite content. Mo et al. [20] developed cement mortar by partially replacing sand with EV. 3.3. Vermiculite Concrete It was found that the density and strength of mortar were reduced, while the water absorption rate, thermal resistance and stability of the developed mortar were increased after incorporating EV. 3.4. Perlite Concrete The later age strength is generally improved for the products made using perlite aggregate due to the high water absorption capacity of expanded perlite. The morphology of the perlite aggregate is shown in Figure 8. Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015), 2021, Elsevier). Karakoc and Demirboga [19] studied the properties of concrete mixtures with varia- ble expanded perlite aggregate (EPA)–fine aggregate ratios. The mixtures have 0–30% EPA. Under dry and wet curing conditions, compressive strength at 28 days ranged from 40 to 57 and 54 to 81 MPa, respectively. At 30% EPA content, thermal conductivity was found minimum. Sengul et al. [67] investigated the mechanical and thermal properties of LWC containing EPA. Mixtures were prepared by partially substituting natural aggregate by EPA, and it was observed that the unit weight of LWC in fresh state varied between 700 and 2000 kg/m3. The uniaxial compressive strength and Young’s modulus of LWC were reduced by increasing EPA amount. Water absorption and sorptivity coefficient in- crease at high EPA content. From a thermal performance point of view, thermal conduc- tivity was improved by adding EPA content. Gandage et al. [68] used class C FA and per- lite as cement and fine aggregates, respectively. Thermal conductivity was measured at five different temperatures readings. It was concluded that as the test temperature was increased, the thermal conductivity decreased. Comparative high 28-day strengths were obtained at 5% perlite content. Oktay et al. [30] investigated the properties of concrete containing SF, super plasticizer (SP) and AEAs with a fixed w/c ratio. Normal aggregates were replaced by EPA at different volume fractions. A reduction in bulk density and thermal performance was observed. Zulkifeli and Saman [31] studied the compressive and flexural strength of mortar using EPA. It was found that the modulus of rupture and strength was increased at a temperature of 200 ◦C. Karatas [12] studied the thermal resistance of self-compacting mortars (SCM) that were developed by normal and LWAs such as EPA and pumice. SF and FA were used as mineral additives. 3.4. Perlite Concrete Siliceous volcanic rocks are the naturally occurring resources, which are also known as perlite. Perlite is formed naturally after the hydration of obsidian and has amorphous nature. It has the unusual characteristic of largely expanding when heated, which is the main cause of its lower density. The specific feature that differentiates perlite from other volcanic glasses is that when rapidly heated at a temperature ranging between 900 ◦C and 1200 ◦C, it expands about 5 to 20 times of its original volume. This makes it lightweight and insulating. It is suitable to produce LWC using expanded perlite due to its low density. The major applications of perlite LWA are LWC blocks, masonry mortar, plasters, ceiling tiles, etc. Globally, the largest producer of perlite is Turkey, with total reserves of about 4.5 billion tons. The later age strength is generally improved for the products made using perlite aggregate due to the high water absorption capacity of expanded perlite. The morphology of the perlite aggregate is shown in Figure 8. p gg g g Karakoc and Demirboga [19] studied the properties of concrete mixtures with variable expanded perlite aggregate (EPA)–fine aggregate ratios. The mixtures have 0–30% EPA. Under dry and wet curing conditions, compressive strength at 28 days ranged from 40 to 57 and 54 to 81 MPa, respectively. At 30% EPA content, thermal conductivity was found minimum. Sengul et al. [67] investigated the mechanical and thermal properties of LWC containing EPA. Mixtures were prepared by partially substituting natural aggregate by EPA, and it was observed that the unit weight of LWC in fresh state varied between 700 and 2000 kg/m3. The uniaxial compressive strength and Young’s modulus of LWC were reduced by increasing EPA amount. Water absorption and sorptivity coefficient increase at high EPA content. From a thermal performance point of view, thermal conductivity was improved by adding EPA content. Gandage et al. [68] used class C FA and perlite as cement Sustainability 2022, 14, 331 11 of 21 C and tweight 11 of 21 C and tweight and fine aggregates, respectively. Thermal conductivity was measured at five different temperatures readings. It was concluded that as the test temperature was increased, the thermal conductivity decreased. Comparative high 28-day strengths were obtained at 5% perlite content. y j pp p y p ceiling tiles, etc. Globally, the largest producer of perlite is Turkey, with total reserves of about 4.5 billion tons. 3.5. Pumice Concrete 3.5. Pumice Concrete Pumice, also known as pumicite in its fine powder form, is categorized as a volcanic rock that contains highly vesicular volcanic glass and generally looks light in color. Due to its durability and hardness, pumice has been used in LWC for years. Pumice aggregates produce lightweight fire-resistant and sound insulating LWC blocks. The pumice can be used to manufacture low-density LWC that can be used in high-rise buildings structures with significantly improved functional properties. It does not increase the total load of the structure; thereby, it is a very useful material for the repair of the old monuments. Fine pumice is used to develop mortars and provides good thermal insulating properties. Gunduz and Ugur [34] developed the LWAC with fine pumice (FPA) and coarse pumice Pumice, also known as pumicite in its fine powder form, is categorized as a volcanic rock that contains highly vesicular volcanic glass and generally looks light in color. Due to its durability and hardness, pumice has been used in LWC for years. Pumice aggregates produce lightweight fire-resistant and sound insulating LWC blocks. The pumice can be used to manufacture low-density LWC that can be used in high-rise buildings structures with significantly improved functional properties. It does not increase the total load of the structure; thereby, it is a very useful material for the repair of the old monuments. Fine pumice is used to develop mortars and provides good thermal insulating properties. Gunduz and Ugur [34] developed the LWAC with fine pumice (FPA) and coarse pumice aggregates (CPA). The study showed that the pumice aggregate concrete possesses lower thermal conductivity. It was also concluded by the authors that the major influencing parameters for thermal performance were the cement/fines ratio and dry unit weight. p p y g Gunduz [73] studied different structural and functional properties of pumice ag- gregate lightweight concrete (PALWC). Lower aggregate/cement ratio showed higher compressive strength, modulus of elasticity and density, while on the other hand, higher ag- gregate/cement ratio showed lower water absorption, shrinkage and thermal performance. Parhizkar et al. [13] investigated the properties of volcanic pumice. The results showed that the volcanic pumice lightweight concretes fulfilled the requirements of lightweight structural concrete. Tasdemir et al. [74] compared the properties of seven different LWC, developed by using pumice aggregates, fine natural aggregate and coarse limestone ag- gregate. 3.5. Pumice Concrete 3.5. Pumice Concrete The compressive strength of the concretes was compared, which showed that the strengths of the expanded polystyrene beads were lower than the pumice or EPA. Kilincar- slan et al. [75] used the pumice aggregates of four different kinds. The physico-mechanical properties and thermal performance of the developed concretes were also investigated. The result showed that the Nevsehir kind of pumice aggregate was the most suitable type of LWAs to be used in foam concrete. 3.4. Perlite Concrete Bulk density, porosity and water absorption results were determined for the hardened SCM. Results of the experiments showed that the use of LWAs increased the total water absorption and porosity of mortars. The compressive strength of specimens (which were exposed to 300 ◦C) was increased due to the incorporation of EPA up to 10%. Kotwica et al. [69] studied the methodology for utilizing waste EPA as a valuable, high-performance pozzolanic supplementary building material. The results showed that the addition of ground waste EPA leads to gains in strength up to 50%. Polat et al. [70] developed lightweight mortar and concrete using EPA. It was concluded through experimentation that as compared to controlled samples, strength, ultrasonic pulse velocity (UPV) and shrinkage were reduced with increasing EPA content, while the degree of hydration was increased with increment in EPA content. Wan et al. [71] used three LWAs, i.e., EPA, scoria and polystyrene, to produce lightweight self-compacting concrete (LWSCC), and then different fresh and hardened properties were determined. It was reported that all LWAs imparted a negative effect on the workability and compressive strength of LWSCC. Demir and Baspinar [72] confirmed the inert behavior of expanded perlite aggregates for all test conditions. No crystalline phase was formed around the perlite grains as shown in Figure 9. 12 of 21 ehavior formed 12 of 21 ehavior formed Sustainability 2022, 14, 331 Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted with permission from Demir and Baspinar (2008), 2021, Elsevier). Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted with permission from Demir and Baspinar (2008), 2021, Elsevier). Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted with permission from Demir and Baspinar (2008), 2021, Elsevier). Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted with permission from Demir and Baspinar (2008), 2021, Elsevier). 3.6. Sintered Fly Ash Concrete Commercially, sintered FA aggregates are known as lytag. It is manufactured by pyro-processing of fly ash, which is a waste product generated from thermal power plants. The main process to develop sintered FA is sintering and pelletization at a temperature range from 1150 to 1350 ◦C. The sintered FA aggregates are found in brown color with a black internal core (Figure 10). The pore sizes range between 10 and 200 µm, as depicted in Figure 11. 13 of 21 wn color µm, as n color µm, as 13 of 21 wn color µm, as n color µm, as Sustainability 2022, 14, 331 Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per- mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with permission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per- mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per- mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with permission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per- mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from Swamy and Lambert (1981), 2021, Elsevier). Kockal and Ozturan [78] developed LWC using various kinds of sintered FA ag- gregates. The results showed that low specific gravity of cold-bonded and sintered FA aggregate was helpful for the production of LWC with densities within the range of 1860–1943 kg/m3. 3.6. Sintered Fly Ash Concrete Twenty-eight days uniaxial compressive strength and elastic modulus ranged from 42.3 to 55.8 MPa and 22.4 and 28.6 MPa, respectively. Kockal and Oztu- ran [15] investigated the effect of aggregate properties on the strength and durability of lightweight FA aggregate concrete. In the development of LWAC, three different types of LWA, i.e., sintered lightweight ash aggregates, cold bonded lightweight FA aggregate and normal-weight aggregate, were used. Then, the effects of all three aggregates on physico-mechanical and durability properties of developed concrete were determined. Sin- tered FA aggregate concretes had more strength and elasticity modulus than cold-bonded ash aggregate concretes. Nadesan and Dinakar [76] studied the utilization of sintered FA aggregate to produce structural LWC. The results showed that the sintered FA aggregate concrete satisfies the requirement of structural LWC. 4. The Comparisons among Different LWAs/LWACs on the Basis of Various Parameters Researchers reported different properties of LWC, which are shown in Table 3. Majorly, the investigated properties were uniaxial compressive strength, split tensile strength, modu- lus of rupture or flexural strength, thermal conductivity, elastic modulus, water absorption, etc. For comparison purposes of compressive strength, splitting tensile strength, density and thermal conductivity, graphs containing six major LWAs are depicted in Figures 12–15. The control concrete (CC) showed the maximum compressive strength among all LWC, i.e., 38 MPa, as presented in Figure 12. As the content of LWA in concrete increased, the compres- sive strength decreased. Here, 50% pumice used in concrete gave maximum compressive strength up to 36 MPa, while vermiculite LWC showed minimum compressive strength. The tensile strength of conventional concrete was varied in the range from 4 to 4.7 MPa, as depicted in Figure 13. Adding LWA in the mix proportion showed a gradual decrement Sustainability 2022, 14, 331 14 of 21 14 of 21 in tensile strength. Forty percent FA sintered aggregate replacement showed maximum tensile strength, while 40% OPS replacement provided minimum strength. Figure 14 shows that perlite used as an aggregate showed a maximum density around 2300 kg/m3, while pumice concrete showed a minimum density of 1300 kg/m3. The thermal conductivity of LWC was lower than the NWCs. As shown in Figure 15, LECA concrete showed minimum thermal conductivity when used as a whole in place of coarse aggregate while control concrete showed maximum k value, i.e., 2.3 W/mK. concrete showed maximum k value, i.e., 2.3 W/mK. Table 3. Fresh, hardened and durability properties of LWAs, as per the literature. Aggregate Type References Compressive Strength Splitting Tensile Strength Flexural Strength Thermal Conductivity Modulus of Elasticity Water Absorption OPS Mannan and Ganapathy [22] ✓ ✓ ✓ ✓ Sobuz et al. [23] ✓ ✓ ✓ ✓ Mo et al. [51] ✓ ✓ ✓ Mo et al. [52] ✓ ✓ LECA Maghsoudi et al. [57] ✓ ✓ Zohrabi et al. [58] ✓ ✓ Kumar and Prakash [79] ✓ ✓ Heiza et al. [60] ✓ ✓ Reddy et al. [61] ✓ Al-Jabri et al. [64] ✓ ✓ Vermiculite Schackow et al. [26] ✓ Koksal et al. [1] ✓ ✓ ✓ ✓ Divya et al. [27] ✓ ✓ ✓ Mo et al. [20] ✓ ✓ Arun et al. [28] ✓ ✓ ✓ Karakoc and Demirboga [19] ✓ ✓ Perlite Sengul et al. [67] ✓ ✓ ✓ ✓ Gandage et al. 4. The Comparisons among Different LWAs/LWACs on the Basis of Various Parameters [68] ✓ ✓ ✓ Polat et al. [70] ✓ Wan et al. [71] ✓ ✓ Gunduz and Ugur [34] ✓ ✓ ✓ ✓ ✓ Pumice Gunduz [73] ✓ ✓ ✓ ✓ Parhizkar et al. [13] ✓ ✓ ✓ Tasdemir et al. [74] ✓ ✓ ✓ Kilincarslan et al. [75] ✓ ✓ Sintered fly ash Kockal and Ozturan [78] ✓ ✓ ✓ Kockal and Ozturan [15] ✓ ✓ ✓ ✓ Nadesan and Dinakar [76] ✓ ✓ ✓ Sustainability 2022, 14, x FOR PEER REVIEW 15 of 22 Karakoc and Demirboga [19]   Perlite Sengul et al. [67]     Gandage et al. [68]    Polat et al. [70]  Wan et al. [71]   Gunduz and Ugur [34]      Pumice Gunduz [73]     Parhizkar et al. [13]    Tasdemir et al. [74]    Kilincarslan et al. [75]   Sintered fly ash Kockal and Ozturan [78]    Kockal and Ozturan [15]     Nadesan and Dinakar [76]    Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. Table 3. Fresh, hardened and durability properties of LWAs, as per the literature. Aggregate Type References Compressive Strength Splitting Tensile Strength Flexural Strength Thermal Conductivity Modulus of Elasticity Water Absorption OPS Mannan and Ganapathy [22] ✓ ✓ ✓ ✓ Sobuz et al. [23] ✓ ✓ ✓ ✓ Mo et al. [51] ✓ ✓ ✓ Mo et al. [52] ✓ ✓ LECA Maghsoudi et al. [57] ✓ ✓ Zohrabi et al. [58] ✓ ✓ Kumar and Prakash [79] ✓ ✓ Heiza et al. [60] ✓ ✓ Reddy et al. [61] ✓ Al-Jabri et al. [64] ✓ ✓ Vermiculite Schackow et al. [26] ✓ Koksal et al. [1] ✓ ✓ ✓ ✓ Divya et al. [27] ✓ ✓ ✓ Mo et al. [20] ✓ ✓ Arun et al. [28] ✓ ✓ ✓ Karakoc and Demirboga [19] ✓ ✓ Perlite Sengul et al. [67] ✓ ✓ ✓ ✓ Gandage et al. [68] ✓ ✓ ✓ Polat et al. [70] ✓ Wan et al. [71] ✓ ✓ Gunduz and Ugur [34] ✓ ✓ ✓ ✓ ✓ Pumice Gunduz [73] ✓ ✓ ✓ ✓ Parhizkar et al. [13] ✓ ✓ ✓ Tasdemir et al. [74] ✓ ✓ ✓ Kilincarslan et al. 4. The Comparisons among Different LWAs/LWACs on the Basis of Various Parameters [75] ✓ ✓ Sintered fly ash Kockal and Ozturan [78] ✓ ✓ ✓ Kockal and Ozturan [15] ✓ ✓ ✓ ✓ Nadesan and Dinakar [76] ✓ ✓ ✓ Sustainability 2022, 14, x FOR PEER REVIEW 15 of 22 Karakoc and Demirboga [19]   Perlite Sengul et al. [67]     Gandage et al. [68]    Polat et al. [70]  Wan et al. [71]   Gunduz and Ugur [34]      Pumice Gunduz [73]     Parhizkar et al. [13]    Tasdemir et al. [74]    Kilincarslan et al. [75]   Sintered fly ash Kockal and Ozturan [78]    Kockal and Ozturan [15]     N d d Di k [76]    Table 3. Fresh, hardened and durability properties of LWAs, as per the literature. Figure 12 Compressive strength of different LWACs Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. 15 of 21 Sustainability 2022, 14, 331 Figure 12. Compressive strength of different LWACs. Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. ER REVIEW 16 of Figure 14. Density of different LWACs. Figure 15. Thermal conductivity of different LWACs. h h l d h d d Figure 14. Density of different LWACs. ER REVIEW 16 of Figure 14. Density of different LWACs. Figure 15. Thermal conductivity of different LWACs. Figure 15. Thermal conductivity of different LWACs. R REVIEW 16 of Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. R REVIEW 16 of Figure 14. Density of different LWACs. Figure 14 Density of different LWACs R REVIEW 16 of Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. E IE R REVIEW Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. R REVIEW g g Figure 14 Density of different LWACs Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. 5. Research and Development at CSIR-CBRI A systematic R&D work was conducted at CSIR-Central Building Research Institute, Roorkee (Haridwar, India), on the development of LWACs using LECA and Class F FA and dry marble slurry powder (MSP). In this study, optimization of LECA aggregates, cement, w/c ratio was performed. LECA was used as a coarse and fine aggregate in three different sizes: 0–2 mm, 2–8 mm and 8–15 mm. The FA was obtained from NTPC-Dadri (UP), which was classified as class F based on ASTM: C618. The specific gravity of FA was 2.24 and fineness 325 m2/kg. The marble slurry used in this study was obtained from Udaipur, Rajasthan (India). The mean particle size of MS was 9.54 µm. According to Blaine’s apparatus of specific surface area, the specific surface area of MS was 350 m2/kg. MS has a higher surface area than that of OPC and FA, which can fill capillary pores as well as gel pores. In order to increase the workability of LWC, a polycarboxylate ether-based superplasticizer (SP) is used. Ordinary tap water is used in all types of concrete mixes. REVIEW 17 of 22 apparatus of specific surface area, the specific surface area of MS was 350 m2/kg. MS has a higher surface area than that of OPC and FA, which can fill capillary pores as well as gel pores. In order to increase the workability of LWC, a polycarboxylate ether-based super- plasticizer (SP) is used. Ordinary tap water is used in all types of concrete mixes. ff h h l f h d l d l Different physico-mechanical properties of the developed concrete specimens along with thermal conductivity were determined. According to ACI 213R-87, the minimum compressive strength for structural LWC should be 17.2 MPa. Mix contains OPC: 400 kg/m3; sand: 550 kg/m3; 15% MS + 15% FA by replacement of cement; LECA: 325 kg/m3; SP (%): 0.78 with w/c ratio of 0.40 satisfied the requirement of LWC, i.e., 20.4 MPa. According to ASTM C330, a minimum splitting tensile strength of 2.0 MPa is a requirement for structural grade lightweight aggregate concrete. The same mix shows the tensile strength of 2.8 MPa, which satisfies the strength criteria of LWAC. The optimized mix showed the maximum flexural strength out of all concrete mixes, i.e., 3.1 MPa. Thermal conductivity (k value) of concrete was measured using the guarded hot plate method followed by IS: 3346–1980 (Figure 16). 4. The Comparisons among Different LWAs/LWACs on the Basis of Various Parameters Fi 15 Th l d i i f diff LWAC gu e e s y o e e Cs Figure 14. Density of different LWACs. igu e 5. e a co ducti ity o di e e t ACs Figure 14 shows that perlite used as an agg Figure 15. Thermal conductivity of different LWACs. Figure 15. Thermal conductivity of different LWACs. Sustainability 2022, 14, 331 16 of 21 Figure 14 shows that perlite used as an aggregate showed a maximum density around 2300 kg/m3, while pumice concrete showed a minimum density of 1300 kg/m3. The thermal conductivity of LWC was lower than the NWCs. As shown in Figure 15, LECA concrete showed minimum thermal conductivity when used as a whole in place of coarse aggregate while control concrete showed maximum k value, i.e., 2.3 W/mK. 5. Research and Development at CSIR-CBRI Specimens of dimensions 300 mm × 300 mm × 50 mm were cast for testing. The results revealed that the thermal conductivity of the LWC using 15% MS + 15% FA along with LECA shows a lower k value, i.e., 0.21 W/mK. Different physico-mechanical properties of the developed concrete specimens along with thermal conductivity were determined. According to ACI 213R-87, the minimum compressive strength for structural LWC should be 17.2 MPa. Mix contains OPC: 400 kg/m3; sand: 550 kg/m3; 15% MS + 15% FA by replacement of cement; LECA: 325 kg/m3; SP (%): 0.78 with w/c ratio of 0.40 satisfied the requirement of LWC, i.e., 20.4 MPa. Accord- ing to ASTM C330, a minimum splitting tensile strength of 2.0 MPa is a requirement for structural grade lightweight aggregate concrete. The same mix shows the tensile strength of 2.8 MPa, which satisfies the strength criteria of LWAC. The optimized mix showed the maximum flexural strength out of all concrete mixes, i.e., 3.1 MPa. Thermal conductivity (k value) of concrete was measured using the guarded hot plate method followed by IS: 3346–1980 (Figure 16). Specimens of dimensions 300 mm × 300 mm × 50 mm were cast for testing. The results revealed that the thermal conductivity of the LWC using 15% MS + 15% FA along with LECA shows a lower k value, i.e., 0.21 W/mK. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC mix showed denser structure as compared to control concrete with LECA only. The inclu- sion of MS into concrete improved the microstructure due to the filler effect and accelera- tion of hydration. Less crystalline phase was observed in the interfacial transition zone (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC mix showed denser structure as compared to control concrete with LECA only. 5. Research and Development at CSIR-CBRI The inclu- sion of MS into concrete improved the microstructure due to the filler effect and acceleration of hydration. Less crystalline phase was observed in the interfacial transition zone (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement of The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC mix showed denser structure as compared to control concrete with LECA only. The inclu- sion of MS into concrete improved the microstructure due to the filler effect and accelera- tion of hydration. Less crystalline phase was observed in the interfacial transition zone (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement f i d ITZ l l d f h i l i b i f li h The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC mix showed denser structure as compared to control concrete with LECA only. The inclu- sion of MS into concrete improved the microstructure due to the filler effect and acceleration of hydration. Less crystalline phase was observed in the interfacial transition zone (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement of Sustainability 2022, 14, 331 17 of 21 e inclu- celera 17 of 21 e inclu- celera microstructure and ITZ also resulted from the internal curing by pre-wetting of lightweight aggregate (Figure 17a–d). (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement of microstructure and ITZ also resulted from the internal curing by pre-wetting of light- weight aggregate (Figure 17a–d). microstructure and ITZ also resulted from the internal curing by pre-wetting of lightweight aggregate (Figure 17a–d). (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement of microstructure and ITZ also resulted from the internal curing by pre-wetting of light- weight aggregate (Figure 17a–d). microstructure and ITZ also resulted from the internal curing by pre-wetting of lightweight aggregate (Figure 17a–d). (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement of microstructure and ITZ also resulted from the internal curing by pre-wetting of light- weight aggregate (Figure 17a–d). (a) (b) REVIEW 18 of 22 (c) (d) Figure 17. SEM Results of laboratory prepared LWAC specimens. 6. Conclusions 6. Conclusions The current paper reviews the prior studies conducted on the properties and uses of LWAs to develop sustainable concrete as green building materials. The utilization of sin- tered FA, vermiculite, pumice, OPS, LECA and perlite as LWA to produce LWC in build- ing construction was reviewed through the recent and past literature. The physical, me- chanical, durability, functional and structural behaviors of LWACs were discussed. Now- adays, LWACs have become an important low-cost green building material that imparts high thermal resistance properties with a reduced dead load of the superstructure. As the LWAs have different types of physical and mechanical characteristics, on the basis of end- user applications, available LWACs are used for different construction purposes. The current paper reviews the prior studies conducted on the properties and uses of LWAs to develop sustainable concrete as green building materials. The utilization of sintered FA, vermiculite, pumice, OPS, LECA and perlite as LWA to produce LWC in building construction was reviewed through the recent and past literature. The physical, mechanical, durability, functional and structural behaviors of LWACs were discussed. Nowadays, LWACs have become an important low-cost green building material that imparts high thermal resistance properties with a reduced dead load of the superstructure. As the LWAs have different types of physical and mechanical characteristics, on the basis of end-user applications, available LWACs are used for different construction purposes. 5. Research and Development at CSIR-CBRI (a) Control concrete; (b) FA 30%; (c) MS 30%; (d) FA15% + MS15%. Figure 17. SEM Results of laboratory prepared LWAC specimens. (a) Control concrete; (b) FA 30%; (c) MS 30%; (d) FA15% + MS15%. (a) REVIEW (b) 18 of 22 (b) (a) (c) (d) (c) (d) Figure 17. SEM Results of laboratory prepared LWAC specimens. (a) Control concrete; (b) FA 30%; (c) MS 30%; (d) FA15% + MS15%. Figure 17. SEM Results of laboratory prepared LWAC specimens. (a) Control concrete; (b) FA 30%; (c) MS 30%; (d) FA15% + MS15%. Thus, the usage of LECA with 15% FA + 15% MS as a cement replacement in LWC shows better compressive, tensile, flexural strength and lower thermal conductivity, and hence it can be proposed for structural purposes. Further research is in process in regard to different durability aspects along with different functional properties such as fire re- sistance and sound insulation properties. Thus, the usage of LECA with 15% FA + 15% MS as a cement replacement in LWC shows better compressive, tensile, flexural strength and lower thermal conductivity, and hence it can be proposed for structural purposes. Further research is in process in regard to different durability aspects along with different functional properties such as fire resistance and sound insulation properties. 7 Recommendations for the Further Research 7. Recommendations for the Further Research 7. Recommendations for the Further Research On the basis of the existing literature a few recommendations are proposed below: On the basis of the existing literature, a few recommendations are proposed below: On the basis of the existing literature, a few recommendations are proposed below: 1. Studies of hardened, durable and functional properties for different LWACs in vari- ou I dia li ati o ditio 1. Studies of hardened, durable and functional properties for different LWACs in various Indian climatic conditions; ous Indian climatic conditions; 2. Comprehensive studies of different durability attributes of high-performance LWAC t i i diff t SCM fib d l 2. Comprehensive studies of different durability attributes of high-performance LWAC containing different SCMs, fibers and polymers; containing different SCMs, fibers and polymers; 3. Statistical modeling for durability properties based on theoretical and empirical stud- i l i h h i lid i i h fi ld 3. Statistical modeling for durability properties based on theoretical and empirical studies along with their validation in the fields; ies along with their validation in the fields; 4. Field studied for fire resistance of LWA concrete incorporated with SCMs, fibers, fire- resistant admixtures etc 4. Field studied for fire resistance of LWA concrete incorporated with SCMs, fibers, fire-resistant admixtures, etc.; Sustainability 2022, 14, 331 18 of 21 18 of 21 5. Study investigating the toughness and fracture energy of LWACs (LECA, OPS, sintered fly ash, pumice, perlite, vermiculite) is needed as these properties are necessary while modeling their mechanical behavior. Thus, it is envisaged that LWACs, because of their techno-economic and environmental advantages, are supposed to capture their major share in the building industry in the 21st century. For this optimism, the most important additional motivating factor is the ever- increasing importance of the need for sustainable development. This is the appropriate time to utilize industrial byproducts such as fly ash, GGBS, silica fume, etc., by thinking more futuristically and investing in R&D for the sake of our world, its environment and its construction industry. Author Contributions: Conceptualization, R.K.; methodology, R.K.; validation, R.K., R.L.; formal analysis, R.K., R.L.; investigation, R.K.; data curation, R.K.; writing—original draft preparation, R.K., A.S.; writing—review and editing, R.K., A.S.; supervision, R.K. All authors have read and agreed to the published version of the manuscript. 7 Recommendations for the Further Research 7. Recommendations for the Further Research Funding: The authors are grateful to ‘Ministry of Environment, Forest and Climate Change, New Delhi, Government of India’ (File Number: 19–44/2018/RE; Project No.: GAP0120) and ‘Indo-US Science and Technology Forum (IUSSTF), New Delhi, India’ (Grant No. GAP: 806) for the sustained financial support to the project. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not Applicable. Data Availability Statement: Not Applicable. Data Availability Statement: Not Applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Grabois, T.M.; Cordeiro, G.C.; Filho, R.D.T. Fresh and hardened-state properties of self-compacting lightweight concrete reinforced with steel fibers. Constr. Build. Mater. 2016, 104, 284–292. [CrossRef] 6. Grabois, T.M.; Cordeiro, G.C.; Filho, R.D.T. 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Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats
Brazilian Journal of Pharmaceutical Sciences
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Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats Bianchuan Cao1†, Qiuju Li1†, Zhe Huang2, Xiufang Huang1, Yihong Zhu1, Ke Xiao1, Fuli Huang1 and Li Zhong iD 1* 1Department of Infectious Disease, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China, 2Department of Paediatrics, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China T lymphocytes, cytokines, and macrophages play important roles in the clearance of Mycobacterium tuberculosis (Mtb) by the immune system. This study aimed to investigate the effects of isoniazid on the functions of both innate and adaptive immune cells. Healthy rats were randomly divided into experimental and control groups. Each group was randomly divided into three subgroups and named according to the duration of drug feeding, 1, 3, and 3 months followed by drug withdrawal for 1 month. The experimental groups were fed with isoniazid (12 mg/mL) and the control groups with normal saline. The percentage of CD4+ and CD8+T lymphocytes, level of interleukin (IL)-12 and interferon (IFN)-γ, and function of macrophages were determined at these three time points. Isoniazid significantly increased the percentage of CD4+T lymphocytes and the CD4+/CD8+T lymphocyte cell ratio (P < 0.05). It transiently (<1 month) enhanced the functions of rat macrophages significantly (P < 0.05). In summary, isoniazid could increase the percentage of CD4+T lymphocytes, CD4+/CD8+T lymphocyte cell ratio, and enhance macrophage function in healthy rats. Keywords: Cytokines. Immune. Isoniazid. Macrophages. T lymphocytes. *Correspondence: L. Zhong, Department of Infectious Disease, The Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street, Luzhou, Sichuan, 646000, China. E-mail: zhongli620@163.com. †These authors contributed equally to this work. *Correspondence: L. Zhong, Department of Infectious Disease, The Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street, Luzhou, Sichuan, 646000, China. E-mail: zhongli620@163.com. †These authors contributed equally to this work. Article Brazilian Journal of Pharmaceutical Sciences Brazilian Journal of Pharmaceutical Sciences http://dx.doi.org/10.1590/s2175-97902019000418547 INTRODUCTION incidence of drug-resistant TB, the severity of TB cases has increased (Das, Satapathy, Murmu, 2016; Ebonyi et al., 2016; Lima et al., 2017). Therefore, TB has become a serious public health problem in China. Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) bacteria which may damage multiple organ systems and poses a serious harm to human health. According to the World Health Organization’s report on the global tuberculosis epidemic, about one-third of the world’s population was infected with Mtb in 2015, 10.4 million new TB patients were diagnosed, and more than 1.4 million patients died of the disease (Sisay et al., 2016). China has a high incidence of TB, ranking second in the world. At present, due to the increase in human immunodeficiency virus/acquired immunodeficiency syndrome, the use of immunosuppressive and cytotoxic drugs, and the T lymphocytes, cytokines, and macrophages play important roles in the clearance of Mtb-related antigens. Insufficient number or dysfunction of T lymphocytes and macrophages, and the lack of related cytokines may lead to decreased immunity to Mtb. Therefore, immune status is closely related to the occurrence, development, and therapeutic response to TB (Grover et al., 2014; Mosquera-Restrepo et al., 2017). In recent years, it has been found that some antibacterial drugs not only kill pathogens but also regulate immune function during treatment (Hamilton-Miller, 2001; Schultz, Speelman, van der Poll, 2001; Tauber, Nau, 2008). Some b-lactam antibiotics, such as cefodizime, can improve immune function (Wang et al., 2015). Although anti-tuberculosis drugs have been used for many years, studies on Page 1/9 Page 1/9 Braz. J. Pharm. Sci. 2020;56: e18547 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong their immune regulation are lacking. Therefore, it is important to investigate the effects of anti-tuberculosis drugs on immune function, especially those related to the clearance of Mtb. powder was dissolved in saline at a concentration of 12 mg/mL (Sichuan Meida Kangjiale Pharmaceutical Co. Ltd., Chengdu, China). Rats were administered isoniazid solution (1 mL/100 g body weight, once every other day) intragastrically. Isoniazid is one of the most important first-line drugs for treating TB. It has strong antibacterial effects; thus, it is used throughout the period of reinforcement and consolidation. The side effects of isoniazid, such as liver damage, may be related to the high level of immune response (Usui et al., 2017; Vinnard et al., 2017). Grouping A total of 60 rats were randomly divided into experimental and control groups (30 rats in each group). Each group was randomly divided into three subgroups (10 rats in each subgroup) and named according to the time points of drug feeding 1, 3, and 3 months, followed by drug withdrawal for 1 month. MATERIAL AND METHODS All the experimental animals and procedures were approved by the animal experiment ethics committee of the Southwest Medical University, China (approval number: 201703192). Sample collection The rats were anesthetized with 3% pentobarbital sodium solution (30 mg/kg). Next, 2 mL blood samples were obtained from the hearts. Anticoagulated whole blood (300 μL) was used to determine the percentage of T lymphocytes. The serum of the remaining blood was used to detect the levels of cytokines after letting it stand and centrifugation. Detection of T lymphocyte percentage by flow cytometry The anticoagulated whole blood (100 µL) was placed in the test tube for flow cytometry. The samples were mixed with 20 μL of Allophycocyanin (APC) Mouse anti-Rat CD3 (BD, NJ, USA), 20 μL of Fluorescein isothiocyanate (FITC) Mouse anti-Rat CD8a (BD), and 20 μL of Phycoerythrin (PE) Mouse anti-Rat CD4 (BD). The samples (100 µL of anticoagulated whole blood) in the isotype control group were mixed with isotype control. All samples were analyzed to detect the percentage and ratio of CD4+ and CD8+T lymphocytes under a BD fluorescence-activated cell sorter (FACS) Verse II flow cytometer (BD). INTRODUCTION At present, it is not clear whether isoniazid has any effect on immune functions related to the clearance of Mtb. Therefore, we explored the effects of isoniazid on the immune functions in healthy rats, and found that isoniazid can serve as a potential immune booster. Control group: The rats were administered normal saline (1 mL/100 g body weight, once every other day) intragastrically. Feeding conditions were the same as those in the experimental group. Detection of the levels of interleukin-12 and interferon-γ by enzyme-linked immunosorbent assay The levels of serum interleukin (IL)-12 and interferon (IFN)-γ were detected using a microplate reader (Thermo Fisher Scientific Inc., MA, USA), IL- 12 enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, CA, USA), and IFN-γ ELISA kit (eBioscience, CA, USA). All the reagent samples and standards were prepared as instructed. The plate was washed, and the standard and sample were added, followed by antibody. Then the stop solution was added, Animals Two-month-old male Sprague-Dawley rats of clean grade were purchased from Chongqing Tengxin Bio- Technology Co., Ltd. [Chongqing, China; production license: SCXK (Yu) 2012-0008]. All the rats were fed under the same standard conditions with a temperature of 23 °C ± 3 °C and relative humidity of 50% ± 10%. RESULTS AND DISCUSSION Most people infected with Mtb develop a latent TB infection without any clinical symptoms that is not cleared by the immune system. Only a small number of patients develop an active TB infection. When various factors cause a decrease in the body’s immune function, the latent Mtb can be activated, allowing it to develop into active TB (Petruccioli et al., 2016; Riou et al., 2016). Therefore, the inhibition and clearance of Mtb are closely related to immune function. This study selected normal healthy rats as subjects to avoid the effects of other factors on the immune system. Detection of acid phosphatase activity in rat peritoneal macrophages by Gomori assay Rats were injected intraperitoneally with 0.7 mL of 6% sterile starch broth, and 0.1-0.2 mL of abdominal fluid was extracted after 3 days. The peritoneal fluid was dripped on a precooled slide and fixed with 10% neutral formalin. After adding acid phosphatase solution and 1% ammonium sulfide successively, the cells were stained with Giemsa stain. The slides were examined under ordinary optical microscope to observe brown particles or plaques in macrophages (Anhe, Lima- Oliveira, Azeredo-Oliveira, 2007). Drug delivery method Experimental group: Isoniazid tablets (Chengdu Jinhua Pharmaceutical Co. Ltd., China) were ground into a powder, and the equivalent dose ratio was calculated according to human and rat body surface area (rats received six times the adult human dosage). Isoniazid Page 2/9 Page 2/9 Braz. J. Pharm. Sci. 2020;56: e18547 Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats and the plate was read. (The detailed instructions on the relevant ELISA kit was followed). using two independent-sample t test, and t’ test was used to assess the heterogeneity of variance. The results were expressed as mean ± standard deviation (x ± s), and a P value < 0.05 was considered statistically significant. Acid phosphatase test: At the same time point, the experimental and control groups were compared using the rank-sum test. The results were expressed as mean rank, and a P value < 0.05 was considered statistically significant. Test of carbon clearance capacity and phagocytic capacity Diluted ink (0.1 mL/20 g body weight) was injected into the tail vein of the rats. Blood samples (30 μL) were obtained from the hearts after 5 (t1) and 15 (t2) min. The optical density (OD) values of the samples obtained at t1 (OD1) and t2 (OD2) were measured at 650 nm wavelength. The rats were sacrificed by cervical dislocation, and the liver and spleen were weighed. The carbon clearance index K was calculated according to the formula K = (1 g ODl - l g OD2)/(t2 - t1). The phagocytic index a was calculated according to the formula a = body weight/(liver weight + spleen weight) × K1/3 (Singh, Yadav, Noolvi, 2012). Comparison of CD4+T, CD8+T lymphocytes and CD4+/CD8+T lymphocyte cell ratio After feeding drug for 1 and 3 months, the percentage of CD4+T lymphocytes and the CD4+/CD8+T lymphocyte cell ratio was higher in the experimental group than in the control group, and the differences were statistically significant (P < 0.05). Moreover, after feeding drug for 3 months followed by drug withdrawal for 1 month, no statistically significant difference was found in the CD4+T lymphocytes or CD4+/CD8+T lymphocyte cell ratio between the experimental and control groups (P > 0.05; Table I). No statistically significant difference was found in the CD8+T lymphocytes between the experimental and control groups at any time point (Figure 1, Table I). Statistical analysis The experimental data were analyzed using the SPSS 19.0 software (SPSS Inc., IL, USA). T lymphocytes, cytokines test, carbon clearance capacity and phagocytic capacity test: At the same time point, the experimental and control groups were compared Page 3/9 Braz. J. Pharm. Sci. 2020;56: e18547 Page 3/9 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong FIGURE 1 - CD4+T lymphocytes in (A) experimental group after drug feeding for 1 month, (B) control group after drug feedi or 1 month, (C) experimental group after drug feeding for 3 months, (D) control group after drug feeding for 3 months, ( xperimental group after drug feeding for 3 months followed by drug withdrawal for 1 month, (F) control group after dr eeding for 3 months followed by drug withdrawal for 1 month. CD8+T lymphocytes in (G) experimental group after dr eeding for 1 month, (H) control group after drug feeding for 1 month, (I) experimental group after drug feeding for 3 month J) control group after drug feeding for 3 months, (K) experimental group after drug feeding for 3 months followed by dr FIGURE 1 - CD4+T lymphocytes in (A) experimental group after drug feeding for 1 month, (B) control group after drug feeding for 1 month, (C) experimental group after drug feeding for 3 months, (D) control group after drug feeding for 3 months, (E) experimental group after drug feeding for 3 months followed by drug withdrawal for 1 month, (F) control group after drug feeding for 3 months followed by drug withdrawal for 1 month. CD8+T lymphocytes in (G) experimental group after drug feeding for 1 month, (H) control group after drug feeding for 1 month, (I) experimental group after drug feeding for 3 months, (J) control group after drug feeding for 3 months, (K) experimental group after drug feeding for 3 months followed by drug withdrawal for 1 month, (L) control group after drug feeding for 3 months followed by drug withdrawal for 1 month. Page 4/9 Page 4/9 Braz. J. Pharm. Sci. Statistical analysis 2020;56: e18547 Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats TABLE I - Comparison of CD4+, CD8+T lymphocytes and CD4+/CD8+ ratio in the experimental and control groups at the three time points Time point Experimental group(n=10) Control group (n=10) P-value CD4+T lymphocytes (%) Feeding drug for 1 month 48.332 ± 5.486 37.625 ± 4.192 0.000 a Feeding drug for 3 months 42.169 ± 6.041 35.421 ± 7.882 0.045 a Feeding drug for 3 months followed by drug withdrawal for 1 month 36.656 ± 5.639 32.642 ± 5.325 0.119 a CD8+T lymphocytes (%) Feeding drug for 1 month 11.243 ± 1.572 13.642 ± 3.292 0.058 a Feeding drug for 3 months 12.280 ± 4.398 15.611 ± 5.557 0.154 a Feeding drug for 3 months followed by drug withdrawal for 1 month 15.780 ± 6.048 15.963 ± 3.814 0.936 a CD4+/CD8+ ratio Feeding drug for 1 month 4.372 ± 0.780 2.880 ± 0.637 0.000 a Feeding drug for 3 months 3.903 ± 1.579 2.552 ± 1.065 0.038 a Feeding drug for 3 months followed by drug withdrawal for 1 month 2.604 ± 0.869 2.185 ± 0.723 0.256 a a = Two independent-sample t test. P < 0.05 denoted statistical significant difference TABLE I - Comparison of CD4+, CD8+T lymphocytes and CD4+/CD8+ ratio in the experimental and control groups at the three time points TABLE I - Comparison of CD4+, CD8+T lymphocytes and CD4+/CD8+ ratio in the experimental and control groups at the three time points a = Two independent-sample t test. P < 0.05 denoted statistical significant difference Significance of T lymphocytes in the Mtb clearance CD4+T lymphocytes and CD4+/CD8+T lymphocyte cell ratio in the peripheral blood of rats, and the percentages could be restored after isoniazid withdrawal. Moreover, isoniazid had no significant effect on CD8+T lymphocytes in the peripheral blood of rats. Mtb is an intracellular bacterium; CD4+ and CD8+T lymphocytes play important roles in the immune response against Mtb infection. Previous studies found that for patients with TB, the number of CD4+T lymphocytes decreased and the number of CD8+T lymphocytes increased. The CD4+/CD8+T lymphocyte cell ratio, which could be used to reflect the severity of TB, decreased or even inverted (Arroyo et al., 2016; Reiley et al., 2010; Scanga et al., 2000). Braz. J. Pharm. Sci. 2020;56: e18547 Significance of cytokines in the treatment of TB of IL-12 could be restored after isoniazid withdrawal. These results were not statistically significant, possibly due to small sample size and short-term administration. Therefore, the effects of isoniazid on the level of IL-12 in the serum of rats may be further examined in future studies by increasing the sample size and extending the administration time. The data presented here suggest that isoniazid may have no significant effect on IFN-γ level in the serum of rats. Cytokines, such as IL-12 and IFN-γ, also play important roles in the immune response to Mtb infection (Chen et al., 2017; Mortaz et al., 2016). IL-12 can promote the proliferation and activation of T lymphocytes (Hamilton et al., 2016) and stimulate the production of IFN-γ. IL-12 can also activate macrophages and enhance the ability of anti-Mtb with IFN-γ (Thada et al., 2016). Recent studies showed that cytokine imbalance was a key factor in the occurrence and development of TB, and was closely related to TB prognosis (Anuradha et al., 2016; Kreins et al., 2015). Comparison of IL-12 and IFN-γ level After feeding drug for 1, 3, and 3 months followed by drug withdrawal for 1 month, no statistically significant difference was found in the IL-12 and IFN-γ level between the experimental and control groups (P > 0.05; Table II). The effects of isoniazid on T lymphocytes in healthy male rats were investigated in this study. The results showed that isoniazid could increase the percentage of Page 5/9 Page 5/9 Braz. J. Pharm. Sci. 2020;56: e18547 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong TABLE II - Comparison of IL-12 and IFN-γ level in the experimental and control groups at the three time points Time point Experimental group (n=10) Control group (n=10) P-value IL-12 (pg/mL) Feeding drug for 1 month 134.409 ± 25.128 131.752 ± 41.084 0.874 a Feeding drug for 3 months 137.034 ± 65.937 96.211 ± 18.836 0.088 a Feeding drug for 3 months followed by drug withdrawal for 1 month 113.241 ± 38.698 96.019 ± 20.463 0.234 a IFN-γ (pg/mL) Feeding drug for 1 month 121.552 ± 21.847 107.609 ± 34.765 0.297 a Feeding drug for 3 months 118.444 ± 8.158 127.379 ± 24.003 0.289 a Feeding drug for 3 months followed by drug withdrawal for 1 month 114.280 ± 18.796 105.517 ± 19.753 0.323 a a = Two independent-sample t test. P < 0.05 denoted statistical significant difference a = Two independent-sample t test. P < 0.05 denoted statistical significant difference Comparison of carbon clearance index, phagocytic index and acid phosphatase level P < 0.05 denoted statistical significant difference TABLE III - Comparison of carbon clearance, phagocytic index and acid phosphatase level in the experimental and control groups at the three time points TABLE III - Comparison of carbon clearance, phagocytic index and acid phosphatase level in the experimental and control groups at the three time points arbon clearance, phagocytic index and acid phosphatase level in the experimental and control ts Braz. J. Pharm. Sci. 2020;56: e18547 Comparison of carbon clearance index, phagocytic index and acid phosphatase level After 1 month of drug feeding, the carbon clearance index, phagocytic index, and acid phosphatase level in the experimental group was higher than in the control group, and the difference was statistically significant (P < 0.05). Moreover, after feeding drug for 3 and 3 months followed by drug withdrawal for 1 month, no statistically significant difference was found in the carbon clearance index, phagocytic index, or acid phosphatase level between the experimental and control groups (P > 0.05; Table III). The effects of isoniazid on the levels of IL-12 and IFN-γ in healthy male rats were investigated in this study. The results showed that the level of IL-12 in the serum of rats decreased as the rat’s age increased. However, the change in IL-12 level in the experimental group was not the same as in the control group. It did not decrease as the rat’s age increased, indicating that isoniazid might increase the level of IL-12 in the serum of rats, and this effect may become more significant as the administration time is extended. Also, the level Page 6/9 Braz. J. Pharm. Sci. 2020;56: e18547 Page 6/9 Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats TABLE III - Comparison of carbon clearance, phagocytic index and acid phosphatase level in the experimental and control groups at the three time points Time point Experimental group (n=10) Control group (n=10) P-value Carbon clearance index Feeding drug for 1 month 0.017 ± 0.004 0.006 ± 0.001 0.000 a Feeding drug for 3 months 0.011 ± 0.001 0.012 ± 0.002 0.798 a Feeding drug for 3 months followed by drug withdrawal for 1 month 0.024 ± 0.009 0.029 ± 0.009 0.248 a Phagocytic index Feeding drug for 1 month 6.383 ± 0.602 4.637 ± 0.374 0.000 a Feeding drug for 3 months 6.366 ± 0.578 6.377 ± 0.491 0.964 a Feeding drug for 3 months followed by drug withdrawal for 1 month 8.391 ± 1.526 8.142 ± 0.989 0.671 a Acid phosphatase level (Mean rank) Feeding drug for 1 month 13.40 7.60 0.029 b Feeding drug for 3 months 10.00 11.00 0.739 b Feeding drug for 3 months followed by drug withdrawal for 1 month 11.25 9.75 0.579 b a = Two independent-sample t test; b = Rank-sum test. REFERENCES Lima GC, Silva EV, Magalhaes PO, Naves JS. Efficacy and safety of a four-drug fixed-dose combination regimen versus separate drugs for treatment of pulmonary tuberculosis: a systematic review and meta-analysis. Braz J Microbiol. 2017;48(2):198-207. Anhe AC, Lima-Oliveira AP, Azeredo-Oliveira MT. Acid phosphatase activity distribution in salivary glands of triatomines (Heteroptera, Reduviidae, Triatominae). Genet Mol Res. 2007;6(1):197-205. Mortaz E, Masjedi MR, Abedini A, Matroodi S, Kiani A, Soroush D, et al. Common features of tuberculosis and sarcoidosis. Int J Mycobacteriol. 2016;5(Suppl 1):S240-S241. Anuradha R, Munisankar S, Bhootra Y, Dolla C, Kumaran P, Babu S. High body mass index is associated with heightened systemic and mycobacterial antigen - Specific pro- inflammatory cytokines in latent tuberculosis. Tuberculosis (Edinb). 2016;101:56-61. Anuradha R, Munisankar S, Bhootra Y, Dolla C, Kumaran P, Babu S. High body mass index is associated with heightened systemic and mycobacterial antigen - Specific pro- inflammatory cytokines in latent tuberculosis. Tuberculosis (Edinb). 2016;101:56-61. Mosquera-Restrepo SF, Caro AC, Garcia LF, Pelaez-Jaramillo CA, Rojas M. Fatty acid derivative, chemokine, and cytokine profiles in exhaled breath condensates can differentiate adult and children paucibacillary tuberculosis patients. J Breath Res. 2017;11(1):016003. Arroyo L, Rojas M, Franken KL, Ottenhoff TH, Barrera LF. Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia. Clin Vaccine Immunol. 2016;23(10):813-824. Petruccioli E, Vanini V, Chiacchio T, Cirillo DM, Palmieri F, Ippolito G, et al. Modulation of interferon-gamma response to QuantiFERON-TB-plus detected by enzyme-linked immunosorbent assay in patients with active and latent tuberculosis infection. Int J Mycobacteriol. 2016;5(Suppl 1):S143-S144. Auricchio G, Garg SK, Martino A, Volpe E, Ciaramella A, De Vito P, et al. Role of macrophage phospholipase D in natural and CpG-induced antimycobacterial activity. Cell Microbiol. 2003;5(12):913-920. Reiley WW, Shafiani S, Wittmer ST, Tucker-Heard G, Moon JJ, Jenkins MK, et al. Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection. Proc Natl Acad Sci USA. 2010;107(45):19408-19413. Chen D, Li G, Fu X, Li P, Zhang J, Luo L. Wnt5a deficiency regulates inflammatory cytokine secretion, polarization, and apoptosis in Mycobacterium tuberculosis-infected macrophages. DNA Cell Biol. 2017;36(1):58-66. Riou C, Bunjun R, Muller TL, Kiravu A, Ginbot Z, Oni T, et al. Selective reduction of IFN-gamma single positive mycobacteria-specific CD4+ T cells in HIV-1 infected individuals with latent tuberculosis infection. Tuberculosis (Edinb). 2016;101:25-30. Das D, Satapathy P, Murmu B. First line anti-TB drug resistance in an urban area of Odisha, India. SOURCES OF SUPPORT Kreins AY, Ciancanelli MJ, Okada S, Kong XF, Ramirez-Alejo N, Kilic SS, et al. Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome. J Exp Med. 2015;212(10):1641-1662. This study was supported by the Sichuan Provincial Health Department (No. 090209). Significance of macrophages in anti-tuberculosis not obvious. A possible explanation is that as innate immune cells, macrophages often respond swiftly to external stimuli then produce high levels of inflammatory mediators. On the other hand, the inflammatory activity of macrophages needs to be tightly regulated in order to prevent the inflammation-induced tissue damage. Many anti-inflammatory cytokines are also produced by activated macrophages to terminate their excessive activation. In contrast, the effects of adaptive immune cells, such as T lymphocytes, are generally persistent over a relatively long time period. Therefore, isoniazid treatment-induced enhancement of macrophage function may be antagonized by negative feed-back mechanisms, thus decreasing the long-term effect of isoniazid. Macrophages play a role in innate immunity, and are critical cells in the immune response to Mtb infection. Activated macrophages can inhibit or even kill Mtb, which plays an important role in the control of Mtb infection (Auricchio et al., 2003). Carbon clearance capacity, phagocytic capacity, and acid phosphatase of macrophages can reflect their function. The effects of isoniazid on macrophage function in healthy male rats were explored in this study. Isoniazid enhanced the carbon clearance capacity, phagocytic capacity, and acid phosphatase activity of macrophages for a short period of time, but the long-term effects were Page 7/9 Page 7/9 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong Hamilton CA, Mahan S, Entrican G, Hope JC. Interactions between natural killer cells and dendritic cells favour T helper1-type responses to BCG in calves. Vet Res. 2016;47(1):85. Hamilton CA, Mahan S, Entrican G, Hope JC. Interactions between natural killer cells and dendritic cells favour T helper1-type responses to BCG in calves. Vet Res. 2016;47(1):85. CONCLUSION In summary, isoniazid could increase the percentage of CD4+T lymphocytes and CD4+/CD8+T lymphocyte cell ratio in peripheral blood and enhance macrophage function in healthy male rats. Hamilton-Miller JM. Immunopharmacology of antibiotics: direct and indirect immunomodulation of defence mechanisms. J Chemother. 2001;13(2):107-111. Braz. J. Pharm. Sci. 2020;56: e18547 Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats REFERENCES J Clin Diagn Res. 2016;10(11):DC04-DC06. Ebonyi AO, Oguche S, Agbaji OO, Sagay AS, Okonkwo PI, Idoko JA, et al. Mortality among pulmonary tuberculosis and HIV-1 co-infected Nigerian children being treated for pulmonary tuberculosis and on antiretroviral therapy: a retrospective cohort study. Germs. 2016;6(4):139-150. Scanga CA, Mohan VP, Yu K, Joseph H, Tanaka K, Chan J, et al. Depletion of CD4(+) T cells causes reactivation of murine persistent tuberculosis despite continued expression of interferon gamma and nitric oxide synthase 2. J Exp Med. 2000;192(3):347-358. Grover A, Troudt J, Foster C, Basaraba R, Izzo A. High mobility group box 1 acts as an adjuvant for tuberculosis subunit vaccines. Immunology. 2014;142(1):111-123. Schultz MJ, Speelman P, van der Poll T. Erythromycin inhibits Pseudomonas aeruginosa-induced tumour necrosis Page 8/9 Braz. J. Pharm. Sci. 2020;56: e18547 This is an open-access article distributed under the terms of the Creative Commons Attribution License. Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats factor-alpha production in human whole blood. J Antimicrob Chemother. 2001;48(2):275-278. Usui T, Meng X, Saide K, Farrell J, Thomson P, Whitaker P, et al. From the cover: characterization of isoniazid-specific T-cell clones in patients with anti-tuberculosis drug-related liver and skin injury. Toxicol Sci. 2017;155(2):420-431. Singh S, Yadav CPS, Noolvi MN. Immunomodulatory activity of butanol fraction of Gentiana olivieri Griseb. on Balb/C mice. Asian Pac J Trop Biomed. 2012;2(6):433-437. Vinnard C, Ravimohan S, Tamuhla N, Ivaturi V, Pasipanodya J, Srivastava S, et al. Isoniazid clearance is impaired among human immunodeficiency virus/tuberculosis patients with high levels of immune activation. Br J Clin Pharmacol. 2017;83(4):801-811. Sisay S, Mengistu B, Erku W, Woldeyohannes D. Ten years’ experience of Directly Observed Treatment Short-course (DOTS) in Gambella Regional State, Ethiopia: An evaluation of tuberculosis control program. Int J Mycobacteriol. 2016;5(Suppl 1):S117-S118. Wang B, Bian R, Wei C, Sun S, Gao P. Cefodizime increases peripheral blood CD4/CD8 and Th1/Th2 ratios in senile patients with bacterial pneumonia. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015;31(4):528-531. (in Chinese) Wang B, Bian R, Wei C, Sun S, Gao P. Cefodizime increases peripheral blood CD4/CD8 and Th1/Th2 ratios in senile patients with bacterial pneumonia. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015;31(4):528-531. (in Chinese) Tauber SC, Nau R. Immunomodulatory properties of antibiotics. Curr Mol Pharmacol. 2008;1(1):68-79. Thada S, Ponnana M, Sivangala R, Joshi L, Alasandagutti M, Ansari MS, et al. Polymorphisms of IFN-gamma (+874A/T) and IL-12 (+1188A/C) in tuberculosis patients and their household contacts in Hyderabad, India. Hum Immunol. 2016;77(7):559-565. Received for publication on 24th July 2018 Accepted for publication on 02nd January 2019 Received for publication on 24th July 2018 Accepted for publication on 02nd January 2019 Braz. J. Pharm. Sci. 2020;56: e18547 Page 9/9
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Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes
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1. Introduction Salt deposits in the crop field are a result of the use of saline underground irrigation water. Discharge of industrial effluents into irrigation canals is also a potential source of salts in agricultural soil. Saline water reduces the transpiration rate of plants by disrupting the evapotranspiration system thus reducing crop yield [6]. A high percentage of salt in the root zone affects root density, root turgor pressure, and water absorption, which eventually affects plant growth and development. The okra plant is sensitive to salinity especially in the early stage of its growth [7] where salinity affects water and nutrient uptake of the plant, and ionic stress reduces leaf expansion. Altered morphological traits in the canola plant [8], reduced plant dry matter and leaf area in soybeans [9], and reduced yield of canola [10] due to salinity have been reported. In the root zone, high salt concentration decreases soil water potential and water availability, which causes dehydration at the cellular level, eventually leading to osmotic stress [11]. Salt deposits in the crop field are a result of the use of saline underground irrigation water. Discharge of industrial effluents into irrigation canals is also a potential source of salts in agricultural soil. Saline water reduces the transpiration rate of plants by disrupting the evapotranspiration system thus reducing crop yield [6]. A high percentage of salt in the root zone affects root density, root turgor pressure, and water absorption, which eventually affects plant growth and development. The okra plant is sensitive to salinity especially in the early stage of its growth [7] where salinity affects water and nutrient uptake of the plant, and ionic stress reduces leaf expansion. Altered morphological traits in the canola plant [8], reduced plant dry matter and leaf area in soybeans [9], and reduced yield of canola [10] due to salinity have been reported. In the root zone, high salt concentration decreases soil water potential and water availability, which causes dehydration at the cellular level, eventually leading to osmotic stress [11]. Soil salinity is a major problem in agriculture that limits plant growth and causes significant loss of crop productivity worldwide [1, 2]. Salinity affects up to 20% and 50% of the total cultivated and irrigated land in the world, respectively [3]. However, the use of saline water in agriculture is gradually increasing owing to shortage of fresh water. Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 6284547, 10 pages http://dx.doi.org/10.1155/2016/6284547 Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 6284547, 10 pages http://dx.doi.org/10.1155/2016/6284547 Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 6284547, 10 pages http://dx.doi.org/10.1155/2016/6284547 Sheikh Hasna Habib,1,2 Hossain Kausar,3,4 and Halimi Mohd Saud1 1Department of Agricultural Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2Oilseed Research Centre, Bangladesh Agricultural Research Institute (BARI), Gazipur 1701, Bangladesh 3Laboratory of Food Crops, Institute of Tropical Agriculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 4Department of Agroforestry and Environmental Science, Sher-E-Bangla Agricultural University, Dhaka, Banglade Correspondence should be addressed to Halimi Mohd Saud; halimi@upm.edu.my Received 10 September 2015; Revised 29 December 2015; Accepted 30 December 2015 Academic Editor: Qaisar Mahmood Copyright © 2016 Sheikh Hasna Habib 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. Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth- promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and examined their effect on salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme activities (SOD, APX, and CAT) and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR) were observed in PGPR inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under salinity stress. 2. Materials and Methods All plates were incubated for 48 h and the optical density (OD) was measured at 600 nm with the Thermo Scientific Multiskan™GO microplate spectropho- tometer (Thermo Fisher Scientific Inc., USA).h (hi , ) The OD values of ACC- and (NH4)2SO4-containing wells were compared with the MgSO4-containing wells to determine the ability of PGPR isolates to utilize ACC for their growth. The PGPR isolates were categorized into three groups as isolates with higher, medium, and lower ACC utilizing rate depending upon the ratio of their OD values at 600 nm for ACC substrate as compared to (NH4)2SO4. Isolates with higher ACC-utilization rate showed OD values for wells with ACC substrate close to OD values for wells with (NH4)2SO4 in the initial 48 h of growth. Similarly, isolates with medium ACC-utilization rate showed lower OD values for ACC wells as compared to those for (NH4)2SO4 in the initial 48 h of growth. Isolates with lower ACC-metabolic rate possessed the lowest OD values for ACC wells (close to OD values of wells with MgSO4) in the same time. f The plant growth-promoting effects of the interactions of Pseudomonas fluorescens YsS6, P. migulae 8R6, and their ACC deaminase deficient mutants on the growth of tomato plants were investigated under 165 mM and 185 mM of salt stress. The plants treated with ACC deaminase-containing bacterial isolates exhibited higher fresh and dry biomass, higher chlorophyll content, and a greater number of flowers and buds than the ACC deaminase deficient bacteria and control plants [27]. Similarly, Saravanakumar and Samiyappan [22] reported that the ACC deaminase-containing P. fluorescens strain TDK1 increased the vigor index of groundnut seedlings significantly under 120 mM of salt stress condition as com- pared to plants pretreated with a Pseudomonas strain lacking ACC deaminase activity. ACC deaminase-containing Bacillus subtilis also induced salinity stress tolerance in hydropon- ically grown tomato plants [28]. Bacteria containing high amounts of SOD and CAT play an important protective role against the deleterious effect of ROS under stress conditions [16, 29]. Moreover, induction of salt stress tolerance using PGPR is an efficient and inexpensive method. However, there are very few reports on PGPR induced salinity tolerance in the okra plant caused by changes in ROS-scavenging enzymes. Therefore, in this study, we characterized the ACC deaminase-containing PGPR with respect to its effect on 2.3. Identification of PGPR Isolates. 2. Materials and Methods 2.1. Source of Plant Growth-Promoting Rhizobacteria. Fifteen PGPR isolates were used in this study. All PGPR isolates were initially isolated from crop fields of the Universiti Putra Malaysia (UPM), Serdang, Selangor, and Semerak, Pasir Puteh, Kelantan, Malaysia, by using the dilution plate technique. All selected PGPR isolates possessed nitrogen fixation, phosphate solubilization, indoleacetic acid (IAA) synthesis, and salt tolerant properties [30]. Genetic engineering is an attractive approach that can generate plants resistant to salt stress [18]. AtNHX1 overex- pressing transgenic Brassica napus plants were found to grow, flower, and produce seeds under 200 mM salt stress [19]. However, the transgenic approach is time-consuming and expensive. ACC deaminase-containing PGPR can reduce the deleterious effects of environmental stress and can enhance stress tolerance of plants by a variety of mechanisms such as the synthesis of phytohormones, mineral solubilization, nutrient uptake, increased leaf area, increased chlorophyll and soluble protein content, and antioxidant enzyme activi- ties [20]. Ethylene is important for plant growth and devel- opment, as well as in the fruit ripening process, but an excess amount of ethylene might decrease seed germination and root growth [21, 22]. It is also reported that ethylene production increases under stress conditions and results in inhibitory effects on plants [23]. However, ACC deaminase- containing PGPR can hydrolyse ACC, the precursor of ethylene, thereby reducing the excess ethylene and rescue plants from inhibitory effects [24–26]. 2.2. ACC Deaminase Activity of Plant Growth-Promoting Rhizobacteria. All 15 PGPR isolates were tested for ACC deaminase activity following the method described by Jacob- son et al. [31]. Bacterial isolates were grown on Nutrient Broth (NB, Difco™) for 48 h at 28 ± 2∘C. The cultures were diluted 10-fold with sterile MgSO4 (0.1 M) solution. ACC (3 mM) was filter sterilized with 0.2 𝜇m filter membrane and stored at −20∘C until further use. In a 96-well plate, 120 𝜇L of minimal salt medium (MSM) was added to each well. To the first 4 lanes, 15 𝜇L of MgSO4 (0.1 M) was added and, to the next 4 lanes, 15 𝜇L of (NH4)2SO4 (0.1 M) was added. Thawed ACC (15 𝜇L) was added to the remaining 4 lanes. Each of the PGPR isolates (15 𝜇L) was added into each well separately. For the untreated control, 15 𝜇L of MgSO4 (0.1 M) was used instead of the PGPR culture. 1. Introduction Consequently, on one hand, salt-affected areas are constantly increasing, and, on the other hand, a significant amount of arable land is being abandoned every year because of salinity [4]. Okra (Abelmoschus esculentus L.) is an annual vegetable crop cultivated in tropical and subtropical regions. It is considered a high-value vegetable crop owing to its high levels of vitamins, minerals, carbohydrates, and fats [5]. Although it has good nutritional value as well as high con- sumer demand, the yield of okra per hectare is very low, and this lower productivity arises mainly from soil salinity. 2 BioMed Research International growth, antioxidant enzyme activities, and expression profiles of ROS pathway genes in the okra plant under high salt stress. Salinity stress generates reactive oxygen species (ROS), namely, H2O2, O−2, and OH−that damage the DNA, RNA, and proteins [12, 13]. These ROS compounds also cause chlorophyll destruction and damage the root meristem activ- ity [14]. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) have the ability to scavenge the ROS and maintain them at low levels. Superoxide dismutase is a metalloenzyme that plays an important role in protecting cells from oxidative damage, by catalyzing the conversion of the superoxide radical to H2O2 [15, 16]. Ascorbate peroxidase has vital defensive role against ROS [17] and can catalyze the breakdown of H2O2 that is produced by SOD. Catalase reduces ROS levels by catalyzing the breakdown of H2O2 into H2O and O2 [12, 13]. 2. Materials and Methods PGPR isolates, UPMR2 and UPMR18, with the highest ACC-metabolizing activities were identified by 16S rRNA gene sequencing as Bacillus megaterium and Enterobacter sp., respectively (Figure 1(a)). In brief, each PGPR isolate was grown at 28∘C for 48–72 h in 5 mL of NB. Of each culture, 1 mL was transferred into separate 1.5 mL microcentrifuge tubes. Bacterial cells were pelleted by centrifugation at 12000 g for 5 min (Eppendorf Centrifuge 5810 R, Hamburg, Germany). Bacterial genomic DNA was extracted with the Genomic DNA Mini Kit BioMed Research International 3 1 2 M 1400bp (a) Bacillus nealsonii KC329823 Bacillus circulans strain KM349203 UPMR2 Bacillus megaterium KF956591 UPMR18 Enterobacter sp. GQ871449 Enterobacter sp. LC014954 Bacillus sp. AB425363 100 100 100 73 78 0.1 (b) Figure 1: (a) Agarose gel electrophoresis of 16S rDNA PCR products of bacterial isolates. Lane M: 100 bP DNA ladder. Lanes 1 and 2: bacterial isolates UPMR2 and UPMR18. (b) The neighbor-joining tree shows the phylogenetic relationship of the isolates UPMR2 and UPMR18 with related isolates from the NCBI database. 1 2 M 1400bp Bacillus nealsonii KC329823 Bacillus circulans strain KM349203 UPMR2 Bacillus megaterium KF956591 UPMR18 Enterobacter sp. GQ871449 Enterobacter sp. LC014954 Bacillus sp. AB425363 100 100 100 73 78 0.1 1400bp Bacillus nealsonii KC329823 Bacillus circulans strain KM349203 UPMR2 Bacillus megaterium KF956591 UPMR18 Enterobacter sp. GQ871449 Enterobacter sp. LC014954 Bacillus sp. AB425363 100 100 100 73 78 0.1 (b) 1 2 M (a) (b) (a) Figure 1: (a) Agarose gel electrophoresis of 16S rDNA PCR products of bacterial isolates. Lane M: 100 bP DNA ladder. Lanes 1 and 2: bacterial isolates UPMR2 and UPMR18. (b) The neighbor-joining tree shows the phylogenetic relationship of the isolates UPMR2 and UPMR18 with related isolates from the NCBI database. (25, 50, 75, and 100 mM) and kept at 25∘C in the dark. After three days, the emergence of the radicle from the seed was considered as an index of germination. The germination percentage was calculated as follows: (Yeastern Biotech Co., Ltd.) according to the manufacturer’s protocol.h p The universal primer pairs 27f (5󸀠-AGAGTTTGATCM- TGGCTCAG-3󸀠) and 1492R (5󸀠-GGTTACCTTGTTACG- ACTT-3󸀠) were used to amplify the 16S rRNA coding region. 2. Materials and Methods The soil surface was covered with black plastic to prevent salt loss. Four treatment groups were defined as follows: T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt treated plants. Each treatment group comprised 16 plants. Plants were grown for 15 days under salt stress conditions. Growth parameters, that is, plant height, root length, fresh weight of leaf, stem, and root, and dry weight of leaf, stem, and root, were recorded at 15 days after salt treatment. 2.5. Evaluation of PGPR Isolates on Growth Promotion of Okra under Salt Stress. Okra seeds were surface sterilized and sown in Petri dishes. After emergence of the radicle and plumule, the seedlings were planted in plastic pots (one seedling/pot) filled with sterilized soil. The soil belongs to Serdang soil series, which was sandy-loam in texture (order Ultisols). The soil contained total N (0.78%), available P (54.92 ppm), potassium (218 ppm), and total C (2.94%) and had a pH of 5.32. Twenty-five milliliters of PGPR suspension (108 cfu/mL) was applied to the roots of potted okra plants at 15 days after transplantation (DAT) and 22 DAT. Control plants were treated with the same amount of water. The plants were subjected to salt stress two weeks after the second inoculation by watering with 75 mM of NaCl solution. To prevent the plants from undergoing osmotic shock, NaCl concentration was imposed in 25 mM increments per day until the final concentration was attained after three days [33]. The soil surface was covered with black plastic to prevent salt loss. Four treatment groups were defined as follows: T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt treated plants. Each treatment group comprised 16 plants. Plants were grown for 15 days under salt stress conditions. Growth parameters, that is, plant height, root length, fresh weight of leaf, stem, and root, and dry weight of leaf, stem, and root, were recorded at 15 days after salt treatment. 2.4. Evaluation of PGPR Isolates on Okra Seed Germination under Salt Stress. Two PGPR isolates UPMR2 and UPMR18 were selected based on their growth promotion, halotoler- ance, and ACC deaminase activities and were investigated in the context of seed germination in okra under different levels of NaCl stress. 2. Materials and Methods The 25 𝜇L PCR reaction mixture consisted of the following components: 1 𝜇L of each extracted PGPR genomic DNA, 0.15 𝜇M of each primer, 1x PCR reaction buffer (Fermentas, USA), 0.2 mM of dNTP mix, 2.5 U of Taq polymerase (Fermentas, USA), 25 mM of MgCl2, and nucleic acid- free water to make up final volume. The thermal cycling conditions were as follows: one cycle for 5 min at 94∘C, 30 cycles at 94∘C for 30 sec, 55∘C for 30 sec, and 72∘C for 1 min followed by an additional cycle at 72∘C for 5 min. The amplified PCR products were purified using QIAquick Gel Extraction Kit (QIAGEN Inc., USA) and sent for sequencing. The obtained sequences were analyzed and compared with sequences obtained from the GenBank database using the BLAST program to determine the percent similarity. A molecular phylogenetic tree was constructed in Mega version 4 software following the neighbor-joining method [32]. The BLASTX analysis showed that the isolates UPMR2 and UPMR18 matched with B. megaterium and Enterobacter sp., respectively, with 99% similarity. Phylogenetic analysis of the bacterial isolates was also performed on the basis of the neighborhood joining method with 100 bootstrap sampling replicates (Figure 1(b)). Germination % = (No. of seeds germinated No. of seeds sown ) × 100. (1) Germination % = (No. of seeds germinated No. of seeds sown ) × 100. (1) (1) 2.5. Evaluation of PGPR Isolates on Growth Promotion of Okra under Salt Stress. Okra seeds were surface sterilized and sown in Petri dishes. After emergence of the radicle and plumule, the seedlings were planted in plastic pots (one seedling/pot) filled with sterilized soil. The soil belongs to Serdang soil series, which was sandy-loam in texture (order Ultisols). The soil contained total N (0.78%), available P (54.92 ppm), potassium (218 ppm), and total C (2.94%) and had a pH of 5.32. Twenty-five milliliters of PGPR suspension (108 cfu/mL) was applied to the roots of potted okra plants at 15 days after transplantation (DAT) and 22 DAT. Control plants were treated with the same amount of water. The plants were subjected to salt stress two weeks after the second inoculation by watering with 75 mM of NaCl solution. To prevent the plants from undergoing osmotic shock, NaCl concentration was imposed in 25 mM increments per day until the final concentration was attained after three days [33]. 2. Materials and Methods patterns of genes of the ROS pathway (APX, CAT, DHAR, and GR) were analyzed by semiquantitative RT-PCR using the primer pairs [40] mentioned in Table 1 with actin as a control. 2.5.2. Enzymatic Assay. Leaf protein was extracted from frozen leaflet samples ground in liquid nitrogen using ice- cold mortar and pestle. Protein was extracted in 3 mL of extraction buffer containing 100 mM K-phosphate buffer (pH 7.8), 0.1 mM EDTA, 14 mM 2-mercaptoethanol, and 0.1% (v/v) Triton X-100 for SOD (EC 1.15.1.1) activity or 50 mM K-phosphate buffer (pH 7.0), 2 mM EDTA, 20 mM ascorbate, and 0.1% (v/v) Triton X-100 for CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) activities. The homogenate was than centrifuged at 15000 ×g for 15 min at 4∘C. The supernatant was used for total protein [36] and enzymatic assays. RT-PCR was performed using aliquots of 1 𝜇L cDNA samples. The PCR conditions were set as follows: one cycle at 95∘C for 5 min, followed by 35 cycles at 95∘C for 30 sec, 52∘C for 30 sec, and 72∘C for 15 sec followed by an additional cycle of five minutes at 72∘C. The PCR products were separated on 1.5% agarose gel and were observed under UV light. 2.6. Statistical Analysis. All experiments were conducted using completely randomized design (CRD) with four repli- cates. The data were subjected to analysis of variance (ANOVA) and tested for significance using the least signif- icant difference (LSD) by PC-SAS software (SAS Institute, Cary, NC, USA, 2001). 2.6. Statistical Analysis. All experiments were conducted using completely randomized design (CRD) with four repli- cates. The data were subjected to analysis of variance (ANOVA) and tested for significance using the least signif- icant difference (LSD) by PC-SAS software (SAS Institute, Cary, NC, USA, 2001). SOD activity was determined according to the method described by Giannopolitis and Ries [37]. The reaction mixture contained 50 mM K-phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 75 𝜇M nitrobluetetra- zolium (NBT), 2 𝜇M riboflavin, and 100 𝜇L enzyme extract. SOD activity was determined by the ability of the enzyme to inhibit photochemical reduction of NBT on blue formazan, followed by monitoring absorbance of the reaction mixture at 560 nm.h 2. Materials and Methods Seeds of the local Malaysian okra variety five anchor were used in this study. The seeds were surface sterilized with 0.5% (v/v) sodium hypochlorite for 20 min followed by repetitive washes with distilled water. The seeds were then soaked in PGPR suspension (108 cfu/mL) or in distilled water (control) for 24 h. A total of 10 seeds were sown in Petri dishes (9 mm diameter) with two sheets of Whatman number 1 filter papers. The seeds were moistened either with distilled water (control) or with solutions of varying NaCl concentration 2.5.1. Leaf Chlorophyll Measurements. The total leaf chloro- phyll and chlorophyll a (chla) and chlorophyll b (chlb) content and the chlorophyll a/b ratio were determined at 15 days after salt treatment by using the method described by Moran and Porath [34]. Frozen leaflet samples of 0.2 g from each treatment were cut into small pieces and placed in a glass vial containing 2 mL of N,N-dimethylformamide 4 BioMed Research International Table 1: Primer pairs used for RT-PCR in this study. NCBI accession number Primer name Sequence (5󸀠-3󸀠) X55749 Actin F: CTGGTGGTGCAACAACCTTA R: GAATGGAAGCAGCTGGAATC AB041343 APX F: ACCAATTGGCTGGTGTTGTT R: TCACAAACACGTCCCTCAAA AY442179 CAT F: TGCCCTTCTATTGTGGTTCC R: GATGAGCACACTTTGGAGGA X76533 GR F: GGATCCTCATACGGTGGATG R: TTAGGCTTCGTTGGCAAATC DQ512964 DHAR F: AGGTGAACCCAGAAGGGAAA R: TATTTTCGAGCCCACAGAGG Table 1: Primer pairs used for RT-PCR in this study. (DMF) and were covered with aluminum foil. The vials were then incubated at 4∘C for 48 h. The absorbance readings of chlorophyll a and b were taken at wave lengths of 663 nm and 645 nm, respectively, with a UV-visible spectrophotometer (Genesys 10 UV, Thermo Fisher Scientific, USA) using DMF as a blank. The concentrations of total chlorophyll, chla, and chlb were calculated according to the following equations [35]: Chlorophyll a = (0.0127𝐷663 −0.00269𝐷645) , Chlorophyll b = (0.0229𝐷645 −0.0468𝐷663) , Total chlorophyll = Chlorophyll a + Chlorophyll b, (2) Chlorophyll a = (0.0127𝐷663 −0.00269𝐷645) , Chlorophyll b = (0.0229𝐷645 −0.0468𝐷663) , (2) Total chlorophyll = Chlorophyll a + Chlorophyll b, where 𝐷663 is absorbance at 663 nm wave length and 𝐷645 is absorbance at 645 nm wave length. patterns of genes of the ROS pathway (APX, CAT, DHAR, and GR) were analyzed by semiquantitative RT-PCR using the primer pairs [40] mentioned in Table 1 with actin as a control. 3. Results The total CAT activity in the leaf was assayed based on the rate of H2O2 consumption at 240 nm [38]. The assay mixture of 3 mL contained 100 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.1% H2O2, and 20 𝜇L enzyme extract. After addition of the enzyme extract to the reaction mixture, decrease in H2O2 levels was determined by measuring the absorbance at 240 nm with a UV1000 spectrophotometer and quantified by using the extinction coefficient (36 M21 cm21). 3.1. ACC Deaminase Activity. The ACC deaminase activity of the PGPR isolates was determined qualitatively to char- acterize the isolates for their ability to use ACC as the sole nitrogen source. The PGPR isolates were categorized into three groups, as isolates with higher (>0.7), medium (0.5– 0.69), and lower (<0.5) ACC metabolism rate depending on their growth which was measured in terms of cell density at OD600 (data not shown). On the basis of their high ACC deaminase activity, two PGPR isolates B. megaterium UPMR2 and Enterobacter sp. UPMR18 were selected to assess their salt tolerance potency in okra plants. fi Total leaf APX activity was estimated at 290 nm by the method described by Chen and Asada [39]. The 3 mL APX assay mixture contained 50 mM K-phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM ascorbate, and 20 𝜇L enzyme extract. The amount of ascorbate oxidized was calculated using extinction coefficient 𝐸= 2.8 mM−1 cm−1. 3.2. Effect of PGPR Isolates on Germination of Okra Seeds under Salt Stress. The results showed that the germination of okra seeds was significantly (𝑝≤0.05) affected by the PGPR isolates under different salt concentrations (Table 2). Both the PGPR isolates either separately or in combination showed 100% seed germination up to 75 mM NaCl con- centrations. Seed germination declined to 80% at 100 mM NaCl concentration. On the other hand in the noninoculated group, seed germination percentage was 70% at 75 mM NaCl 2.5.3. Expression of ROS Pathway Genes. Total RNA was extracted from approximately 0.1 g of frozen okra leaf sam- ples using TRIzolⓇreagent (Invitrogen, USA). The first- strand cDNA was synthesized from 1 𝜇g of total RNA using QuantiTectⓇReverse Transcription Kit (Qiagen, USA) according to the manufacturer’s instructions. The expression 5 BioMed Research International BioMed Research International Table 2: Effect of PGPR inoculation on germination of okra seeds under salt stress. concentration while the lowest germination percentage (50%) was recorded at 100 mM NaCl concentration. 3.3. Effect of PGPR Isolates on Growth of Okra Seedlings under Salt Stress. The effects of PGPR inoculation on okra plants were assessed at 75 mM NaCl concentration. The results demonstrate that the application of PGPR isolates B. megaterium UPMR2 and Enterobacter sp. UPMR18 sig- nificantly (𝑝≤0.05) increased shoot and root growth of okra as compared to noninoculated plants. Significantly (𝑝≤ 0.05), the highest leaf fresh (4.04 g) and dry (0.469 g) weight were obtained in Enterobacter sp. UPMR18 (T2) treated plants compared to other treatments. As with leaf biomass, significantly (𝑝≤0.05) highest shoot dry weight (0.28 g) and root fresh weight (0.78 g) were also observed in the plants receiving Enterobacter sp. UPMR18 (T2) under salt stress. In addition, significantly (𝑝≤0.05) highest shoot fresh weight and root dry weight were recorded both in Enterobacter sp. UPMR18 (T2) treatment and in the combined application of B. megaterium UPMR2 and Enterobacter sp. UPMR18 (T3) treated plants under salinity stress conditions (Table 3). 3. Results Treatments Salinity levels 0 mM 25 mM 50 mM 75 mM 100 mM Control 100 100 100 70b 50b Bacillusmegaterium (UPMR2) 100 100 100 100a 80a Enterobacter sp. (UPMR18) 100 100 100 100a 80a Coinoculation of UPMR2 & UPMR18 100 100 100 100a 80a Means within columns with the same letters are not significantly different at 𝑝< 0.05. Table 2: Effect of PGPR inoculation on germination of okra seeds under salt stress. Table 2: Effect of PGPR inoculation on germination of okra seeds under salt stress. inoculated with Enterobacter sp. UPMR18 (T2), combined PGPR (T3) application, and B. megaterium UPMR2 (T1), respectively, as compared to control plants under salt stress. Similarly, the activity of SOD was also increased significantly (𝑝≤0.05) (approximately 2 and 1.5 times higher) in the plants inoculated with Enterobacter sp. UPMR18 (T2) and with the combined application of both PGPR (T3), compared to the noninoculated stressed plants. 3.6. Expression Analysis of ROS Pathway Genes by RT-PCR. The ROS-scavenging enzymes, CAT, SOD, and APX, were assessed in okra plants after salinity stress, after inocu- lation, or without inoculation with PGPR B. megaterium UPMR2 and Enterobacter sp. UPMR18. The PGPR inoculated salinized plants exhibited the maximum percent increase in ROS-scavenging enzymes with respect to the noninocu- lated salinized control plants. APX activity was significantly increased in B. megaterium UPMR2 inoculated salinized plants and was approximately 13 times higher than that of noninoculated salinized plants (Figure 4). Significantly increased CAT activity was also observed in all PGPR inoc- ulated salinized plants than that of control (noninoculated salinized plants). CAT activity was recorded as approxi- mately 3.4, 5.4, and 4.8 times higher in plants inoculated with B. megaterium UPMR2, Enterobacter sp. UPMR18, and combined application of PGPR isolates, respectively, compared to that of noninoculated plants under the same stress conditions (Figure 4). Similarly, SOD activity was also increased significantly (approximately 2 and 1.5 times higher) in plants inoculated with Enterobacter sp. UPMR18 and with combined application of PGPR, respectively, growing under stress condition compared to noninoculated salt stressed plants (Figure 4). 4. Discussion Application of ACC deaminase-containing PGPR as a soil amendment resulted in enhanced seed germination, chloro- phyll content, and growth of okra plants under salinity stress by maintaining low stress ethylene levels and increasing the ROS-scavenging enzymes. Ethylene, a plant growth regulator, is involved in various physiological responses [41]. However, it is regarded as a stress hormone since it is synthesized at a rapid rate under stress [42]. Stress ethylene decreases seed germination and root development and eventually hinders plant growth [21, 22]. Microorganisms synthesizing the ACC deaminase enzyme can cleave ACC to 𝛼-ketobutyrate and ammonia, thereby decreasing ethylene stress in plants [43– 45]. In this study, two PGPR B. megaterium UPMR2 and Enterobacter sp. UPMR18 that possessed characteristics of ACC deaminase activity demonstrated their effectiveness in inducing salt tolerance and consequent improvement in the growth of okra plants under salt stress. 3.4. Determination of Okra Leaf Chlorophyll Content. The data obtained for plant pigments showed that chlorophyll a was significantly (𝑝≤0.05) highest in plants treated with Enterobacter sp. UPMR18 (T2) and the combined application of both the PGPR (T3) compared to other treatments. Chlorophyll b and chlorophyll a/b ratio were increased significantly (𝑝≤0.05) in all inoculated plants compared to noninoculated salinized plants. Similarly, all the PGPR treated plants showed significantly (𝑝≤0.05) higher values for total chlorophyll content than the control (Figure 2). 3.5. Determination of Activities of ROS-Scavenging Enzymes. All PGPR inoculated plants exhibited higher percent increase in ROS-scavenging enzymes in comparison to the noninocu- lated control plants (Figure 3). APX activity was significantly (𝑝≤0.05) highest in B. megaterium UPMR2 (T1) inoculated salinized plants where it was approximately 13 times higher than the noninoculated plants. Significantly (𝑝≤0.05) higher CAT activity was also observed in all PGPR inoculated plants as compared to control. CAT activity was recorded to be approximately 5.4, 4.8, and 3.4 times higher in the plants Okra is a salt sensitive crop, especially in its early growth stage [46]. Seed germination of okra was reduced at a higher rate with increasing level of salinity in the noninoculated group (Table 2). Salinity increases the osmotic potential of growth medium and, as a result, seeds require more energy to absorb water, resulting in decreased germination BioMed Research International 6 Table 3: Effect of PGPR inoculation on growth attributes of okra plants under salinity stress. 4. Discussion Treatment LFW/plant LDW/plant SFW/plant SDW/plant RFW/plant RDW/plant (g) (g) (g) (g) (g) (g) T0 2.63 ± 0.13c 0.26 ± 0.03c 1.57 ± 0.14c 0.14 ± 0.02c 0.34 ± 0.03d 0.04 ± 0.01b T1 2.83 ± 0.15c 0.28 ± 0.03c 1.90 ± 0.08b 0.16 ± 0.02c 0.47 ± 0.05c 0.03 ± 0.00b T2 3.92 ± 0.17a 0.46 ± 0.01a 2.56 ± 0.29a 0.28 ± 0.01a 0.79 ± 0.08a 0.07 ± 0.02a T3 3.28 ± 0.25b 0.37 ± 0.01b 2.65 ± 0.07a 0.22 ± 0.01b 0.65 ± 0.06b 0.06 ± 0.00a T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: UPMR2 and UPMR18 inoculated salt treated plants. LFW: leaf fresh weight, LDW: leaf dry weight, SFW: shoot fresh weight, SDW: shoot dry weight, RFW: root fresh weight, and RDW: root dry weight. Means within columns with the same letters are not significantly different at 𝑝< 0.05. Table 3: Effect of PGPR inoculation on growth attributes of okra plants under salinity stress. Treatment LFW/plant LDW/plant SFW/plant SDW/plant RFW/plant RDW/plant (g) (g) (g) (g) (g) (g) T0 2.63 ± 0.13c 0.26 ± 0.03c 1.57 ± 0.14c 0.14 ± 0.02c 0.34 ± 0.03d 0.04 ± 0.01b T1 2.83 ± 0.15c 0.28 ± 0.03c 1.90 ± 0.08b 0.16 ± 0.02c 0.47 ± 0.05c 0.03 ± 0.00b T2 3.92 ± 0.17a 0.46 ± 0.01a 2.56 ± 0.29a 0.28 ± 0.01a 0.79 ± 0.08a 0.07 ± 0.02a T3 3.28 ± 0.25b 0.37 ± 0.01b 2.65 ± 0.07a 0.22 ± 0.01b 0.65 ± 0.06b 0.06 ± 0.00a T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: UPMR2 and UPMR18 inoculated salt treated plants. LFW: leaf fresh weight, LDW: leaf dry weight, SFW: shoot fresh weight, SDW: shoot dry weight, RFW: root fresh weight, and RDW: root dry weight. Means within columns with the same letters are not significantly different at 𝑝< 0.05. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: UPMR2 and UPMR18 inoculated salt treated plants. LFW: leaf fresh weight, LDW: leaf dry weight, SFW: shoot fresh weight, SDW: shoot dry weight, RFW: root fresh weight, and RDW: root dry weight. Means within columns with the same letters are not significantly different at 𝑝< 0.05. 4. Discussion Treatments T0 T1 T2 T3 c b a a 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Chlorophyll a mg/g fresh leaf Treatments T0 T1 T2 T3 b a a a 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Chlorophyll b mg/g fresh leaf Treatments T0 T1 T2 T3 c b a a 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Chlorophyll a mg/g fresh leaf Treatments T0 T1 T2 T3 b a a a 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Chlorophyll b mg/g fresh leaf Treatments T0 T1 T2 T3 c b a a 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Chlorophyll a/b mg/g fresh leaf Treatments T0 T1 T2 T3 c b a a 0.000 1.000 2.000 3.000 4.000 5.000 6.000 7.000 Total chlorophyll mg/g fresh leaf Figure 2: Effect of PGPR inoculation on chlorophyll content of okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3; both UPMR2 and UPMR18 inoculated salt treated plants. Error bars refer to standard error of means of four replicates. Means within columns with the same letters are not significantly different at 𝑝< 0.05. Treatments T0 T1 T2 T3 b a a a 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Chlorophyll b mg/g fresh leaf Treatments Treatments Treatments T0 T1 T2 T3 c b a a 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Chlorophyll a/b mg/g fresh leaf Treatments T0 T1 T2 T3 c b a a 0.000 1.000 2.000 3.000 4.000 5.000 6.000 7.000 Total chlorophyll mg/g fresh leaf Treatments T0 T1 T2 T3 c b a a 0.000 1.000 2.000 3.000 4.000 5.000 6.000 7.000 Total chlorophyll mg/g fresh leaf Figure 2: Effect of PGPR inoculation on chlorophyll content of okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3; both UPMR2 and UPMR18 inoculated salt treated plants. Error bars refer to standard error of means of four replicates. Means within columns with the same letters are not significantly different at 𝑝< 0.05. [47, 48]. Our results were consistent with the results of previous studies [21, 22]. These studies demonstrated that increased salt concentrations decrease seed germination and root growth in dicotyledonous plants. 4. Discussion BioMed Research International 7 Treatments T0 T1 T2 T3 c d b a CAT activity 0 300 600 900 1200 (𝜇mol min−1 mg protein−1) Treatments T0 T1 T2 T3 c d b a APX activity 0 100 200 300 400 500 600 Treatments T0 T1 T2 T3 c d b a CAT activity 0 300 600 900 1200 (𝜇mol min−1 mg protein−1) (𝜇mol min−1 mg protein−1) Treatments T0 T1 T2 T3 c c b a SOD activity 0 10 20 30 40 50 60 70 80 (𝜇mol min−1 mg protein−1) Treatments T0 T1 T2 T3 c c b a SOD activity 0 10 20 30 40 50 60 70 80 (𝜇mol min−1 mg protein−1) Treatments Figure 3: Effect of PGPR inoculation on antioxidant enzymes activities in okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt treated plants. Error bars refer to standard error of means of four replicates. APX: ascorbate peroxidase, CAT: catalase, and SOD: superoxide dismutase. Means within columns with the same letters are not significantly different at 𝑝< 0.05. observed that inoculation of plants with wild-type P. flu- orescens YsS6 and P. migulae 8R6 significantly increased the total chlorophyll content of tomato plants compared with their ACC deaminase deficient mutants and control plants under salt stress. Higher chlorophyll content was also reported in ACC deaminase-containing PGPR inoculated salt stressed rice [59] and cucumber [60] compared to noninoculated plants. [52]. Cucumber plants inoculated with wild-type P. putida UW4 and Gigaspora rosea BEG9 showed significantly higher root and shoot fresh biomass than the plants that received ACC deaminase deficient bacteria and untreated control plants under 72 mM salt stress [53]. When canola seeds were inoculated with ACC deaminase-containing salt tolerant bacteria under the 150 mM NaCl condition, the biomass of treated plants increased by up to 47% of the control plants [54]. Red pepper seedlings inoculated with ACC deaminase- containing salt tolerant bacteria reduced 57% stress ethylene production and produced similar amounts of biomass as to those in no salt treatment control plant [55]. Salinity stress leads to the formation of ROS, namely, superoxide (O2 −), singlet oxygen (O2), hydroxyl (OH−), and hydrogen peroxide (H2O2), which cause severe damage to cell structures by exerting oxidation of cell membranes in a process known as oxidative stress [17, 61]. 4. Discussion In addition, ger- mination of Limonium stocksii and Suaeda fruticosa seeds was inhibited with increasing NaCl concentrations [49]. In contrast, when the seeds were inoculated with the PGPR suspension, seed germination was reduced at a lower rate despite increasing salinity. This may indicate that the ACC deaminase-containing PGPR isolates, B. megaterium UPMR2 and Enterobacter sp. UPMR18, are able to ameliorate the effect of NaCl on growth medium. The PGPR got attached to the seed surface and synthesized phytohormones in response to amino acids produced by the seeds perhaps alleviating the salinity stress [50]. Our results were also supported by the findings of Jalili et al. [51] who demonstrated that the rate of germinating canola seeds (Brassica napus L.) inoculated with ACC deaminase-containing plant growth-promoting P. fluorescens and P. putida was significantly higher under salinity stress. y ACC deaminase-containing PGPR inoculated okra plants showed higher root and shoot biomass than noninoculated plants under salinity stress. This might be due to the presence of PGPR isolates in the growth medium, which alleviate the effects of salinity on okra plants by producing ACC deaminase. ACC deaminase-containing plant growth- promoting bacteria have been documented to facilitate the growth of a variety of plants under high salinity conditions BioMed Research International 7 Treatments T0 T1 T2 T3 c d b a APX activity 0 100 200 300 400 500 600 Treatments T0 T1 T2 T3 c d b a CAT activity 0 300 600 900 1200 Treatments T0 T1 T2 T3 c c b a SOD activity 0 10 20 30 40 50 60 70 80 (𝜇mol min−1 mg protein−1) (𝜇mol min−1 mg protein−1) (𝜇mol min−1 mg protein−1) Figure 3: Effect of PGPR inoculation on antioxidant enzymes activities in okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt treated plants. Error bars refer to standard error of means of four replicates. APX: ascorbate peroxidase, CAT: catalase, and SOD: superoxide dismutase. Means within columns with the same letters are not significantly different at 𝑝< 0.05. References [1] S. I. Allakhverdiev, A. Sakamoto, Y. Nishiyama, M. Inaba, and N. Murata, “Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcussp.,” Plant Physiology, vol. 123, no. 3, pp. 1047–1056, 2000. Figure 4: Effect of PGPR inoculation on DHAR, GR, APX, and CAT transcript levels by semiquantitative RT-PCR analysis of okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt treated plants. Actin: positive control, APX: ascorbate peroxidase, CAT: catalase, GR: glutathione reductase, and DHAR: dehydroascorbate reductase. [2] R. Munns, “Comparative physiology of salt and water stress,” Plant, Cell & Environment, vol. 25, no. 2, pp. 239–250, 2002. [3] Z. Cheng, O. Z. Woody, B. J. McConkey, and B. R. Glick, “Combined effects of the plant growth-promoting bacterium Pseudomonas putida UW4 and salinity stress on the Brassica napus proteome,” Applied Soil Ecology, vol. 61, pp. 255–263, 2012. [4] W. B. Frommer, U. Ludewig, and D. Rentsch, “Taking transgenic plants with a pinch of salt,” Science, vol. 285, no. 5431, pp. 1222– 1223, 1999. inoculated plants were adapted to saline conditions by elim- inating ROS through APX, CAT, and SOD activities. Our results are also supported by the findings of Gururani et al. [63], who reported enhanced activities of different ROS- scavenging enzymes in PGPR inoculated potato plants under stress [63]. Moreover, in our study semiquantitative RT-PCR results (Figure 4) revealed that expression levels of different ROS pathway genes encoding CAT, APX, GR, and DHAR were increased in the ACC deaminase-containing PGPR treated salinized plants compared to the untreated controls. This result confirmed that the plants acquired protection from salt challenge as a consequence of PGPR inoculation. Our results were in agreement with the findings of Gururani et al. [63] who reported enhanced mRNA expression of different ROS pathway genes under salt, drought, and heavy- metal stress in PGPR inoculated potato plants. [5] F. N. Emuh, A. E. Ofuoku, and E. Oyefia, “Effect of intercrop- ping okra (Hibiscus esclentus)with pumpkin (Curcubita maxima Dutch ex Lam) on some growth parameters and economic yield of maize (Zea mays) and maximization of land use in a fadama soil,” Research Journal of Biological Sciences, vol. 1, no. 1–4, pp. 50–54, 2006. [6] L. M. Dudley, A. Ben-Gal, and U. References Shani, “Influence of plant, soil, and water on the leaching fraction,” Vadose Zone Journal, vol. 7, no. 2, pp. 420–425, 2008. [7] A. Cerd´a, J. Pardines, M. A. Botella, and V. Martinez, “Effect of potassium on growth, water relations, and the inorganic and organic solute contents for two maize cultivars grown under saline conditions,” Journal of Plant Nutrition, vol. 18, no. 4, pp. 839–851, 1995. [8] H. M. Zadeh and M. B. Naeini, “Effects of salinity stress on the morphology and yield of two cultivars of canola (Brassica napus L.),” Journal of Agronomy, vol. 6, no. 3, pp. 409–414, 2007.f Acknowledgment APX The authors thank the Universiti Putra Malaysia for financial and technical support through the research Grant “Geran (GP-IBT/2013/9407000) Universiti Putra Malaysia.” CAT 4. Discussion However, a defensive system called the antioxidant enzyme system is also activated under stress conditions. This system consists of several ROS-scavenging enzymes such as superoxide dismu- tase (SOD), peroxidase (POD), glutathione reductase (GR), monohydroascorbate reductase (MDHAR), ascorbate perox- idase (APX), and catalase (CAT). These antioxidant enzymes have the ability to remove the free radicals produced during abiotic stress conditions in the cell [13, 58, 62]. The okra plants inoculated with ACC deaminase-containing PGPR exhibited significant elevation of antioxidant enzyme activities (APX, CAT, and SOD) compared to noninoculated plants under saline conditions (Figure 3), thus confirming that PGPR Leaf chlorophyll concentration is an indicator of salt tolerance and responds to increasing salinity [56]. Chloro- phyll content was significantly higher in okra plants receiving bacterial suspension compared with control plant. In control plants, chlorophyll is destroyed due to excessive amount of salts, ions (Na and Cl), or reactive oxygen species (ROS) which disturb the cellular metabolism and result in the degeneration of cell organelles in the leaf tissue [57, 58]. On the other hand, the inoculated salt stressed okra plants exhibited higher chlorophyll content and dark green leaves owing to the presence of ACC deaminase-containing PGPR isolates that maintain the photosynthetic efficiency of plants by reducing ethylene biosynthesis. Ali and colleagues [27] BioMed Research International 8 Actin DHAR GR APX CAT T3 T2 T1 T0 Figure 4: Effect of PGPR inoculation on DHAR, GR, APX, and CAT transcript levels by semiquantitative RT-PCR analysis of okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt treated plants. Actin: positive control, APX: ascorbate peroxidase, CAT: catalase, GR: glutathione reductase, and DHAR: dehydroascorbate reductase. Actin T3 T2 T1 T0 in areas where salinity is a major constraint. However, further research is required to validate the effectiveness of this PGPR isolate, Enterobacter sp. UPMR18, in field conditions before recommendation for large scale okra cultivation at the agricultural level. DHAR Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. GR 5. Conclusion [30] S. H. Habib, H. M. Saud, M. R. Ismail, and H. Kausar, “Bimolecular characterization of stress tolerant plant growth promoting rhizobacteria (PGPR) for growth enhancement of rice,” International Journal of Agriculture and Biology, In press. [16] R. Santos, D. H´erouart, A. Puppo, and D. Touati, “Critical protective role of bacterial superoxide dismutase in Rhizobium- legume symbiosis,” Molecular Microbiology, vol. 38, no. 4, pp. 750–759, 2000. [31] C. B. Jacobson, J. J. Pasternak, and B. R. Glick, “Partial purification and characterization of 1-aminocyclopropane-1- carboxylate deaminase from the plant growth promoting rhi- zobacterium Pseudomonas putida GR12-2,” Canadian Journal of Microbiology, vol. 40, no. 12, pp. 1019–1025, 1994. [17] K. Apel and H. Hirt, “Reactive oxygen species: metabolism, oxidative stress, and signal transduction,” Annual Review of Plant Biology, vol. 55, pp. 373–399, 2004. [32] K. Tamura, J. Dudley, M. Nei, and S. Kumar, “MEGA4: Molec- ular Evolutionary Genetics Analysis (MEGA) software version 4.0,” Molecular Biology and Evolution, vol. 24, no. 8, pp. 1596– 1599, 2007. [18] E. Sergeeva, S. Shah, and B. R. Glick, “Growth of transgenic canola (Brassica napus cv. Westar) expressing a bacterial 1- aminocyclopropane-1-carboxylate (ACC) deaminase gene on high concentrations of salt,” World Journal of Microbiology and Biotechnology, vol. 22, no. 3, pp. 277–282, 2006. [33] M. A. Shahid, M. A. Pervez, R. M. Balal et al., “Salt stress effects on some morphological and physiological characteristics of okra (Abelmoschus esculentus L.),” Soil and Environment, vol. 30, no. 1, pp. 66–73, 2011. [19] H.-X. Zhang, J. N. Hodson, J. P. Williams, and E. Blumwald, “Engineering salt-tolerant Brassicaplants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 22, pp. 12832–12836, 2001. [34] R. Moran and D. Porath, “Chlorophyll determination in intact tissues using N,N-dimethylformamide,” Plant Physiology, vol. 65, no. 3, pp. 478–479, 1980. [35] D. I. Arnon, “Copper enzymes in isolated chloroplasts. Polyphe- nol oxidase in Beta vulgaris,” Plant Physiology, vol. 24, no. 1, pp. 1–15, 1949. [20] S. Dobbelaere, J. Vanderleyden, and Y. Okon, “Plant growth- promoting effects of diazotrophs in the rhizosphere,” Critical Reviews in Plant Sciences, vol. 22, no. 2, pp. 107–149, 2003. [36] M. M. Bradford, “A rapid and sensitive method for the quanti- tation of microgram quantities of protein utilizing the principle of protein-dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976. 5. Conclusion [9] M. R. Amirjani, “Effect of salinity stress on growth, mineral composition, proline content, antioxidant enzymes of soybean,” American Journal of Plant Physiology, vol. 5, no. 6, pp. 350–360, 2010. Little is known about enhanced salinity tolerance in okra due to ACC deaminase-containing PGPR. The current study showed that ACC deaminase-containing Enterobacter sp. UPMR18 emerged as the best treatment agent for enhancing seed germination and growth of okra seedlings under salinity stress. This may perhaps be due to a reduction in the growth inhibitory effect of salt on okra plants through the enhanced activity of antioxidant enzymes and expression of ROS pathway genes induced by the PGPR. Therefore, Enterobacter sp. UPMR18 could be used for okra cultivation [10] A. Bybordi and E. Ebrahimian, “Effect of salinity stress on activity of enzymes involved in nitrogen and phosphorous metabolism case study: canola (Brassica napus L.),” Asian Journal of Agricultural Research, vol. 5, no. 3, pp. 208–214, 2011. [11] J. Lloyd, P. E. Kriedemann, and D. Aspinall, “Comparative sensitivity of ‘Prior Lisbon’ lemon and ‘Valencia’ orange trees to foliar sodium and chloride concentrations,” Plant, Cell & Environment, vol. 12, no. 5, pp. 529–540, 1989. BioMed Research International 9 [12] C. A. Jaleel, K. Riadh, R. Gopi et al., “Antioxidant defense responses: physiological plasticity in higher plants under abiotic constraints,” Acta Physiologiae Plantarum, vol. 31, no. 3, pp. 427– 436, 2009. endophytes that contain ACC deaminase,” Plant Physiology and Biochemistry, vol. 80, pp. 160–167, 2014. [28] M. Woitke, H. Junge, and W. H. Schnitzler, “Bacillus subtilis as growth promotor in hydroponically grown tomatoes under saline conditions,” Acta Horticulturae, vol. 659, pp. 363–369, 2004. [13] R. Mittler, “Oxidative stress, antioxidants and stress tolerance,” Trends in Plant Science, vol. 7, no. 9, pp. 405–410, 2002. [29] M. Becana, F. J. Paris, L. M. Sandalio, and L. A. Del R´ıo, “Isoenzymes of superoxide dismutase in nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) walp,” Plant Physiology, vol. 90, no. 4, pp. 1286–1292, 1989. [14] J. Foreman, V. Demidchik, J. H. F. Bothwell et al., “Reactive oxygen species produced by NADPH oxidase regulate plant cell growth,” Nature, vol. 422, no. 6930, pp. 442–446, 2003. [15] J. F. Moran, E. K. James, M. C. Rubio, G. Sarath, R. V. Klucas, and M. Becana, “Functional characterization and expression of a cytosolic iron-superoxide dismutase from cowpea root nodules,” Plant Physiology, vol. 133, no. 2, pp. 773–782, 2003. 5. Conclusion [21] A. A. Belimov, V. I. Safronova, T. A. Sergeyeva et al., “Char- acterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1- carboxylate deaminase,” Canadian Journal of Microbiology, vol. 47, no. 7, pp. 642–652, 2001. [37] C. N. Giannopolitis and S. K. Ries, “Superoxide dismutases: I. Occurrence in higher plants,” Plant Physiology, vol. 59, no. 2, pp. 309–314, 1977. [22] D. Saravanakumar and R. Samiyappan, “ACC deaminase from Pseudomonas fluorescens mediated saline resistance in ground- nut (Arachis hypogea) plants,” Journal of Applied Microbiology, vol. 102, no. 5, pp. 1283–1292, 2007. [38] E. A. Havir and N. A. McHale, “Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves,” Plant Physiology, vol. 84, no. 2, pp. 450–455, 1987. [23] J. Yang, J. W. Kloepper, and C.-M. Ryu, “Rhizosphere bacteria help plants tolerate abiotic stress,” Trends in Plant Science, vol. 14, no. 1, pp. 1–4, 2009. [39] G.-X. Chen and K. Asada, “Ascorbate peroxidase in tea leaves: occurrence of two isozymes and the differences in their enzy- matic and molecular properties,” Plant and Cell Physiology, vol. 30, no. 7, pp. 987–998, 1989. [24] S. Mayak, T. Tirosh, and B. R. Glick, “Effect of wild-type and mutant plant growth-promoting rhizobacteria on the rooting of mung bean cuttings,” Journal of Plant Growth Regulation, vol. 18, no. 2, pp. 49–53, 1999. [40] Hemavathi, C. P. Upadhyaya, N. Akula et al., “Biochemical analysis of enhanced tolerance in transgenic potato plants overexpressing d-galacturonic acid reductase gene in response to various abiotic stresses,” Molecular Breeding, vol. 28, no. 1, pp. 105–115, 2011. [25] B. R. Glick, C. B. Jacobson, M. K. Schwarze, and J. J. Pasternak, “1-Aminocyclopropane-1-carboxylic acid deaminase mutants of the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 do not stimulate canola root elongation,” Cana- dian Journal of Microbiology, vol. 40, no. 11, pp. 911–915, 1994. [41] F. B. Abeles, P. W. Morgan, and M. E. J. Salveit, Ethylene in Plant Biology, Academic Press, San Diego, Calif, USA, 2nd edition, 1992. [26] B. R. Glick, D. M. Penrose, and J. Li, “A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria,” Journal of Theoretical Biology, vol. 190, no. 1, pp. 63–68, 1998. [42] J. C. Stearns and B. R. Glick, “Transgenic plants with altered ethylene biosynthesis or perception,” Biotechnology Advances, vol. 21, no. 3, pp. 193–210, 2003. [43] B. R. 5. Conclusion Glick, “Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase,” FEMS Microbiology Letters, vol. 251, no. 1, pp. 1–7, 2005. [27] S. Ali, T. C. Charles, and B. R. Glick, “Amelioration of high salinity stress damage by plant growth-promoting bacterial BioMed Research International 10 [44] S. Rashid, T. C. Charles, and B. R. Glick, “Isolation and char- acterization of new plant growth-promoting bacterial endo- phytes,” Applied Soil Ecology, vol. 61, pp. 217–224, 2012. [58] T. Abbas, M. A. Pervez, C. M. Ayyub, and R. Ahmad, “Assess- ment of morphological, antioxidant, biochemical and ionic responses of salttolerant and salt-sensitive okra (Abelmoschus esculentus) under saline regime,” Pakistan Journal of Life and Social Sciences, vol. 11, no. 2, pp. 147–153, 2013. [45] Y. Sun, Z. Cheng, and B. R. 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Waqas et al., “Plant growth- promoting rhizobacteria reduce adverse effects of salinity and osmotic stress by regulating phytohormones and antioxidants in Cucumis sativus,” Journal of Plant Interactions, vol. 9, no. 1, pp. 673–682, 2014. [47] M. Jamil and E. H. Rha, “The effect of salinity (NaCI) on the germination and seedling of sugar beet (Beta vulgaris L.) and cabbage (Brassica oleracea L.),” Plant Resources, vol. 7, no. 3, pp. 226–232, 2004. [61] S. Hussain, A. Khaliq, A. Matloob, M. A. Wahid, and I. Afzal, “Germination and growth response of three wheat cultivars to NaCl salinity,” Soil & Environment, vol. 32, no. 1, pp. 36–43, 2013. [48] M. Kafi and M. Goldani, “Effect of water potential and type of osmoticum on seed germination of three crop species of wheat, sugarbeet, and chickpea,” Agricultural Sciences and Technology, vol. 15, no. 1, pp. 121–133, 2001. [62] G. M. 5. 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Tuning anhydrous proton conduction in single-ion polymers by crystalline ion channels
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ARTICLE OPEN ARTICLE Results g p y The design of proton-conducting polymers in the form of so- called single-ion polymers would resolve the above issue, since the current is carried only by protons, and no anion depletion should theoretically be observed.28 However, compared to con- ventional polymer electrolytes, where both protons and counter anions exhibit mobility, single ion-polymers exhibit lower con- ductivities by orders of magnitude.17–19,29,30 With this in mind, most studies on single-ion polymers typically aim at expediting the relaxation of polymer chains and ions.30,31 The presence of crystalline phases in such materials, commonly formed by clus- tered ions (dimers, quadrupoles, and so on), is avoided because it tends to slow down polymer chain relaxation. Glass transition temperature (Tg) of PSS:Im in PSS:Im-b-PMB was 40 °C, which decreased to 34 °C, 29 °C, and 15 °C with the addition of ZImS, ZImS/Im, and ZImS2/Im, respectively (Sup- plementary Fig. 1). The low Tg value is the main reason for employing low molecular weight polymer in this study to achieve thermodynamic equilibrium without extensive thermal annealing. However, one needs to ask the question of whether the above reasoning is always correct. The apparent advantage of crystalline electrolytes is the gain of molecularly arranged ion-binding sites, which may favor the structural diffusion of protons over vehicular diffusion.32 Therefore, the formation of well-organized crystalline protogenic channels from single-ion conducting polymers should allow the fabrication of highly efficient proton- conducting materials featuring long-range hydrogen bonding networks.33 Structural analysis of PSS:Im-b-PMB comprising additives. Neat ZImS and neutral Im are commonly obtained as monoclinic crystals of the P21/c space group.34,35 We found that the co- presence of ZImS and Im results in the formation of different crystal structures. For example, ZImS/Im afforded monoclinic crystals of the P1m1 space group (Supplementary Fig. 2 and Supplementary Table 1). The crystallization behavior of ZImS and Im was remarkably altered in the presence of polymer sulfonate groups. ZImS in PSS: Im phases did not crystallize when incorporated into PSS:Im-b- PMB, as confirmed by the wide-angle X-ray scattering (WAXS) profile shown in Fig. 2a. A typical amorphous halo, lacking the evidence of crystalline phases, was clearly revealed. Results Synthesis of single-ion conducting polymers. Poly(styrene-b- methylbutylene) (PS-b-PMB, 1.0-b-1.0 kg mol–1) block copoly- mer was synthesized as a precursor polymer. Degree of poly- merization (N) of PS and PMB were 10 and 14, respectively. 20 mol.% of styrene units were randomly sulfonated to yield poly (styrene sulfonate-b-methylbutylene) (PSS-b-PMB, 1.2-b-1.0 kg mol–1) that was subsequently doped with one equivalent of imi- dazole (Im) relative to the quantity of sulfonic acid groups. The above sample was denoted as PSS:Im-b-PMB, and its chemical structure is shown in Fig. 1. PSS homopolymer with N = 10 and a sulfonation level of 20 mol.% was synthesized and doped by Im to prepare PSS:Im as a control sample. In addition to these efforts, much attention has been paid to the development of anhydrous proton conductors in view of the increased importance of achieving high proton conductivity at elevated temperatures.17–22 As part of this standpoint, much effort has been directed at the investigation of ionic liquid–containing polymers, which have been identified as pro- mising next-generation ion conductors owing to the good ther- mal/chemical stability and high anhydrous conductivity at elevated temperatures.17–26 Although these studies have altered the stream of research on proton-conducting materials, the prevention of anion diffusion in ionic liquid–containing polymers remains a major challenge.27 Likewise, the same concern has been raised for other types of ionic conductors, i.e., metal-organic frameworks and covalent organic frameworks, owing to the substantial quantities of counter anions contained therein.10,11 Thus, although various high-conductivity materials have been developed, cases of their successful applications remain rare given that the actual current values observed during device operation are very low.11,27 In proton-conducting polymers such as PSS:Im-b-PMB and PSS:Im, the covalent attachment of anions to the polymer backbone eliminates anion migration. Considering the fact that most single-ion polymers suffer from low ionic conductivity, PSS: Im-b-PMB and PSS:Im were additionally modified by the introduction of a zwitterion, 3-(1-methyl-3-imidazolium) propa- nesulfonate (ZImS), and neutral Im to allow the modulation of ion dissociation degree and charge number density. The chemical similarities of Im and ZImS with PSS:Im ensure good thermo- dynamic compatibility with polymers. As denoted in Fig. 1, ZImS: Im molar ratio was varied as 1:0, 1:1, and 2:1 (per mole of –SO3–- Im+ moieties in the polymer), which were referred to as ZImS, ZImS/Im, and ZImS2/Im, respectively. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 2 S O HN NH m n N N N SO3 – + NH + + O O 8 14 Fig. 1 Molecular structures. Molecular structures of PSS:Im-b-PMB samples containing different molar ratios of ZImS:Im (1:0, 1:1, and 2:1 per mole of –SO3–-Im+ moieties in the polymer) I I n the past few decades, much effort has been devoted to the fabrication of proton-conducting materials1,2 ranging from protogenic polymers,3−6 liquid crystals,7,8 to porous organic frameworks.9–11 Although these studies were focused on different substances, they shared the same goal of achieving high proton conductivity via the creation of well-defined less tortuous ionic channels in mechanically stable supports.2,12,13 In addition, the suppression of anion diffusion in such materials has become increasingly important, since this diffusion is related to device polarization under applied dc voltage.14 The formation of well-defined ionic channels in mechanical supports can be achieved by covalent linking of ionophilic polymers to an ionophobic matrix,15,16 which results in sponta- neous nanometer-scale self-assembly with adjustable geometry. Several studies have dealt with the design of microphase- separated ion-containing polymers of various architectures13,17 to establish morphology-transport relationship. Fig. 1 Molecular structures. Molecular structures of PSS:Im-b-PMB samples containing different molar ratios of ZImS:Im (1:0, 1:1, and 2:1 per mole of –SO3–-Im+ moieties in the polymer) Tuning anhydrous proton conduction in single-ion polymers by crystalline ion channels Onnuri Kim 1, Kyoungwook Kim 2, U. Hyeok Choi3 & Moon Jeong Park 1,2 The synthesis of high-conductivity solid-state electrolyte materials with eliminated polar- ization loss is a great challenge. Here we show a promising potential of single-ion block copolymers with crystalline protogenic channels as efficient proton conductors. Through the self-organization of zwitterion, imidazole, and polystyrene sulfonate with controlled dipolar interactions therein, the distance between neighboring proton donors and acceptors in ionic crystals, as well as the dipolar orientation in nanoscale ionic phases was precisely tuned. This allowed a markedly high static dielectric constant comparable to water and fast structural diffusion of protons with a low potential barrier for single-ion polymers. The optimized sample exhibited a high proton diffusion coefficient of 2.4 × 10–6 cm2 s–1 under anhydrous conditions at 90 °C. 1 Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. 2 Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. 3 Department of of Polymer Engineering, Pukyong National University, Busan 608-737, Korea. These authors contributed equally: Onnuri Kim, Kyoungwook Kim. Correspondence and requests for materials should be addressed to M.J.P. (email: moonpark@postech.ac.kr) NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 1 1 Results Distances between the neighboring Im+ and Im were marked in each crystal ZImS Scattering intensity (a.u.) q (nm–1) ZImS2/Im ZImS/Im (002) (020) (–1–13) (1–21) (021 ) (020) (200) (002) (121) (–211) (–1–21) (1–31) (–121) (211) (220) (021) a (1–20) (102) (130) (0–12) (0–22) (–111) (111) (210) 10 12 14 16 18 (–102) (–121) q (nm–1) 10 12 14 16 18 ZImS/Im ZImS2/Im b b Scattering intensity (a.u.) c c 3 2 w/ZImS/Im 1 z y 1: 5.113 Å 2: 6.060 Å 3: 5.998 Å C H N O S w/ZImS2/Im 1 2 3 z x 1: 7.972 Å C H N O S 2: 6.548 Å 3: 5.372 Å Fig. 2 Self-organization of charged/dipolar moieties. a WAXS profiles of PSS:Im-b-PMB comprising various additives, measured at 25 °C, indexed with triclinic P1 space group (ZImS/Im) and monoclinic P2 1/c space group (ZImS2/Im). b Le Bail refinement of the WAXS patterns of PSS:Im-b-PMB with ZImS/Im and ZImS2/Im. c Molecular arrangements of SS:Im with ZImS/Im and ZImS2/Im. C, H, N, O, and S atoms are shown in black, pink, blue, red, and yellow, respectively. Distances between the neighboring Im+ and Im were marked in each crystal Fig. 3). Dissimilar dipolar orientations were expected for the samples depending on the molar ratio of ZImS:Im. Based on these structures and refined lattice parameters, the molecular arrange- ments of SS:Im, ZImS, and Im with different molar ratios are depicted in Fig. 2c. Table 1 Ionic crystals formed in PSS:Im-b-PMB upon the addition of ZImS/Im and ZImS2/Im PSS:Im-b-PMB w/ ZImS/Im w/ ZImS2/Im Crystal structure Triclinic Monoclinic Space group P 1 P 2 1/c a (Å) (α) 5.70 (62.5°) 9.82 (90.0°) b (Å) (β) 11.93 (104.7°) 11.78 (94.9°) c (Å) (γ) 12.66 (99.6°) 8.02 (90.0°) Table 1 Ionic crystals formed in PSS:Im-b-PMB upon the addition of ZImS/Im and ZImS2/Im While both crystals displayed an analogous distance of ~ 2.7 Å between Im+ and –SO3– moieties in unit cells, the distances between the neighboring Im+/Im moieties were noticeably different, which is of importance for determining the structural diffusion of protons. For the sample comprising ZImS/Im, the short distances of 5.11 Å, 6.06 Å, and 5.99 Å were obtained in the a-, b-, and c-directions of the triclinic lattice. On the contrary, this distance increased to 5.37 Å, 7.97 Å, and 6.55 Å for the sample with ZImS2/Im showing the monoclinic lattice. Results On the contrary, the addition of ZImS/Im or ZImS2/Im resulted in the appearance of a series of diffraction peaks indexed to triclinic crystal of the P1 space group or monoclinic crystal of the P2 1/c Herein, we report a peculiar type of proton conductors based on single-ion conducting block copolymers, where anion migra- tion is fundamentally prevented. Notably, the self-organization of dipolar moieties in the above conductors results in the formation of ionic crystals confined in the nanoscale ionic phases of block copolymers, which allows us to control the distance between neighboring proton donors and acceptors, as well as the dipolar orientation in ionic phases to increase the rate of structural dif- fusion of protons along protogenic channels. NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 ZImS Scattering intensity (a.u.) q (nm–1) q (nm–1) ZImS2/Im ZImS/Im (002) (020) (–1–13) (1–21) (021 ) (020) (200) (002) (121) (–211) (–1–21) (1–31) (–121) (211) (220) (021) a (1–20) c (102) (130) (0–12) (0–22) (–111) (111) (210) 10 12 14 16 18 10 12 14 16 18 (–102) (–121) ZImS/Im 3 2 w/ZImS/Im 1 z y 1: 5.113 Å 2: 6.060 Å 3: 5.998 Å ZImS2/Im C H N O S w/ZImS2/Im 1 2 3 z x 1: 7.972 Å C H N O S 2: 6.548 Å 3: 5.372 Å b Fig. 2 Self-organization of charged/dipolar moieties. a WAXS profiles of PSS:Im-b-PMB comprising various additives, measured at 25 °C, indexed with triclinic P1 space group (ZImS/Im) and monoclinic P2 1/c space group (ZImS2/Im). b Le Bail refinement of the WAXS patterns of PSS:Im-b-PMB with ZImS/Im and ZImS2/Im. c Molecular arrangements of SS:Im with ZImS/Im and ZImS2/Im. C, H, N, O, and S atoms are shown in black, pink, blue, red, and yellow, respectively. NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 PSS:Im-b-PMB displayed lamellar morphology, as shown by the Bragg peaks (inverted black triangles) at 1q* and 2q* (q* = 2π/ d100 and d100 = 6.4 nm). The presence of ZImS in PSS:Im phases of PSS:Im-b-PMB destabilized the microphase-separated mor- phology, which was attributed to the weakening of interactions between Im+ and SO3- units of polymer by the presence of neighboring ZImS moieties. The well-defined lamellar structure was restored upon the introduction of ZImS/Im and ZImS2/Im, and both of these samples were shown to contain ionic crystals. The addition of ZImS, ZImS/Im, and ZImS2/Im into PSS:Im-b- PMB progressively increased the sample domain size to 6.9, 7.1, and 7.7 nm, respectively. The swelling of PSS:Im phases with additives can be envisaged by the reductions in the Tg value (Supplementary Fig. 1). Scattering intensity (a.u.) ZImS2/Im ZImS/Im ZImS 7 nm 103 102 101 100 10–1 0.5 1.0 1.5 2.0 2.5 Neat q (nm–1) Fig. 3 Effects of additives on self-assembly of PSS:Im-b-PMB. SAXS profiles of PSS:Im-b-PMB comprising various additives, measured at 25 °C. Bragg peaks at 1q*, 2q* (q* = 2π/d100) indicate lamellar morphology with ca. 7 nm domain size, as schematically depicted in the inset Scattering intensity (a.u.) ZImS2/Im ZImS/Im ZImS 7 nm 103 102 101 100 10–1 0.5 1.0 1.5 2.0 2.5 Neat q (nm–1) The size and location of ionic crystals were further examined by transmission electron microscopy (TEM) imaging. Figure 4a and Fig. 4b display representative bright-field TEM and high- resolution TEM micrographs of ZImS/Im– and ZImS2/ Im–containing PSS:Im-b-PMB, respectively, revealing that 2–3 nm-sized crystalline structures were selectively confined within PSS:Im phases (darkened area) of lamellae for both samples. For clarity, ionic crystals are marked by circles in each high-resolution TEM image. The lattice parameters of ionic crystals obtained by fast Fourier transform (FFT) analysis were in good agreement with WAXS results. Figure 4c schematically depicts the formation of ionic crystal in ionic phases of lamellar grains for ZImS/Im–containing PSS:Im- b-PMB, as a representative example. PS moieties are not shown for clarity. The crystal is formed by the co-organization of SS:Im, ZImS, and Im molecules in amorphous polymer matrix with controlled dipolar interactions therein. Proton transport properties. Is the formation of nano-confined ionic crystals with long-range hydrogen-bonding networks truly beneficial for the enhancement of structural diffusion of proton in single-ion conducting polymers? Results Therefore, it was concluded that long-range proton conduction networks can be easily formed in PSS:Im phases comprising ZImS/Im. space group, respectively. Miller indices of the reflection planes (hkl) of each crystal were obtained by Mercury DSD 3.9 software, as given in the figure. Refinement of lattice parameters and structure factor ampli- tudes were obtained by Le Bail refinement of WAXS intensities and (hkl) Miller indices using a JANA2006 crystallographic program.36 Fig. 2b shows the refinement of data for ZImS/ Im–containing and ZImS2/Im–containing PSS:Im-b-PMB and the results are summarized in Table 1. It is apparent that the lattice parameters of these crystals were broadly variable, depending on the ZImS:Im molar ratio. y g It should be noted here that no evidence of crystal formation was observed for PSS:Im homopolymers with embedded ZImS, ZImS/Im, and ZImS2/Im (Supplementary Fig. 4), contrary to the cases of block copolymers described above. Thus, the confine- ment of dipolar moieties in nanoscale PSS domains was concluded to play a key role in facilitating crystal formation. The incorporation of ZImS and/or Im into PSS:Im-b-PMB affected its self-assembled morphology. Figure 3 shows repre- sentative small angle X-ray scattering (SAXS) profiles of the investigated samples, which were measured at room temperature and remained unchanged over the temperature range 25–130 °C. To delineate how PSS:Im, ZImS, and neutral Im combined into crystalline phases, the conformations of Im-doped styrene sulfonate (SS:Im) with incorporated ZImS and neutral Im were optimized by ab initio calculations based on the density functional theory (DFT, B3LYP/6-31 G(d)) (Supplementary 3 3 ARTICLE a Proton diffusion coefficients, b ionic conductivities, obtained for PSS:Im-b-PMB comprising additives, measured at different temperatures. In a and b the role of ionic crystals in determining the ion transport properties of single-ion polymers was clearly shown. c Schematics depicting dominant proton hopping mechanisms in PSS:Im-b-PMB containing ZImS/Im 2.7 a b 1 8 101 101 100 10–1 150 120 90 60 30 T-Tg (K) 10–1 10–2 100 6 4 2 0 DH+ × 1010 (filled symbols, m2 s–1) Conductivity (mS cm–1) DH+/DC–H (open symbols) 2.8 2.9 3.0 1000/T (K–1) 1000/T (K–1) 3.1 2.2 2.4 2.6 2.8 3.0 w/ZImS w/ZImS/Im w/ZImS2/Im ZImS ZImS/Im ZImS2/Im 2.7 a 1 8 6 4 2 0 DH+ × 1010 (filled symbols, m2 s–1) DH+/DC–H (open symbols) 2.8 2.9 3.0 1000/T (K–1) 3.1 w/ZImS w/ZImS/Im w/ZImS2/Im b 101 101 100 10–1 150 120 90 60 30 T-Tg (K) 10–1 10–2 100 Conductivity (mS cm–1) 1000/T (K–1) 2.2 2.4 2.6 2.8 3.0 ZImS ZImS/Im ZImS2/Im b c Ionic phases Nonionic phases H+ hopping PSS:Im-b-PMB Fig. 5 Proton transport properties. a Proton diffusion coefficients, b ionic conductivities, obtained for PSS:Im-b-PMB comprising additives, measured at different temperatures. In a and b the role of ionic crystals in determining the ion transport properties of single-ion polymers was clearly shown. c Schematics depicting dominant proton hopping mechanisms in PSS:Im-b-PMB containing ZImS/Im c Ionic phases Nonionic phases PSS:Im-b-PMB c Ionic phases Nonionic phases PSS:Im-b-PMB PSS:Im-b-PMB Fig. 5 Proton transport properties. a Proton diffusion coefficients, b ionic conductivities, obtained for PSS:Im-b-PMB comprising additives, measured at different temperatures. In a and b the role of ionic crystals in determining the ion transport properties of single-ion polymers was clearly shown. c Schematics depicting dominant proton hopping mechanisms in PSS:Im-b-PMB containing ZImS/Im Tammann equation (solid lines in Fig. 5b) further suggested dissimilar activation barriers to ion conduction, i.e., 1127 K for ZImS, 692 K for ZImS/Im, and 990 K for ZImS2/Im. The higher normalized conductivity and lower activation barrier obtained with ZImS/Im are notable. spin-echo (PGSE) NMR spectroscopy. Proton diffusion coeffi- cient (DH+) was then calculated based on proton dissociation degree (x) of the samples, determined as 52% (with ZImS), 65% (with ZImS/Im), and 54% (with ZImS2/Im) by confocal Raman spectroscopy (Supplementary Fig. 5). The considerably high x value observed for PSS:Im-b-PMB with ZImS/Im is particularly noteworthy. Based on proton diffusion coefficients in Fig. 5a and ionic conductivities in Fig. ARTICLE To answer this question, self- diffusion coefficients associated with N–H (DN–H) and C–H (DC–H) protons of Im+ and neutral Im were measured for PSS: Im-b-PMB with different additives using 1H pulsed-field gradient Fig. 3 Effects of additives on self-assembly of PSS:Im-b-PMB. SAXS profiles of PSS:Im-b-PMB comprising various additives, measured at 25 °C. Bragg peaks at 1q*, 2q* (q* = 2π/d100) indicate lamellar morphology with ca. 7 nm domain size, as schematically depicted in the inset 0.21 nm (002) (020) (002)  = 96° (020) (022) (022) (022) (022) c b (022) (002) (020) (002) 0.24 nm (020) (022)  = 61° a ZImS/Im ZImS2/Im Non-ionic Non-ionic Non-ionic Ionic Ionic Ionic Fig. 4 Ionic crystals in self-assembled lamellar grains. Bright-field TEM, high-resolution TEM micrographs, and FFT patterns obtained from PSS:Im-b-PMB with the addition of a ZImS/Im and b ZImS2/Im. The darkened area of lamellae indicates PSS:Im phases for both samples and ionic crystals are marked by circles in each TEM image for clarity. Scale bars in bright-field TEM and high-resolution TEM images are 20 nm and 2 nm, respectively. c Schematic illustration of the formation of ionic crystal in ionic phases of lamellar grains for PSS:Im-b-PMB with ZImS/Im (022) (002) (020) (002) 0.24 nm (020) (022)  = 61° a ZImS/Im a c Non-ionic Non-ionic Non-ionic Ionic Ionic Ionic 0.21 nm Fig. 4 Ionic crystals in self-assembled lamellar grains. Bright-field TEM, high-resolution TEM micrographs, and FFT patterns obtained from PSS:Im-b-PMB with the addition of a ZImS/Im and b ZImS2/Im. The darkened area of lamellae indicates PSS:Im phases for both samples and ionic crystals are marked by circles in each TEM image for clarity. Scale bars in bright-field TEM and high-resolution TEM images are 20 nm and 2 nm, respectively. c Schematic illustration of the formation of ionic crystal in ionic phases of lamellar grains for PSS:Im-b-PMB with ZImS/Im NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 2.7 c a b 1 8 101 101 100 10–1 150 120 90 60 30 T-Tg (K) 10–1 10–2 100 6 4 2 0 DH+ × 1010 (filled symbols, m2 s–1) Conductivity (mS cm–1) DH+/DC–H (open symbols) 2.8 2.9 3.0 1000/T (K–1) Ionic phases Nonionic phases H+ hopping PSS:Im-b-PMB 1000/T (K–1) 3.1 2.2 2.4 2.6 2.8 3.0 w/ZImS w/ZImS/Im w/ZImS2/Im ZImS ZImS/Im ZImS2/Im Fig. 5 Proton transport properties. ARTICLE 5b, Haven ratios were calculated for each sample at 60 °C and 90 °C. This is to quantify the dissimilar ion dissociation behavior of each sample by evaluating how much it deviates from unity. At 60 °C, the Haven ratios were 7.7, 7.1, and 5.2 for PSS:Im-b-PMB samples with ZImS, ZImS/Im and ZImS2/ Im, respectively. Upon heating the samples to 90 °C, at the temperature well-above the Tg values, the Haven ratios decreased to 6.2 (ZImS), 2.3 (ZImS/Im), and 1.2 (ZImS2/Im). Significant reductions in the Haven ratios seen only for crystal-forming samples are particularly noteworthy, indicating high dissociation degree of ions bound in the crystal structure to contribute to efficient proton transport, as schematically illustrated in Fig. 5c. Note in passing that the grain size of lamellar structures decreased in the order of ZImS2/Im > ZImS/Im > ZImS. This would also have an impact on the Haven ratio, but the concrete under- pinning remains as a future study. y Figure 5a shows the DH+ values and DH+ / DC-H ratios of investigated samples, clearly displaying the role of ionic crystals in determining the proton diffusion behavior in single-ion polymers. Note that the accessible temperature window of the NMR spectrometer was 25–90 °C because it was calibrated using ethylene glycol standard in water. Overall, DH+ values obtained for the sample comprising ZImS were lower than those obtained with ZImS/Im and ZImS2/Im by factors of 2–5. Notably, the DH+ / DC-H ratio was as high as ~ 7.6 for the sample comprising ZImS/ Im, whereas much lower values of 4.5 and 1.9 were obtained for ZImS2/Im-containing and ZImS-containing analogs, respectively. This finding suggests that proton hopping was most pronounced in the sample comprising ZImS/Im because of the shortest distances between protogenic moieties therein (Fig. 2c). Figure 5b shows the temperature-dependent ionic conductivity of PSS:Im-b-PMB with different additives, revealing that this quantity decreased in the order of ZImS2/Im > ZImS/Im > ZImS, in good agreement with the results of diffusivity measurements. With excluded influence of Tg values of the samples on conductivity, as shown in inset plot of Fig. 5b, proton transport efficiency was highest for the ZImS/Im-containing sample if the sample temperature was lower than 90 °C. The lowest normalized conductivity was seen for the ZImS-embedded one for entire temperature window examined. NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications Discussion W l We explored the dielectric relaxation behavior of the above- mentioned samples. Figure 6a shows static dielectric constant (εs) of the samples, measured at different temperatures (dielectric permittivity spectra are given in Supplementary Fig. 7). ZImS/ Im–containing PSS:Im-b-PMB exhibited high εs values over 78 while the lowest εs value of 13 was observed with the incor- poration of ZImS. It should be noted that εs value of neat PSS:Im- b-PMB was as low as 5. The sample with ZImS2/Im exhibited g ≈1 and Vp/Vm > 1, ascribed to the increased density of dipoles, resulting in a sig- nificant dipole overlap to restrict dipolar correlation. This leads us to conclude that the dipole correlation affects the structure (amorphous vs. crystalline) and proton hopping distance (ordered dipolar moieties reduce the distance). The origin of the differences in εs values was elucidated by analyzing local directional correlations between neighboring dipoles for each sample. Based on DFT calculations, we deter- mined three major dipole moments of ion pair (SO3--Im+, 7 D), ZImS (17.5 D), and neutral pair (Im-ZImS, 19.3 D), as listed in Supplementary Table 2. This computational method also enabled us to estimate the optimized dipole structures, as provided in Supplementary Fig. 3. The Kirkwood-Fröhlich g-factor was cal- culated by equation (1)30 using the number density of dipolar moieties (vi) and the dipole moment (mi) (listed in Supplemen- tary Table 2). The overlap of dipole polarizability volumes, defined as the ratio of Debye’s polarizability volume Vp to molecular volume Vm, was calculated by equation (2). Repre- sentative Vp/Vm values of each sample obtained at 60 °C are listed in Supplementary Table 2. It is worth noting that no substantial enhancement of εs was observed for PSS:Im homopolymers with various additives (Supplementary Fig. 8). This finding implies that synergistic dipole alignments occurred only in samples containing nano- confined ionic crystals, the formation of which is thus of key importance for improving proton transport properties. g We would like to conclude this paper by commenting on the single-ion properties of PSS:Im-b-PMB comprising additives. Because zwitterion is composed of covalently connected cation and anion, it is electroneutral. Nevertheless, the excessive use of zwitterion in polymer electrolytes may result in charge depletion at electrolyte/electrode interfaces at a given voltage. However, this was avoided in our polymers because the ZImS is packed in ionic crystals and further confined within PSS:Im domains, as con- firmed by polarization experiments (Supplementary Fig. ARTICLE Model fits by Vogel-Fulcher- It is worth noting that we confirmed no substantial effects of the molecular weight of PSS:Im-b-PMB on ionic crystal formation and conductivity enhancement. Supplementary Fig. 6 shows scattering profiles and temperature-dependent conductiv- ity data obtained from PSS:Im-b-PMB (20–39 kg mol−1, a sulfonation level of 25 mol.%, lamellae, d100=56 nm) with additives. Ionic crystals were again formed only with ZImS/Im and ZImS2/Im, leading to markedly enhanced ionic conductivity, compared with amorphous sample comprising ZImS. This NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 90 a b 2.0 1.5 g factor 1.0 0.5 0.0 60 s 30 0 2.6 2.8 3.0 1000/T (K–1) 3.2 w/ZImS w/ZImS/Im w/ZImS2/Im w/ZImS w/ZImS/Im w/ZImS2/Im 3.4 2.6 2.8 3.0 1000/T (K–1) 3.2 Fig. 6 Dielectric properties. a Static dielectric constant and b Kirkwood-Fröhlich g factor obtained for PSS:Im-b-PMB comprising additives, measured at different temperatures 90 a 60 s 30 0 2.6 2.8 3.0 1000/T (K–1) 3.2 w/ZImS w/ZImS/Im w/ZImS2/Im 3.4 b 2.0 1.5 g factor 1.0 0.5 0.0 w/ZImS w/ZImS/Im w/ZImS2/Im 4 2.6 2.8 3.0 1000/T (K–1) 3.2 hlich g factor obtained for PSS:Im-b-PMB comprising additives measured at b tatic dielectric constant and b Kirkwood-Fröhlich g factor obtained for PSS:Im-b-PMB comprising additives, measured Fig. 6 Dielectric properties. a Static dielectric constant and b Kirkwood-Fröhlich g factor obtained for PSS:Im-b-PMB comprising additives, measured at different temperatures samples owing to the presence of SO3––Im+ ion pairs. With ZImS addition, the low g-factor of < 1 and Vp/Vm < 1 were obtained, which indicated that the ion pair (SO3––Im+) and ZImS tend to form a quadrupole (i.e., two dipoles are antiparallel each other), leading to offset of dipole moment by dipole-dipole attraction. This is consistent with the lowest εs value of PSS:Im-b- PMB containing ZImS (Fig. 6a). finding suggests the crucial role of co-organization of neutral Im and ZImS in developing the unique crystalline structures in PSS: Im phases of PSS:Im-b-PMB, which is intimately related to the improved ion transport properties. Recent work of Winey and coworkers37 may be relevant to our results given that ionic moieties (–SO3-–H3O+) are confined by surrounding crystalline matrix (polyethylene backbones). How- ever, the fundamental difference lies in the fact that PSS:Im-b- PMB block copolymers have essentially amorphous PS backbones and PSS chains directly involved in the ionic crystal formation for participation in proton transport. ARTICLE On the contrary, the highest g-factor of ~ 2 was obtained for PSS:Im-b-PMB comprising ZImS/Im to enhance the εs value. This can be rationalized by the DFT calculations (Supplementary Fig. 3), displaying that neutral Im breaks the quadrupole to make surrounding dipoles are arranged parallelly, thereby providing the system with a net dipole moment. The net dipole moment is intimately connected to close packing of neighboring dipolar moieties and the dipole-dipole interaction acts as an enthalpic contribution to the ionic crystal formation. This intriguing dipolar orientation was further envisaged by the Vp/Vm ratio of ~1 (Supplementary Table 2), signaling that ion pairs were forced to strongly interact at the overlap point.30 NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications References 1. Jiménez-García, L., Kaltbeitzel, A., Pisula, W., Gutmann, J. S., Klapper, M. & Müllen, K. Phosphonated hexaphenylbenzene: a crystalline proton conductor. Angew. Chem. Int. Ed. 48, 9951 (2009). 2. Braun, A. & Chen, Q. Experimental neutron scattering evidence for proton polaron in hydrated metal oxide proton conductors. Nat. Commun. 8, 15830 (2017). Preparation of single-ion conducting polymers. Inhibitor-free anhydrous THF ( ≥99.9%) and anhydrous methanol (99.8%) were purchased from Sigma-Aldrich and used without further purification. Predetermined quantities of PSS:Im-b-PMB (or PSS:Im), ZImS, and neutral Im were dissolved into 80/20 vol.% methanol/THF mixtures to prepare ca. 5 wt% solutions using the balance (Mettler Toledo, ME 204) inside the argon-filled glove box. Under argon atmosphere, solvents were slowly evaporated at room temperature for 2 days and were further exposed to vacuum at 70 °C for 7 days. 3. Kreuer, K. D. On the development of proton conducting polymer membranes for hydrogen and methanol fuel cells. J. Membr. Sci. 185, 29–39 (2001). 4. Hickner, M. A., Ghassemi, H., Kim, Y. S., Einsla, B. R. & McGrath, J. E. Alternative polymer systems for proton exchange membranes (PEMs). Chem. Rev. 104, 4587–4612 (2004). 5. Çelik, S. Ü., Bozkurt, A. & Hosseini, S. S. Alternatives toward proton conductive anhydrous membranes for fuel cells: Heterocyclic protogenic solvents comprising polymer electrolytes. Prog. Polym. Sci. 37, 1265–1291 (2012). X-ray scattering (SAXS and WAXS) experiments. Synchrotron SAXS and WAXS measurements on the single-ion polymers were performed using the PLS-II 4 C and 9 A beamlines at the Pohang Accelerator Laboratory (PAL), equipped with temperature-controlled sample stage and two-dimensional detector. The samples were laminated into an air-tight cell inside argon-filled glove box to avoid the issue of water contamination. Three different sample-to-detector distances of 2.0 m, 0.2 m, and 0.1 m were used to cover a wide scattering wave vector q (q = 4π sin(θ/2)/λ, where θ is the scattering angle). The scattering data were azimuthally averaged to obtain intensity versus q. 6. Kusoglu, A. & Weber, A. Z. New insights into perfluorinated sulfonic-acid ionomers. Chem. Rev. 117, 987–1104 (2017). 7. Soberats, B., Yoshio, M., Ichikawa, T., Taguchi, S., Ohno, H. & Kato, T. 3D anhydrous proton-transporting nanochannels formed by self-assembly of liquid crystals composed of a sulfobetaine and a sulfonic Acid. J. Am. Chem. Soc. 135, 15286–15289 (2013). 8. Kobayashi, T., Ichikawa, T., Kato, T. & Ohno, H. Development of glassy bicontinuous cubic liquid crystals for solid proton-conductive materials. Adv. Method S th i Synthesis of sulfonated polymers. A poly(styrene-b-methylbutylene) (PS-b- PMB, 1.0-b-1.0 kg mol-1) precursor block copolymer was synthesized by sequential anionic polymerization of styrene (ReagentPlus®, 99.9%, Sigma-Aldrich) and iso- prene ( ≥99%, Sigma-Aldrich) using sec-butyllithium initiator in cyclohexane, followed by selective hydrogenation of isoprene units using Ni-Al catalyst. A polystyrene (PS, 1.0 kg mol-1) homopolymer was also prepared by anionic poly- merization as a control sample. The absolute molecular weights of PS-b-PMB block copolymer and PS homopolymer were characterized by end-group analysis using 1H Nuclear Magnetic Resonance (1H-NMR, Bruker AVB-300) spectroscopy in CDCl3. Size exclusion chromatography (SEC, Waters Breeze 2 HPLC) was used to characterize polydispersity indices of PS-b-PMB and PS, which were determined to be 1.03 and 1.02, respectively, with PS standards and tetrahydrofuran (THF) eluent. Subsequent sulfonation reaction of PS-b-PMB block copolymer and PS homo- polymer using acetic sulfate in anhydrous dichloromethane ( ≥99.8%, Sigma- Aldrich) yielded PSS-b-PMB block copolymer and PSS homopolymer. The sulfo- nation level was determined to be 20 mol.% by 1H-NMR in acetone-d6. The PSS-b- PMB and PSS were doped with equivalent imidazole ( ≥99%, Sigma-Aldrich) to the mole of sulfonic acid group of polymer using methanol to yield PSS:Im-b-PMB block copolymer and PSS:Im homopolymer. Dielectric relaxation spectroscopy. The dielectric permittivity spectra of the samples (the same samples employed for impedance spectroscopy measurements) were acquired using a Novocontrol GmbH Concept 40 broadband dielectric spectrometer, equipped with Novocontrol sample chamber, under a sinusoidal voltage of 0.1 V in a frequency range of 10–107 Hz. The sample cell consists of two polished brass electrodes (10 mm-diameter top electrode and 30 mm diameter bottom electrode) and a silica spacer. Samples were annealed at 120 °C for 1 h prior to the measurements to achieve good contact of the sample with electrodes. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 ARTICLE interactions and effective confinements of ionic moieties in polymer matrix can inhibit leaching of constituents over time, our polymers have potential advantages over conventional ionic liquid-containing polymer electrolytes. Our approach should find wide applicability in various electrochemical applications. Raman spectroscope (WITec, Ulm, Germany), equipped with a HeNe laser, under low laser excitation power below 3 mW. The spatial resolution of the spectrometer was 250 nm. The samples were placed on dimple-patterned slide glasses inside glovebox and covered with a window glass to avoid the contact of samples with air. Self-diffusion coefficient measurements. Pulsed gradient spin echo (PGSE) 1H- NMR experiments on PSS:Im-b-PMB block copolymers comprising various additives were performed using a Bruker AVB-300 spectrometer. The samples were loaded into 4 mm (o.d.) NMR microtubes in an argon-filled glovebox and were sealed with caps. Time interval of the field gradient and duration time between two gradient pulses were in the range 1–10 ms and 0.1–0.3 s, respectively. The samples were equilibrated for 1 h at each temperature prior to the measurements, which was calibrated using ethylene glycol standard. In summary, we demonstrate that proton transport rate in single-ion conducting polymers can be enhanced by creating high-dielectric-constant crystalline protogenic channels in an amorphous polymer matrix. Specifically, the strong dipolar interactions in nanoscale ionic phases of block copolymers resulted in the formation of 2–3 nm-sized ionic crystals packed within. The developed approach allowed the realization of dominant structural diffusion of protons, improved ion dis- sociation degree, and significantly enhanced dielectric constant of the single-ion polymers. Ionic conductivity measurements. The through-plane conductivities of PSS:Im-b- PMB comprising additives were measured by impedance spectroscopy using PARSTAT 2273 and Solartron 1260 A. The dimension of solvent-cast membrane was 0.8 cm × 0.8 cm × 200 μm, which was placed inside engraved counter Pt elec- trode and covered by Pt working electrode with Kapton spacers inside the glove box. Samples were annealed for 30 min at each temperature and data were collected in a wide frequency range of 10–105 Hz. Received: 6 August 2018 Accepted: 5 November 2018 Received: 6 August 2018 Accepted: 5 November 2018 Synthesis of zwitterion. 3-(1-methyl-3-imidazolium) propanesulfonate (ZImS) was synthesized by mixing 1-methylimidazole (ReagentPlus®, 99.9%, Sigma- Aldrich) and 1,3-propanesultone ( ≥99%, Sigma-Aldrich) in acetone ( ≥99.5%, Sigma-Aldrich) at room temperature. After 5 days of stirring, the mixtures were purified by repeated filtration and precipitation. White powder was recovered by vacuum drying at 60 °C and the successful synthesis of ZImS was confirmed by combining 1H-NMR and Fourier transform Infrared (FT-IR, a Spectrum Two, PerkinElmer, USA) spectroscopy experiments. Data availability The data supporting the findings of this study are available within the paper and its Supplementary Information. Other datasets analyzed for the present study are available from the corresponding author on reasonable request. Received: 6 August 2018 Accepted: 5 November 2018 Discussion W l 9). Note that the content of ZImS is low as 13 wt% for PSS:Im-b-PMB containing ZImS/Im (neutral Im content is 4 wt%). g ¼ 9ε0kT P i vim2 i εs  ε1 ð Þ 2εs þ ε1 ð Þ εs ε1 þ 2 ð Þ2 ð1Þ Vp Vm ¼ X i vim2 i 12πε0kT ð2Þ ð1Þ ð2Þ Although a large number of ion-containing polymers have been reported in literature, cases of developing well-defined protogenic crystals with in-depth underpinning of conductivity- structure relationship remains rare. Given that strong dipolar Figure 6b illustrates the temperature dependence of the g-fac- tor, showing that ionic orientational polarization is present in all 6 Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 25. Wang, S. W., Liu, W. & Colby, R. H. Counterion dynamics in polyurethane- carboxylate ionomers with ionic liquid counterions. Chem. Mater. 23, 1862–1873 (2011). reprintsandpermissions/ 26. Hoarfrost, M. L., Tyagi, M. S., Segalman, R. A. & Reimer, J. A. Effect of confinement on proton transport mechanisms in block copolymer/ionic liquid membranes. Macromolecules 45, 3112–3120 (2012). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 27. Zhang, H. & Shen, P. K. Recent development of polymer electrolyte membranes for fuel cells. Chem. Rev. 112, 2780–2832 (2012). 28. Kreuer, K.-D., Paddison, S. J., Spohr, E. & Schuster, M. Transport in proton conductors for fuel-cell applications: simulations, elementary reactions, and phenomenology. Chem. Rev. 104, 4637–4678 (2004). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 29. Ryu, S.-W. et al. Effect of counter ion placement on conductivity in single-ion conducting block copolymer electrolytes. J. Electrochem. Soc. 152, A158–A163 (2005). 30. Kim, O., Kim, H., Choi, U. H. & Park, M. J. 1 Volt-driven, superfast polymer actuators based on single ion conductors. Nat. Commun. 7, 13576 (2016). 31. Choi, U. H., Liang, S., Chen, Q., Runt, J. & Colby, R. H. Segmental dynamics and dielectric constant of polysiloxane polar copolymers as plasticizers for polymer electrolytes. ACS Appl. Mater. Interfaces 8, 3215–3225 (2016). p y y pp f 32. Shao, J.-J., Raidongia, K., Koltonow, A. R. & Huang, J. Self-assembled two- dimensional nanofluidic proton channels with high thermal stability. Nat. Commun. 6, 7602 (2015). 33. He, X., Zhu, Y. & Mo, Y. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 13. Kim, O., Kim, S. Y., Lee, J. & Park, M. J. Building less tortuous ion-conduction pathways using block copolymer electrolytes with a well-defined cubic symmetry. Chem. Mater. 28, 318–325 (2016). 34. Craven, B. M., Mcmullan, R. K., Bell, J. D. & Freeman, H. 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Mater. 17, 725–731 (2018). 16. Rubatat, L., Li, C., Dietsch, H., Nykänen, A., Ruokolainen, J. & Mezzenga, R. Structure-properties relationship in proton conductive sulfonated polystyrene- polymethyl methacrylate block copolymers (sPS-PMMA). Macromolecules 41, 8130–8137 (2008). Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2017R1A2B3004763) and by the Korea government (MSIT) (No. NRF-2017R1A5A1015365). We also acknowledge the Creative Materials Discovery Program through the NRF funded by Ministry of Science and ICT (2018M3D1A1058624) and the Global Frontier R&D program on Center for Multiscale Energy System through the NRF funded by the Ministry of Education, Science and Technology. 17. Kim, S. Y., Yoon, E., Joo, T. & Park, M. J. Morphology and conductivity in ionic liquid incorporated sulfonated block copolymers. Macromolecules 44, 5289–5298 (2011). 18. Jangu, C. et al. Imidazole-containing triblock copolymers with a synergy of ether and imidazolium sites. J. Mater. Chem. C. 3, 3891–3901 (2015). 19. Kim, O. et al. Phase behavior and conductivity of sulfonated block copolymers containing heterocyclic diazole-based ionic liquids. Macromolecules 45, 8702–8713 (2012). 20. Jung, H. Y., Kim, O. & Park, M. J. Ion transport in nanostructured phosphonated block copolymers containing ionic liquids. Macromol. Rapid Commun. 37, 1116–1123 (2016). Author contributions M.J.P. conceived the idea and designed the project and O.K. and K.K. carried out the crystalline structures analysis. K.K performed Le Bail refinement and charge-flipping electron density reconstruction. O.K. and U.H.C. carried out the dielectric experiments and analysed data. M.J.P. and O.K. wrote the paper. 21. Kim, S. Y., Kim, S. & Park, M. J. Enhanced proton transport in nanostructured block copolymer electrolyte/ionic liquid membrane under water free conditions. Nat. Commun. 1, 88 (2010). 22. 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Liu, M. et al. Three-dimensional protonic conductivity in porous organic cage solids. Nat. Commun. 7, 12750 (2016). 12. Jiang, Y. et al. The evolution of cyclopropenium ions into functional polyelectrolytes. Nat. Commun. 6, 5950 (2015). Fourier transform Raman (FT-Raman) experiments. Confocal Raman spectra of PSS:Im-b-PMB comprising additives were measured using a WITEC Alpha 300 R 7 NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 33. He, X., Zhu, Y. & Mo, Y. Origin of fast ion diffusion in super-ionic conductors. Nat. Commun. 8, 15893 (2017). Additional information q 23. Ueki, T. & Watanabe, M. Macromolecules in ionic liquids: progress, challenges, and opportunities. Macromolecules 41, 3739–3749 (2008). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-07503-4. 24. Virgili, J. M., Nedoma, A. J., Segalman, R. A. & Balsara, N. P. Ionic liquid distribution in ordered block copolymer solutions. Macromolecules 43, 3750–3756 (2010). Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. Origin of fast ion diffusion in super-ionic conductors. Nat. Commun. 8, 15893 (2017). © The Author(s) 2018 © The Author(s) 2018 NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 8 8
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Out-of-hospital cardiac arrest in children: An epidemiological study based on the German Resuscitation Registry identifying modifiable factors for return of spontaneous circulation
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Out-of-hospital cardiac arrest in ch epidemiological study based on the Resuscitation Registry identifying m for return of spontaneous circulatio Stephan Katzenschlager  (  stephan.katzenschlager@med.uni-heide Heidelberg University Hospital Inga K. Kelpanides  Oslo University Hospital Patrick Ristau  University Hospital Schleswig-Holstein Matthias Huck  Heidelberg University Hospital Stephan Seewald  University Hospital Schleswig-Holstein Sebastian Brenner  University Clinic Carl Gustav Carus Florian Hoffmann  Dr. von Hauner Children's Hospital, Ludwig- Maximilians-University Jan Wnent  University Hospital Schleswig-Holstein Jo Kramer-Johansen  University of Oslo Ingvild B. M. Tjelmeland  University of Oslo Markus A. Weigand  Heidelberg University Hospital Jan-Thorsten Gräsner  University Hospital Schleswig-Holstein Erik Popp  Heidelberg University Hospital Out-of-hospital cardiac arrest in children: An epidemiological study based on the German Resuscitation Registry identifying modifiable factors for return of spontaneous circulation Matthias Huck  Heidelberg University Hospital Stephan Seewald  University Hospital Schleswig-Holstein Sebastian Brenner  University Clinic Carl Gustav Carus Florian Hoffmann Research Article Research Article Page 1/24 Keywords: Out of hospital cardiac arrest, resuscitation, pediatric cardiac arrest, emergency medical service, epidemiology Keywords: Out of hospital cardiac arrest, resuscitation, pediatric cardiac arrest, emergency medical service, epidemiology Results OHCA in children aged < 1 year had the highest incidence per 100 000 inhabitants, with 23.42, accounting for 42.0% of all included cases. Overall, hypoxia was the leading presumed cause of OHCA; whereas trauma and drowning accounted for a high proportion in children aged > 1 year. Bystander-witnessed OHCA and bystander CPR rate were highest in children aged 1–4 years, with 43.9% and 62.3%, respectively. In reference to EMS-started CPR, bystander CPR with ventilation were associated with an increased odds ratio for ROSC at hospital admission after adjusting for age, sex, year of OHCA and location of OHCA. Conclusion This study provides an epidemiological overview of OHCA in children in Germany and identifies bystander CPR with ventilation as one primary factor for survival. Abstract This work provides an epidemiological overview of out-of-hospital cardiac arrest (OHCA) in children in Germany between 2007 and 2021. We wanted to identify modifiable factors associated with survival. Methods Data from the German Resuscitation Registry (GRR) was used, and we included patients registered between 1st January 2007 and 31st December 2021. We included children aged > 7 days and < 18 years, where cardiopulmonary resuscitation (CPR) was started and treatment was continued by emergency medical services (EMS). Incidences and descriptive analyses are presented for the overall cohort and each age group. Multivariate binary logistic regression was performed on the whole cohort to determine the influence of (1) CPR with/without ventilation started by bystander, (2) OHCA witnessed status, and (3) nighttime on the outcome hospital admission with ROSC. Posted Date: July 14th, 2023 Posted Date: July 14th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3156608/v1 DOI: https://doi.org/10.21203/rs.3.rs-3156608/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Critical Care on September 7th, 2023. See the published version at https://doi.org/10.1186/s13054-023-04630-3. Page 2/24 Trial registrations German Clinical Trial Register: DRKS00030989, December 28th 2022. German Clinical Trial Register: DRKS00030989, December 28th 2022. Study design In this retrospective cross-sectional cohort study, patients under the age of 18 with an OHCA from January 1st 2007 until December 31st 2021 were included. This analysis was performed with data from GRR and followed the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines [16]. Introduction Out-of-hospital cardiac arrest (OHCA) in children is a rare event with a high burden for the victim, caregivers, rescue personnel, and society. Globally, the incidence of OHCA in children remains low, with around 8/100,000 inhabitants per year [1–3]; however, incidence in < 1 year-olds is substantially higher [3–5]. The Page 3/24 low incidence compared to OHCA in adults, combined with ethical issues, has resulted in a lack of interventional studies on OHCA in children [6]. Survival rates have increased for in-hospital cardiac arrests, but this success did not show higher rates of survival with good neurologic outcome [7]. Emergency medical service (EMS) crews are challenged by the heterogeneity of different age groups, facing wide anatomical and physiological ranges. Aetiologies of OHCA in children differ from those in adults, as hypoxia is one of the leading presumed causes [8, 9]. However, the aetiology varies across the paediatric OHCA population, with an increase in trauma in the adolescent group and a decrease in respiratory failure and hypoxemia. Due to those differences, compared to adults, treatment changes and the focus lies more on ventilation and oxygenation rather than early defibrillation [10]. As opposed to the assumption of a cardiac cause in adult OHCA without any other obious causes, an universal presumed cause of cardiac arrest in children has not been identified; leaving caregivers and researchers with another challenge in the endeavour to improve the treatment of OHCA in children. Although an Utstein recommendation on reporting OHCA in children exists [11], comparability between different studies is limited due to the different age groups reported. It is crucial to differentiate between respiratory and cardiopulmonary arrest; however, this is not always possible, especially in retrospective cohort studies. While there is an increase in the analyses of specific treatment strategies [2, 12, 13], recent large epidemiological studies across a whole nation or a continent are lacking. These can help EMS systems to tailor their pediatric advanced life support (PALS) training and provide the best possible care. This paper is, to our knowledge, the first to give an epidemiologic overview of OHCA in children, including patients in Germany between 2007 and 2021, and analyse modifiable factors for the occurrence sustained ROSC at hospital admission. The German Resuscitation Registry (GRR) is one of Europe biggest cardiac arrest registries [14, 15], therefore, significantly contributing to the scarce population data and literature on OHCA in children. EMS system in Germany The EMS system is a two-tier system with ambulance crews consisting of at least a paramedic (German: 'Notfallsanitäter') and an emergency medical technician (EMT) (German: 'Rettungssanitäter'). The second tier that is always dispatched to suspected cardiac arrests, is a physician response unit staffed with an emergency physician and a paramedic. The physician response unit can either be a rapid response car or an air ambulance. In addition, some regions have implemented first responder systems. Paramedics are trained in advanced life support (ALS) treatment of OHCA in adults and paediatrics. Ultimately, the emergency physician makes the treatment decisions on scene. German Resuscitation Registry (GRR) GRR covered approximately 37.4% (31.1 million) of the population of Germany by the end of 2021. Out of the proportion covered within GRR, 16.7% (5.2 million) were under the age of 18 years. This increased from only 1.3 million children covered within the registry in 2007 (Supplement Table 1a). To date, about 100 EMS organisations are participating in GRR on a voluntarily basis. In addition to sites in Germany, some Austrian and Swiss EMS systems choose to participate in the GRR. For the acquisition of pre-hospital data, a dataset with 118 variables in concordance with the Utstein recommendations exists [17]. This dataset focuses on Page 4/24 Page 4/24 time intervals, pre-hospital interventions, patient specific factors such as age, known diseases, pre- emergency state according to the American Society of Anesthesiology physical status classification (ASA classification) [18], and patient status at hospital admission. ASA classification ranged from 1 to 5 and was determined by the treating physician according to the information available on scene. Preexisting conditions were entered into GRR by the treating physician on scene. Due to the structure of the database, up to three existing conditions could be entered. ‘Reference centers’ are EMS systems that provide high data quality for both, pre- and in-hospital data [19]. All pre-hospital data are collected and entered into GRR by the treating emergency physician. Inclusion and exclusion criteria We included all patients >7 days and <18 years of age suffering an OHCA with resuscitation attempts and continued treatment by EMS in Germany. No restrictions for sex or the aetiology of OHCA were applied. Data from participants outside of Germany and data on patients with an incorrect or missing age were excluded. Furthermore, cases where the patient was declared deceased on arrival (DoA) by the EMS team without resuscitation attempts by neither a bystander nor EMS were excluded. Descriptive baseline characteristics of patients <=7 days and children declared DoA are presented in the supplementary material (Supplement Table 2 and 3). EMS time intervals The following time intervals were calculated based on the times reported by the EMS system: The following time intervals were calculated based on the times reported by The following time intervals were calculated based on the times reported by the EMS system: - Response time, defined as the interval from the emergency call started until the first EMS team arrived at the destination defined by dispatch - On-scene time, defined as the interval from the first EMS team on scene until transport to the hospital was either started or the patient was declared dead - Transport time, defined as the interval from the start of the transport by EMS until arrival at the hospital Page 5/24 - EMS treatment time, defined as the interval from the arrival of the first EMS team until arrival at the hospital (= On scene interval + transport interval). When the patient diseased and was not transported, EMS treatment duration was defined as the on-scene interval. Page 5/24 - EMS treatment time, defined as the interval from the arrival of the first EMS team until arrival at the hospital (= On scene interval + transport interval). When the patient diseased and was not transported, EMS treatment duration was defined as the on-scene interval. - Duration to first shock, defined as the interval from the emergency call until anyone delivered the first shock in patients with shockable rhythm Data synthesis and statistical analysis All analyses were performed using SPSS (Statistical Product and Services Solutions, Version 28, SPSS Inc., Chicago, IL, USA). Baseline and demographic characteristics are presented as incidence per 100,000 children and frequencies for the overall cohort and as frequencies only for the age-specific subgroups. Continuous variables are summarised as means with standard deviations (SD) or medians and interquartile ranges (Q1-Q3). Incidence was calculated based on the official population calculations from the German Federal Statistical Office [20]. We assumed that the proportion of children is equally distributed across Germany. The complete data for incidence calculation is available in the supplementary material (Supplement Table 1b). The four age groups were categorised according to the latest Utstein recommendation, with >7 days to <1 year (henceforth named <1 year), 1 to 4 years, 5 to 12 years, and 13 to <18 years [11]. Treatment before EMS arrival was defined as interventions performed either by bystanders or first responders. Nighttime was defined between 10pm to 6am. First rhythm assessed was defined as the first rhythm when CPR was commenced. Bradycardia was defined as a heartrate below 60 beats per minute; due to the structure of GRR, this is only possible in patients below 14 years of age. EMS time intervals were analysed using Mann-Whitney-U due to the non-normal distribution of the data. The effect size was calculated as the differences between pseudo medians with nonparametric 95% confidence intervals (CI) using the Hodges-Lehmann method [21]. Multivariate binary regression analysis was used to calculate the adjusted odds ratio (aOR) for the factors 'chest compression started by', 'chest compression and ventilation started by', 'OHCA witnessed by', and 'nighttime' for the outcome 'ROSC at hospital admission'. Regression analysis was adjustmented for age (per year, continuous variable), sex, year of OHCA (continuous variable) and location of OHCA. Missing data were not imputed. A two-sided level of significance < 0.05 was considered statistically significant. Steering committee and ethical approval According to the regulations of the GRR, a study protocol was drafted and approved by the scientific advisory board before the beginning of the data analysis (Ref. number: 2022-06). This study was approved by the University Heidelberg ethics committee with reference number S-085/2022, and registered at the German Clinical Trial Register (www.drks.de—study number DRKS00030989). Demographic overview Demographic overview Page 6/24 Over the 15 years, 235,537 OHCAs were recorded in GRR. The complete list of excluded cases with reasons is available in Figure 1. Figure 1 – Study flowchart demonstrating included cases and incidences per age group; *1 case with an unknown immediate outcome. Abbreviations: OHCA = out-of-hospital cardiac arrest; GRR = German Resuscitation Registry; CPR = cardiopulmonary resuscitation The main reason for exclusion was age ≥18 years (n = 233,142; 98.9%). Furthermore, 476 cases where no CPR was performed due to obvious signs of death and 97 cases with age ≤7 days were excluded. (Supplement Table 2 and 3). In total, 1,740 datasets of children suffering from OHCA and receiving CPR were included in this study. This results in an overall incidence across all age groups of 3.08/100,000 children. There was slight variance within the incidence per year with a minimum of 1.68 in 2010 and a maximum of 4.26 in 2019. Incidence was highest in children aged <1 year, with 23.42/100,000 (Figure 1). Non shockable rhythms had the highest overall incidence, with 2.46/100,000 for initial rhythm. In contrast, the incidence of ventricular fibrillation (VF) was 0.29/100,000. The highest proportion of VF as initial rhythm was found in children aged 13 to <18 years, with 21.4% (n=74) (Figure 2a, Table 1). Across all age groups, "Home" was the most common location of OHCA with 1.91/100,000. In the age group 13 to <18 years, "Home" and "Public place" had proportions of 49.5% (n=163) and 41.3% (n=136), respectively (Figure 2b). The most common presumed cause of OCHA was hypoxia and cardiac with the overall incidences of 0.91 and 0.43/100,000, respectively. Presumed cause of OHCA differed  between the age groups. For children <1 year, hypoxia and sudden infant death syndrome (SIDS) were the leading presumed causes of OHCA. In older children, trauma, drowning and cardiac causes are increasing (Table 1). Across all age groups, "Home" was the most common location of OHCA with 1.91/100,000. In the age group 13 to <18 years, "Home" and "Public place" had proportions of 49.5% (n=163) and 41.3% (n=136), 13 to 18 years, Home and Public place had proportions of 49.5% (n 163) and 41.3% (n 136), respectively (Figure 2b). The most common presumed cause of OCHA was hypoxia and cardiac with the overall incidences of 0.91 and 0.43/100,000, respectively. Presumed cause of OHCA differed  between the age groups. Demographic overview For children <1 year, hypoxia and sudden infant death syndrome (SIDS) were the leading presumed causes of OHCA. In older children, trauma, drowning and cardiac causes are increasing (Table 1). respectively (Figure 2b). The most common presumed cause of OCHA was hypoxia and cardiac with the overall incidences of 0.91 and 0.43/100,000, respectively. Presumed cause of OHCA differed  between the age groups. For children <1 year, hypoxia and sudden infant death syndrome (SIDS) were the leading presumed causes of OHCA. In older children, trauma, drowning and cardiac causes are increasing (Table 1). Across the entire time span, OHCAs were mainly unwitnessed (1.80/100,000); however, this declined from 2007 (2.19) to 2021 (1.88). Bystander-witnessed OHCAs increased from an incidence of 0.51 in 2007 to 1.11 in 2021. This is equally distributed across all age groups. Across the entire time span, OHCAs were mainly unwitnessed (1.80/100,000); however, this declined from 2007 (2.19) to 2021 (1.88). Bystander-witnessed OHCAs increased from an incidence of 0.51 in 2007 to 1.11 in 2021. This is equally distributed across all age groups. Figure 2 – (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age group; Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing Figure 2 – (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age group; Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing The full demographic results including pre-existing conditions and pre emergency status are presented in Table 1. The full demographic results including pre-existing conditions and pre emergency status are presented in Table 1. Demographic overview # Location of OHCA: 110 cases are missing. Respective case numbers are: <1 Year: 702, 1-4 Years: 336, 5-12 Years: 263, 13-<18 Years: 329. ## Nighttime: 51 cases are missing. Respective case numbers are: 0-<1 Year: 712, 1-4 Years: 356, 5-12 Years 273, 13-<18 Years: 348. ### First rhythm assessed: 49 cases are missing. Respective case numbers are: 0- <1 Year: 712, 1-4 Years: 358, 5-12 Years: 277, 13-<18 Years: 344.* SIDS was only recorded in children up to the age of 2 years. Abbreviations: OHCA = out of hospital cardiac arrest; SIDS = sudden infant death syndrome; EMS = emergency medical service; CPR = cardiopulmonary resuscitation; ASA = American Society of Anaesthesiologists Classification; RACA = Return of spontaneous circulation after cardiac arrest Percentages were rounded, wherefore they might not add up to 100%. # Location of OHCA: 110 cases are missing. Respective case numbers are: <1 Year: 702, 1-4 Years: 336, 5-12 Years: 263, 13-<18 Years: 329. ## Nighttime: 51 cases are missing. Respective case numbers are: 0-<1 Year: 712, 1-4 Years: 356, 5-12 Years 273, 13-<18 Years: 348. ### First rhythm assessed: 49 cases are missing. Respective case numbers are: 0- <1 Year: 712, 1-4 Years: 358, 5-12 Years: 277, 13-<18 Years: 344.* SIDS was only recorded in children up to the age of 2 years. Abbreviations: OHCA = out of hospital cardiac arrest; SIDS = sudden infant death syndrome; EMS = emergency medical service; CPR = cardiopulmonary resuscitation; ASA = American Society of Anaesthesiologists Classification; RACA = Return of spontaneous circulation after cardiac arrest Percentages were rounded, wherefore they might not add up to 100%. # Location of OHCA: 110 cases are missing. Respective case numbers are: <1 Year: 702, 1-4 Years: 336, 5-12 Years: 263, 13-<18 Years: 329. ## Nighttime: 51 cases are missing. Respective case numbers are: 0-<1 Year: 712, 1-4 Years: 356, 5-12 Years 273, 13-<18 Years: 348. ### First rhythm assessed: 49 cases are missing. Respective case numbers are: 0- <1 Year: 712, 1-4 Years: 358, 5-12 Years: 277, 13-<18 Years: 344.* SIDS was only recorded in children up to the age of 2 years. Abbreviations: OHCA = out of hospital cardiac arrest; SIDS = sudden infant death syndrome; EMS = emergency medical service; CPR = cardiopulmonary resuscitation; ASA = American Society of Anaesthesiologists Classification; RACA = Return of spontaneous circulation after cardiac arrest Demographic overview Table 1 – Baseline Characteristics Table 1 – Baseline Characteristics Page 7/24 Characteristic   Incidence/100,000 Overall N = 1,740 3.08/100,000  <1 Year n = 731 23.42/100,000 1 – 4 Years n = 369 2.98/100,000 5 – 12 Years n = 283 1.16/100,000 13 – <18 Years n = 357 2.24/100,000 Age, median (Q1 to Q3) 1.8 years (0.3 to 10.9) 93 days (40 to 171) 2.2 years (1.5 to 3.2) 8.7 years (6.6 to 11.1) 16.2 years (14.9 to 17.1) Sex, n female (%) 686 (39.4%) 288 (39.4%) 160 (43.4%) 107 (37.8%) 131 (36.7%) Location of OHCA, n (%) / Incidence# Healthcare Facility outside hospital Public Place Home Educational Institution / Workplace Not recorded   45 (3%) / 0.07 378 (23.2%) / 0.66 1111 (68.2%) / 1.91 18 (1%) / 0.03 78 (5%) / 0.23   25 (4%) 59 (8%) 587 (83.6%) 3 (0.4%) 28 (4%)   8 (2%) 86 (25.6%) 218 (64.9%) 5 (2%) 19 (6%)   6 (2%) 97 (36.9%) 143 (54.4%) 3 (1%) 14 (5%)   6 (2%) 136 (41.3%) 163 (49.5%) 7 (2%) 17 (5%) Presumed cause, n (%) / Incidence  Cardiac Trauma Drowning Hypoxia Intoxication SIDS Other Unknown   241 (13.9%) / 0.43 193 (11.1%) / 0.37 134 (8%) / 0.22 540 (31.0%) / 0.91 24 (1%) / 0.04 211 (12.1%) / 0.38   20 (3%) 22 (3%) 7 (1%) 229 (31.3%) 3 (0.4%) 202 (27.6%) 51 (7%) 111 (15.2%)   29 (8%) 38 (10.3%) 72 (19.5%) 143 (38.7%) 2 (0.5%) 9 (2%)* 37 (10.0%) 39 (10.5%)   43 (15.2%) 45 (15.9%) 46 (16.2%) 83 (29.3%) 1 (0.4%) - 34 (12.0%) 31 (11.0%)   63 (17.6%) 88 (24.6%) 9 (3%) 85 (23.8%) 18 (5%) - 34 (9%) 60 (16.8%) Page 8/24 Incidence/100,000 , 3.08/100,000 23.42/100,000 2.98/100,000 1.16/100,000 n = 357 2.24/100,000 Age, median (Q1 to Q3) 1.8 years (0.3 to 10.9) 93 days (40 to 171) 2.2 years (1.5 to 3.2) 8.7 years (6.6 to 11.1) 16.2 years (14.9 to 17.1) Sex, n female (%) 686 (39.4%) 288 (39.4%) 160 (43.4%) 107 (37.8%) 131 (36.7%) Location of OHCA, n (%) / Incidence# Healthcare Facility outside hospital Public Place Home Educational Institution / Workplace Not recorded   45 (3%) / 0.07 378 (23.2%) / 0.66 1111 (68.2%) / 1.91 18 (1%) / 0.03 78 (5%) / 0.23   25 (4%) 59 (8%) 587 (83.6%) 3 (0.4%) 28 (4%)   8 (2%) 86 (25.6%) 218 (64.9%) 5 (2%) 19 (6%)   6 (2%) 97 (36.9%) 143 (54.4%) 3 (1%) 14 (5%)   6 (2%) 136 (41.3%) 163 (49.5%) 7 (2%) 17 (5%) Presumed cause, n (%) / Incidence  Cardiac Trauma Drowning Hypoxia Intoxication SIDS Other Unknown   241 (13.9%) / 0.43 193 (11.1%) / 0.37 134 (8%) / 0.22 540 (31.0%) / 0.91 24 (1%) / 0.04 211 (12.1%) / 0.38 156 (9%) / 0.31 241 (13.9%) / 0.43   20 (3%) 22 (3%) 7 (1%) 229 (31.3%) 3 (0.4%) 202 (27.6%) 51 (7%) 111 (15.2%)   29 (8%) 38 (10.3%) 72 (19.5%) 143 (38.7%) 2 (0.5%) 9 (2%)* 37 (10.0%) 39 (10.5%)   43 (15.2%) 45 (15.9%) 46 (16.2%) 83 (29.3%) 1 (0.4%) - 34 (12.0%) 31 (11.0%)   63 (17.6%) 88 (24.6%) 9 (3%) 85 (23.8%) 18 (5%) - 34 (9%) 60 (16.8%) Relation to sports activity, n (%) 40 (2%) 5 (1%) 1 (0.3%) 8 (3%) 26 (7%) Nighttime (10pm to 6pm), n (%)## 369 (21.2%) 166 (23.3%) 63 (17.7%) 55 (20.1%) 85 (24.4%) Characteristic   Incidence/100,000 Overall N = 1,740 3.08/100,000  <1 Year n = 731 23.42/100,000 1 – 4 Years n = 369 2.98/100,000 5 – 12 Years n = 283 1.16/100,000 13 – <18 Years n = 357 2.24/100,000 Witnessed by, n (%) / Incidence Bystander First Responder EMS Unwitnessed   632 (36.3%) / 1.07 22 (1%) / 0.04 113 (7%) / 0.19 973 (55.9%) /1.80   214 (29.3%) 11 (2%) 46 (6%) 460 (62.9%)   162 (43.9%) 5 (1%) 16 (4%) 186 (50.4%)   109 (38.5%) 3 (1%) 22 (8%) 149 (52.7%)   147 (41.2%) 3 (1%) 29 (8%) 178 (49.9%) Bystander CPR, n (%) 911 (52.3%) 368 (50.3%) 230 (62.3%) 149 (52.7%) 164 (45.9%) Telephone assisted CPR, n (%) 444 (25.5%) 210 (28.7%) 109 (29.5%) 55 (19.4%) 70 (19.6%) First assessed rhythm, n (%) / Incidence### Asystole PEA Ventricular Fibrillation Bradycardia   1155 (68.3%) / 2.05 245 (14.5%) / 0.41 153 (9%) / 0.29 138 (8%) / 0.23   529 (74.3%) 87 (12.3%) 33 (5%) 63 (9%)   240 (67.0%) 64 (17.9%) 10 (3%) 44 (12.3%)   186 (67.1%) 32 (11.6%) 36 (13.0%) 23 (8%)   200 (58.1%) 62 (18.1%) 74 (21.4%) 8 (2%) Pre Emergency Status, n (%) / Incidence ASA I ASA II ASA III ASA IV ASA V   690 (39.7%) / 1.02 145 (8%) / 0.21 209 (12.0%) / 0.31 135 (8%) / 0.19   304 (41.6%) 53 (7%) 97 (13.3%) 37 (5%) 1 (0.1%) 239 (32.7%)   145 (39.3%) 27 (7%) 49 (13.3%) 33 (9%) 1 (0.3%) 114 (30.9%)   102 (36.0%) 21 (7%) 39 (13.8%) 36 (12.7%) - 85 (30.0%)   139 (38.9%) 44 (12.3%) 24 (7%) 29 (8%) 1 (0.3%) 120 (33.6%) Page 9/24 Page 9/24 Characteristic   Incidence/100,000 Overall N = 1,740 3.08/100,000  <1 Year n = 731 23.42/100,000 1 – 4 Years n = 369 2.98/100,000 5 – 12 Years n = 283 1.16/100,000 13 – <18 Years n = 357 2.24/100,000 Pre-existing conditions, n (%)/ Incidence Cardiac Pulmonary Metabolic Malignancy Neurologic Immunodeficiency   181 (10.4%) / 0.27 120 (7%) / 0.20 80 (5%) / 0.12 19 (1%) / 0.03 204 (11.7%) / 0.33 21 (1%) / 0.03   99 (13.5%) 56 (8%) 35 (5%) 10 (1%) 46 (6%) 5 (1%)   34 (9%) 29 (8%) 16 (4%) 4 (1%) 56 (15.2%) 6 (2%)   29 (10.2%) 25 (9%) 14 (5%) 3 (1%) 54 (19.1%) 6 (2%)   19 (5%) 18 (5%) 15 (4%) 2 (1%) 48 (13.4%) 4 (1%) Percentages were rounded, wherefore they might not add up to 100%. Pre-hospital treatment Referring to the first link in the chainmail of survival [22], bystanders performed chest compression only (CCO) in 20.0% (n=348) of all cases. Chest compression and ventilation (CCV) were performed in 18.6% (n=324), with a higher proportion compared to CCO in 1 to 4 and 5 to 12 years (27.6% vs 20.1%, and 19.1% vs 17.3%, respectively). Out of 235 performed defibrillations, EMS performed 97.5% (n=229). In total six defibrillations were performed before EMS arrival, four of those by first responders. Refractory VF, defined as >3 shocks, was stated in 21.3% (n=50) of all cases, with the largest proportion in the 13 to <18 years group (26.5%, n=26). A similar proportion was found in the <1 year age group with 23.4% (n=15). Endotracheal intubation (ETI) was the most frequently used strategy for airway management in all age groups. In comparison, bag-mask-ventilation during CPR only was used in 3.8% (n=67) of all cases. Any type of advanced airway management during the course of prehospital treatment achieved higher aOR for hospital admission with ROSC (Figure 3, Supplement Table 4). Regarding supraglottic airway devices, laryngeal tubes (LT) were used in 54.7% (175/320) (Supplement Table 5c). Although an airway management device Page 10/24 Page 10/24 was used in most cases, end-tidal CO2 (etCO2) at hospital admission was not routinely reported. With an increase over the study period (Supplement Table 1b). Administration of adrenaline using intraosseous (i.o.) access was highest in the youngest age group (<1 year) with 42.1% and declined to 21.0% in the oldest age group (13 to <18 years). Full details on pre-hospital treatment are presented in Table 2 and Supplement Table 6. was used in most cases, end-tidal CO2 (etCO2) at hospital admission was not routinely reported. With an increase over the study period (Supplement Table 1b). Administration of adrenaline using intraosseous (i.o.) access was highest in the youngest age group (<1 year) with 42.1% and declined to 21.0% in the oldest age group (13 to <18 years). Full details on pre-hospital treatment are presented in Table 2 and Supplement Table 6. Pre-hospital treatment * Percentage calculated from the total of defibrillations (n = 235). ** Percentage calculated from the total cases where advanced airwaymanagement with SGA or ETI was performed (n = 1390). *** Percentage calculated from the total number of the administered drug (N). Abbreviations: BMV = bag-mask ventilation; SGA = supraglottic airway device; SD = standard deviation; CPR = cardiopulmonary resuscitation; Percentages were rounded, wherefore they might not add up to 100%. * Percentage calculated from the total of defibrillations (n = 235). ** Percentage calculated from the total cases where advanced airwaymanagement with SGA or ETI was performed (n = 1390). *** Percentage calculated from the total number of the administered drug (N). Abbreviations: BMV = bag-mask ventilation; SGA = supraglottic airway device; SD = standard deviation; CPR = cardiopulmonary resuscitation; Pre-hospital treatment Table 2 - Pre-hospital Treatment Page 11/24 Overall N = 1,740 <1 Year n = 731 1 – 4 Years n = 369 5 – 12 Years n = 283 13 – <18 Years n = 357 Treatment before EMS arrival, n (%)  Chest compression only  Ventilation only  Chest compression and ventilation  Defibrillation*   362 (20.8%) 35 (2%) 355 (20.4%) 6 (0.3%)   145 (19.8%) 11 (2%) 150 (20.5%) 1 (0.1%)   76 (20.6%) 10 (3%) 108 (29.3%) -   54 (19.1%) 3 (1%) 60 (21.2%) 2 (1%)   87 (24.4%) 11 (3%) 39 (10.9%) 3 (1%) Airway management, n (%)           BMV only 67 (4%) 26 (4%) 8 (2%) 8 (3%) 25 (7%) SGA** 320 (23%) 150 (26%) 59 (21%) 34 (15%) 77 (25%) Endotracheal Intubation** End-tidal CO2 at admission ** 1070 (77%) 425 (31%) 420 (74%) 119 (21%) 221 (79%) 103 (37%) 193 (85%) 85 (37%) 236 (75%) 118 (38%) Medication   Adrenaline, N i.v. n (%)*** i.o. n (%)*** 1579 873 (55%) 668 (42%) 673 344 (51%) 308 (46%) 360 177 (49%) 173 (48%) 269 156 (58%) 112 (42%) 277 196 (71%) 75 (21%) Atropine, N i.v. n (%)*** i.o. n (%)*** 176 106 (60%) 65 (37%) 52 28 (54%) 23 (44%) 56 31 (55%) 23 (41%) 37 23 (62%) 14 (38%) 31 24 (77%) 5 (16%) Amiodarone, N i.v. n (%)*** i.o. n (%)*** 70 61 (87%) 9 (13%) 17 17 (100%) - 4 2 (50%) 2 (50%) 10 7 (70%) 3 (30%) 39 35 (90%) 4 (10%) Crystalloid Fluids, n (%) 964 (55.4%) 352 (48.2%) 194 (52.6%) 189 (66.8%) 229 (64.1%) Thrombolysis, n (%) 19 (1%) 8 (1%) - - 10 (3%) Overall N = 1,740 <1 Year n = 731 1 – 4 Years n = 369 5 – 12 Years n = 283 13 – <18 Years n = 357 1 Shock, n (%)* 84 (35.7%) 21 (32.8%) 12 (48.0%) 20 (41.7%) 31 (31.6%) 2-3 Shocks, n (%)* 48 (20.4%) 4 (6%) 3 (12.0%) 10 (20.8%) 31 (31.6%) >3 Shocks, n (%)* 50 (21.3%) 15 (23.4%) 2 (8%) 7 (14.6%) 26 (26.5%) unknown, n (%)* 50 (21.3%) 24 (37.5%) 8 (32.0%) 11 (22.9%) 10 (10.2%) Mechanical CPR, n (%) 47 (3%) - - - 47 (13.2%) Percentages were rounded, wherefore they might not add up to 100%. * Percentage calculated from the total Percentages were rounded, wherefore they might not add up to 100%. Bystander influencing immediate outcome Chest compressions and ventilation started by bystanders had an adjusted odds ratio (aOR) for hospital admission with ROSC of 2.78 (95% CI 2.12 to 3.65), compared to CCV started by EMS. When only chest compressions were started by bystanders, aOR was also significant, with 1.72 (95% CI 1.38 to 2.14). OHCA witnessed by bystanders or EMS had increased aOR for hospital admission with ROSC compared to an unwitnessed OHCA. In addition to witness status and bystander CPR, the time of OHCA was analysed. Odds ratio were decresead during nighttime for admission with ROSC (Figure 3, Supplement Table 9). Figure 3 – Forest plot with adjusted odds ratio for hospital admission with ROSC. Abbreviations: CCV = chest compression and ventilation; CCO = chest compression only; EMS = emergency medical service; ROSC = return of spontaneous circulation; CI = confidence interval Outcome Of 1,740 cases, the main proportion achieved no pre-hospital ROSC across all age groups. Of 1,740 cases, the main proportion achieved no pre-hospital ROSC across all age groups. The incidence of children declared dead on scene after unsuccessful resuscitation was 1.20/100,000; similar to those admitted with ROSC to hospital admission (1.13/100,000). In 46.9% (n=343) in the group <1 year, children were declared dead on scene, compared to 28.2% (n=104) in 1 to 4 years, 31.8% (n=90) in 5 to 12 years and 35.0% (n=125) in 13 to <18 years Immediate and short-term outcomes for all age groups are shown in Table 3. In total, 994 children did not achieve ROSC prehospitally; with the majority being unwitnessed arrests and presenting with asystole as initial rhythm (Supplement Table 7). Long term outcome, where available, is presented in Supplement Table 8. EMS time intervals did not differ between any short-term outcome. An overall median response time of seven minutes (Q1 to Q3 5 to 9) was observed. Univariate analysis showed no difference for any ROSC when EMS response time was below 7 minutes compared to above 7 minutes (OR 0.86 [95% CI 0.70 to 1.04]) with a similar rate of bystander CPR (62.4% vs. 62.3%; p = 1.0). Across different outcomes, EMS time intervals did not differ (Supplement Table 9, Supplement Figure 1). Table 3 - Short term outcomes Page 13/24 Overall N = 1,740 0 – <1 Year n = 731 1 – 4 Years n = 369 5 – 12 Years n = 283 13 – <18 Years n = 357 Status at hospital admission, n (%) / Incidence           no admission, dead on scene 662 (38.0%) / 1.20 343 (46.9%) 104 (28.2%) 90 (31.8%) 125 (35.0%) admitted with ongoing CPR 427 (24.5%) / 0.75 170 (23.3%) 108 (29.3%) 74 (26.1%) 75 (21.0%) admitted with ROSC 650 (37.4%) / 1.13 218 (29.8%) 157 (42.5%) 118 (41.7%) 157 (44.0%) Unknown 1 (0.1%) - - 1 (0.4%) - Abbreviations: CPR = cardiopulmonary resuscitation; ROSC = return of spontaneous circulation Abbreviations: CPR = cardiopulmonary resuscitation; ROSC = return of spontaneous circulation Discussion This study identified 1,740 children with OHCA and CPR in Germany from 2007 until 2021. Children aged >7 days to <1 year had the highest incidence of OHCA with 23.42/100,000. While 83.6% of OHCA occurred at home in the youngest age group, occurrence of OHCA in public places had a significant proportion in school children (5 to 12 years) and adolescents (13 to <18 years) with 37% and 41%, respectively. Trauma and drowning constituted a significant proportion of presumed causes in children above 1 year, while cardiac causes accounted for 17.6% in the oldest age group. The overall incidence of any ROSC was low at 1.27/100,000. Adolescents were most frequently admitted with ROSC (44.0%; 157/357). Admittance with ongoing CPR was equally distributed across all age groups. Page 14/24 Bystander CCV and CCO had significantly higher aOR for hospital admission with ROSC. Confidence intervals of CCO and CCV for hospital admission with ROSC barely overlap, indicating a possible significant difference in favour of CCV [23]. To conclude, this points out the relevance of bystander-initiated ventilation Page 14/24 Page 14/24 in children suffering from OHCA even if survival to hospital discharge has not yet improved [24,25]. One confounder might be that bystanders who perform CCV are better trained and provide more sufficient CPR than those who provide chest compressions "only". This finding has also been identified within adults and should be translated into future first aid courses and can also be implemented in childbirth preparation courses [23]. Although the group of first responders was small, it is important to differentiate between bystanders and first responders [26]. Bystanders will perform CPR by chance, whereas first responers perform CPR by system. In this cohort advanced airway management (AAM) had higher aOR of hospital admission with ROSC, whereas  it was not associated with survival in other systems (4.6% vs 5.1%) [2,27]. These findings have been repreated throughout different systems. Due to the low incidence, recruitement of a significant sample size is associated with a very high effort, needing nation wide participation in a prospective trial. Therefore, prospective studies on AAM in children are likely to fail. The herein-presented data on immediate outcome compares to different ROSC rate at hospital admission, ranging from 8.1% [5] to 38.7% [28] in various systems. As awareness for recognition of cardiac arrest has increased over the last couple of years, different study periods might partially explain these differences. Discussion Age groups have not been uniformly defined across recent studies; however, children aged <1 year consistently have the highest proportion of OHCA [29]. Based on this study, future EMS training should focus on children <1 year in general and on the other age groups on treating trauma, drowning, and hypoxia as reversible causes. In adults, when no obvious cause of OHCA is present, ‘cardiac’ is stated as the presumed cause; in children an uniformly presumed cause in the absence of any clear reason for OHCA is undefined. This is mirrored in a combined share of 20.5% "unknown" and "other causes" within this study which corroborates a 30.0% of unknown causes in a recent study by Holgersen et al. [28]. Although one does not influence the timing of OHCA, the decreased aOR for nighttime indicates there is a continuous need to evaluate monitoring devices, such as surveillance matresses, for a potential role in aiding the early detection of cardiac or respiratory detoriation, alerting caregivers to prevent or recognise cardiac arrests immediately [30]. To our knowledge there has not been a study showing the benefit of such devices, however cardiac arrest registries could record the presence of this potentially preventive device. Always keeping the parent stress levels in mind. The high number (>80%) of unknown outcomes at 24 hours, 30 days, and discharge suggests mandatory participation for hospitals receiving children with OHCA. Other registries were able to determine reliable 30- day outcomes [28,31], enabled due to the linkage of different registries through a personal identification number. Data protection regulations should emphazise the usage of anonymized or pseudonymized data to gain a better understanding of scarce patient populations. Page 15/24 Although this study presents previously scarcely reported data from one of the largest cardiac arrest registries in Europe, there are some limitations. (1) The latest Utstein recommendation on reporting OHCA in the paediatric population from 1995 proposes a reporting flowchart [11]. However, as current registry variables do not support the proposed format, such a chart was not applied in our study. (2) Due to the retrospective nature of this study, it was not possible to assess causation for different interventions like airway management [2,27] or dosage of adrenaline [13]. (3) The lack of long-term outcome data limits the significance of  hospital admission with ROSC. Discussion (4) Due to the anonymised data, it was impossible to contact the participating sites to clarify discrepancies or collect missing data. (5) Any information on bystanders is missing. Therefore, it is impossible to differentiate bystanders with and without medical backgrounds. (6) Determining the cause of death and providing this information to EMS providers can improve patient care. Even when a coroner evaluates the cause of death, 11.2% (49/474) remain undetermined, displaying high complexity and uncertainty [32]. Future studies should attempt to establish global collaborations to provide a complete picture of OHCA in the paediatric population. Existing registries should try to collect prospective information on treatment decisions in order to get a better understanding of the rationale behind those decisions in this rare event. Conclusion This study reports the incidence of OHCA in the paediatric population over a 15-year period in Germany. Chest compressions and ventilation by bystander are identidified to have a higher impact on immediate survival compared with chest compression only. This study corroborates the findings of an increased incidence in <1 year old and a low survival rate. Acknowledgements We thank all participants from the German Resuscitation Registry for their contribution to this study. We also thank Alicja Suliman, graphic designer, for creating the visual abstract. We thank all participants from the German Resuscitation Registry for their c also thank Alicja Suliman, graphic designer, for creating the visual abstract. Authors' contributions SK, IKK, IT, JW, JKJ, JTG and EP made substantial contributions to the conception of this work. SK, IKK, IT, JKJ and JTG designed the work. SK and PR performed the data acquisition, MH significantly contributed to the data acquistion. SK performed the data analysis, PR and SS contributed to the data analysis. SB, FH and EP contributed to the interpretation of data. SK drafted the manuscript and all authors have substantively revised it. The final version of this manuscript is read and approved by all authors. Funding This study was done without funding Ethics approval and consent to participate Ethics approval and consent to participate This study was approved by the University Heidelberg ethics committee with reference number S-085/2022 on March 23rd 2022. Due to anonymous data, patients consent was waived. Abbreviations Page 16/24 AAM Advanced airway management ALS Advanced life support aOR Adjusted Odds ratio ASA American Society of Anaesthesiologists Classification CCO Chest compression only CCV Chest compression and ventilation CPC Cerebral performance category CPR Cardiopulmonary resusctiation DoA Deceased on arrival EMS Emergency medical service EMT Emergency medical technician ETI Endotracheal intubation EuReCa European Registriy of Cardiac arrest GRR German Resuscitation Registry i.o. Intraosseous access i.v. Intravenous access LT Laryngeal tube OHCA Out of hospital cardiac arrest PALS Paediatric advanced life support PEA Pulseless electrical activity RACA Return of spontaneous circulation after cardiac arrest ROSC Return of spontaneous circulation SD Standard deviation SIDS Sudden infant death syndrome STROBE Strengthening the Reporting of Observational studies in Epidemiology uaOR Unadjusted Odds ratio VF Ventricular fibrillation VT Ventricular tachycardia AAM Advanced airway management ALS Advanced life support aOR Adjusted Odds ratio ASA American Society of Anaesthesiologists Classification CCO Chest compression only CCV Chest compression and ventilation CPC Cerebral performance category CPR Cardiopulmonary resusctiation DoA Deceased on arrival EMS Emergency medical service EMT Emergency medical technician ETI Endotracheal intubation EuReCa European Registriy of Cardiac arrest GRR German Resuscitation Registry i.o. Intraosseous access i.v. Intravenous access LT Laryngeal tube OHCA Out of hospital cardiac arrest PALS Paediatric advanced life support PEA Pulseless electrical activity RACA Return of spontaneous circulation after cardiac arrest ROSC Return of spontaneous circulation SD Standard deviation SIDS Sudden infant death syndrome STROBE Strengthening the Reporting of Observational studies in Epidemiology uaOR Unadjusted Odds ratio VF Ventricular fibrillation Declarations Consent for publication Availability of data and materials An aggregated dataset used during the current study is available from the corresponding author on reasonable request. JKJ has no COI for the current research work, but has received unrestricted grant from Laerdal foundation or non-related research project References Page 18/24 Page 18/24 1. Fink EL, Prince DK, Kaltman JR, Atkins DL, Austin M, Warden C, et al. Unchanged pediatric out-of- hospital cardiac arrest incidence and survival rates with regional variation in North America. Resuscitation 2016;107:121–8. https://doi.org/10.1016/j.resuscitation.2016.07.244. 2. Le Bastard Q, Rouzioux J, Montassier E, Baert V, Recher M, Hubert H, et al. Endotracheal intubation versus supraglottic procedure in paediatric out-of-hospital cardiac arrest: a registry-based study. Resuscitation 2021;168:191–8. https://doi.org/10.1016/j.resuscitation.2021.08.015. 3. Atkins DL, Everson-Stewart S, Sears GK, Daya M, Osmond MH, Warden CR, et al. Epidemiology and outcomes from out-of-hospital cardiac arrest in children: the Resuscitation Outcomes Consortium Epistry-Cardiac Arrest. Circulation 2009;119:1484–91. https://doi.org/10.1161/CIRCULATIONAHA.108.802678. 4. Herlitz J, Svensson L, Engdahl J, Gelberg J, Silfverstolpe J, Wisten A, et al. Characteristics of cardiac arrest and resuscitation by age group: an analysis from the Swedish Cardiac Arrest Registry. Am J Emerg Med 2007;25:1025–31. https://doi.org/10.1016/j.ajem.2007.03.008. 5. Rajan S, Wissenberg M, Folke F, Hansen CM, Lippert FK, Weeke P, et al. Out-of-hospital cardiac arrests in children and adolescents: Incidences, outcomes, and household socioeconomic status. Resuscitation 2015;88:12–9. https://doi.org/10.1016/j.resuscitation.2014.11.025. 5. Rajan S, Wissenberg M, Folke F, Hansen CM, Lippert FK, Weeke P, et al. Out-of-hospital cardiac arrests in children and adolescents: Incidences, outcomes, and household socioeconomic status. Resuscitation 2015;88:12–9. https://doi.org/10.1016/j.resuscitation.2014.11.025. 6. Wyckoff MH, Greif R, Morley PT, Ng K-C, Olasveengen TM, Singletary EM, et al. 2022 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations: Summary From the Basic Life Support; Advanced Life Support; Pediatric Life Support; Neonatal Life Support; Education, I. 2022. https://doi.org/10.1161/cir.0000000000001095. 7. Girotra S, Spertus JA, Li Y, Berg RA, Nadkarni VM, Chan PS. Survival trends in pediatric in-hospital cardiac arrests an analysis from get with the guidelines-resuscitation. Circ Cardiovasc Qual Outcomes 2013;6:42–9. https://doi.org/10.1161/CIRCOUTCOMES.112.967968. 8. Moler FW, Meert K, Donaldson AE, Nadkarni V, Brilli RJ, Dalton HJ, et al. In-hospital versus out-of- hospital pediatric cardiac arrest: a multicenter cohort study. Crit Care Med 2009;37:2259–67. https://doi.org/10.1097/CCM.0b013e3181a00a6a. 9. Moler FW, Donaldson AE, Meert K, Brilli RJ, Nadkarni V, Shaffner DH, et al. Multicenter cohort study of out-of-hospital pediatric cardiac arrest. Crit Care Med 2011;39:141–9. https://doi.org/10.1097/CCM.0b013e3181fa3c17. 10. Voorde P Van De, Turner NM, Djakow J, Lucas N De, Martinez-mejias A, Biarent D, et al. European Resuscitation Council Guidelines 2021: Paediatric Life Support. Resuscitation 2021:1–61. https://doi.org/10.1016/j.resuscitation.2021.02.015. 10. Voorde P Van De, Turner NM, Djakow J, Lucas N De, Martinez-mejias A, Biarent D, et al. European Resuscitation Council Guidelines 2021: Paediatric Life Support. Resuscitation 2021:1–61. References https://doi.org/10.1016/j.resuscitation.2021.02.015. 11. Zaritsky A, Nadkarni V, Hazinski MF, Foltin G, Quan L, Wright J, et al. Recommended Guidelines for Uniform Reporting of Pediatric Advanced Life Support: The Pediatric Utstein Style. Ann Emerg Med 1995;26:487–503. https://doi.org/10.1016/S0196-0644(95)70119-2. 11. Zaritsky A, Nadkarni V, Hazinski MF, Foltin G, Quan L, Wright J, et al. Recommended Guidelines for Uniform Reporting of Pediatric Advanced Life Support: The Pediatric Utstein Style. Ann Emerg Med 1995;26:487–503. https://doi.org/10.1016/S0196-0644(95)70119-2. 12. Hansen ML, Lin A, Eriksson C, Daya M, McNally B, Fu R, et al. A comparison of pediatric airway management techniques during out-of-hospital cardiac arrest using the CARES database. Resuscitation 2017;120:51–6. https://doi.org/10.1016/j.resuscitation.2017.08.015. 12. Hansen ML, Lin A, Eriksson C, Daya M, McNally B, Fu R, et al. A comparison of pediatric airway management techniques during out-of-hospital cardiac arrest using the CARES database. Resuscitation 2017;120:51–6. https://doi.org/10.1016/j.resuscitation.2017.08.015. Page 19/24 Page 19/24 13. Recher M, Canon V, Lockhart M, Lafrance M, Hubert H, Leteurtre S. High dose of epinephrine does not improve survival of children with out-of-hospital cardiac arrest: Results from the French National Cardiac Arrest Registry. Front Pediatr 2022;10. https://doi.org/10.3389/fped.2022.978742. 14. Gräsner JT, Lefering R, Koster RW, Masterson S, Böttiger BW, Herlitz J, et al. EuReCa ONE—27 Nations, ONE Europe, ONE Registry: A prospective one month analysis of out-of-hospital cardiac arrest outcomes in 27 countries in Europe. Resuscitation 2016;105:188–95. https://doi.org/10.1016/j.resuscitation.2016.06.004. 15. Gräsner JT, Wnent J, Herlitz J, Perkins GD, Lefering R, Tjelmeland I, et al. Survival after out-of-hospital cardiac arrest in Europe - Results of the EuReCa TWO study. Resuscitation 2020;148:218–26. https://doi.org/10.1016/j.resuscitation.2019.12.042. 16. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. PLoS Med 2007;4:e296. https://doi.org/10.1371/journal.pmed.0040296. 16. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. PLoS Med 2007;4:e296. https://doi.org/10.1371/journal.pmed.0040296. 17. Gräsner JT, Meybohm P, Fischer M, Bein B, Wnent J, Franz R, et al. A national resuscitation registry of out-of-hospital cardiac arrest in Germany-A pilot study. Resuscitation 2009;80:199–203. https://doi.org/10.1016/j.resuscitation.2008.10.008. 17. Gräsner JT, Meybohm P, Fischer M, Bein B, Wnent J, Franz R, et al. A national resuscitation registry of out-of-hospital cardiac arrest in Germany-A pilot study. Resuscitation 2009;80:199–203. https://doi.org/10.1016/j.resuscitation.2008.10.008. 18. Ament R. Origin of the ASA Classification. Anesthesiology 1979;51:179. https://doi.org/10.1097/00000542-197908000-00023. 18. Ament R. Origin of the ASA Classification. Anesthesiology 1979;51:179. https://doi.org/10.1097/00000542-197908000-00023. References 19. Fischer, M., Wnent, J., Gräsner, J.-T., Seewald, S., Brenner, S., Bein, B., Ristau P, Bohn A. Annual Report of the German Resuscitation Registry: Out-of-Hospital Resuscitation 2021. Anästhesie Intensivmed 2022;63:V116–22. https://doi.org/10.19224/ai2022.V116. 19. Fischer, M., Wnent, J., Gräsner, J.-T., Seewald, S., Brenner, S., Bein, B., Ristau P, Bohn A. Annual Report of the German Resuscitation Registry: Out-of-Hospital Resuscitation 2021. Anästhesie Intensivmed 2022;63:V116–22. https://doi.org/10.19224/ai2022.V116. 20. Federal Statistical Office. Current population of Germany n.d. https://www.destatis.de/EN/Themes/Society-Environment/Population/Current-Population/_node.html (accessed January 5, 2023). https://www.destatis.de/EN/Themes/Society-Environment/Population/Current-Population/_node.html (accessed January 5, 2023). 21. Hodges JL, Lehmann EL. Estimates of Location Based on Rank Tests. In: Rojo J, editor. Sel. Work. E. L. Lehmann, Boston, MA: Springer US; 2012, p. 287–300. https://doi.org/10.1007/978-1-4614-1412-4_25. 21. Hodges JL, Lehmann EL. Estimates of Location Based on Rank Tests. In: Rojo J, editor. Sel. Work. E. L. Lehmann, Boston, MA: Springer US; 2012, p. 287–300. https://doi.org/10.1007/978-1-4614-1412-4_25. 22. Schnaubelt S, Greif R, Monsieurs K. The chainmail of survival-A modern concept of an adaptive approach towards cardiopulmonary resuscitation. Resuscitation 2023. 22. Schnaubelt S, Greif R, Monsieurs K. The chainmail of survival-A modern concept of an adaptive approach towards cardiopulmonary resuscitation. Resuscitation 2023. https://doi.org/10.1016/j.resuscitation.2023.109707. 22. Schnaubelt S, Greif R, Monsieurs K. The chainmail of survival-A modern concept of an adaptive approach towards cardiopulmonary resuscitation. Resuscitation 2023. https://doi.org/10.1016/j.resuscitation.2023.109707. 23. Wnent J, Tjelmeland I, Lefering R, Koster RW, Maurer H, Masterson S, et al. To ventilate or not to ventilate during bystander CPR — A EuReCa TWO analysis. Resuscitation 2021;166:101–9. https://doi.org/10.1016/j.resuscitation.2021.06.006. 23. Wnent J, Tjelmeland I, Lefering R, Koster RW, Maurer H, Masterson S, et al. To ventilate or not to ventilate during bystander CPR — A EuReCa TWO analysis. Resuscitation 2021;166:101–9. https://doi.org/10.1016/j.resuscitation.2021.06.006. 24. Murasaka K, Yamashita A, Owada H, Wato Y, Inaba H. Association between the types of bystander cardiopulmonary resuscitation and the survival with good neurologic outcome of preschool pediatric out-of-hospital cardiac arrest cases in Japan: A propensity score matching analysis using an extended nationwide. Front Pediatr 2023;10:1–8. https://doi.org/10.3389/fped.2022.1075983. 24. Murasaka K, Yamashita A, Owada H, Wato Y, Inaba H. Association between the types of bystander cardiopulmonary resuscitation and the survival with good neurologic outcome of preschool pediatric out-of-hospital cardiac arrest cases in Japan: A propensity score matching analysis using an extended nationwide. Front Pediatr 2023;10:1–8. https://doi.org/10.3389/fped.2022.1075983. 25. Albargi H, Mallett S, Berhane S, Booth S, Hawkes C, Perkins GD, et al. Bystander cardiopulmonary resuscitation for paediatric out-of-hospital cardiac arrest in England: An observational registry cohort study. Resuscitation 2022;170:17–25. https://doi.org/10.1016/j.resuscitation.2021.10.042. References Page 20/24 Page 20/24 26. Maurer H, Masterson S, Tjelmeland IBM, Gräsner J-T, Lefering R, Böttiger BW, et al. When is a bystander not a bystander any more? A European survey. Resuscitation 2019;136:78–84. https://doi.org/10.1016/j.resuscitation.2018.12.009. 27. Tham LP, Fook-Chong S, Binte Ahmad NS, Ho AFW, Tanaka H, Shin S Do, et al. Pre-hospital airway management and survival outcomes after paediatric out-of-hospital cardiac arrests. Resuscitation 2022;176:9–18. https://doi.org/10.1016/j.resuscitation.2022.04.018. 28. Holgersen MG, Jensen TW, Breindahl N, Kjerulff JLB, Breindahl SH, Blomberg SNF, et al. Pediatric out- of-hospital cardiac arrest in Denmark. Scand J Trauma Resusc Emerg Med 2022;30:58. https://doi.org/10.1186/s13049-022-01045-x. 29. Nehme Z, Namachivayam S, Forrest A, Butt W, Bernard S, Smith K. Trends in the incidence and outcome of paediatric out-of-hospital cardiac arrest: A 17-year observational study. Resuscitation 2018;128:43– 50. https://doi.org/10.1016/j.resuscitation.2018.04.030. 30. Fonseca AM, Horta ET, Sendra S, Rodrigues JJPC, Moutinho JAF. A sudden infant death prevention system for babies. 2014 IEEE 16th Int Conf e-Health Networking, Appl Serv Heal 2014 2014:525–30. https://doi.org/10.1109/HealthCom.2014.7001897. 30. Fonseca AM, Horta ET, Sendra S, Rodrigues JJPC, Moutinho JAF. A sudden infant death prevention system for babies. 2014 IEEE 16th Int Conf e-Health Networking, Appl Serv Heal 2014 2014:525–30. https://doi.org/10.1109/HealthCom.2014.7001897. 31. Lee J, Yang WC, Lee EP, Huang JL, Hsiao HJ, Lin MJ, et al. Clinical Survey and Predictors of Outcomes of Pediatric Out-of-Hospital Cardiac Arrest Admitted to the Emergency Department. Sci Rep 2019;9:1– https://doi.org/10.1038/s41598-019-43020-0. 31. Lee J, Yang WC, Lee EP, Huang JL, Hsiao HJ, Lin MJ, et al. Clinical Survey and Predictors of Outcomes of Pediatric Out-of-Hospital Cardiac Arrest Admitted to the Emergency Department. Sci Rep 2019;9:1–9. https://doi.org/10.1038/s41598-019-43020-0. 32. Ong MEH, Stiell I, Osmond MH, Nesbitt L, Gerein R, Campbell S, et al. Etiology of pediatric out-of- hospital cardiac arrest by coroner’s diagnosis. Resuscitation 2006;68:335–42. https://doi.org/10.1016/j.resuscitation.2005.05.026. Figures Page 21/24 Figure 2 (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age group; Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age group; Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing Page 23/24 Figure 1 Study flowchart demonstrating included cases and incidences per age group; *1 case with an unknown immediate outcome. Abbreviations: OHCA = out-of-hospital cardiac arrest; GRR = German Resuscitation Registry; CPR = cardiopulmonary resuscitation Study flowchart demonstrating included cases and incidences per age group; *1 case with an unknown immediate outcome. Abbreviations: OHCA = out-of-hospital cardiac arrest; GRR = German Resuscitation Registry; CPR = cardiopulmonary resuscitation Page 22/24 Figure 2 Figure 2 Figure 3 Forest plot with adjusted odds ratio for hospital admission with ROSC. Abbreviations: CCV = chest compression and ventilation; CCO = chest compression only; EMS = emergency medical service; ROSC = return of spontaneous circulation; CI = confidence interval Forest plot with adjusted odds ratio for hospital admission with ROSC. Abbreviations: CCV = chest compression and ventilation; CCO = chest compression only; EMS = emergency medical service; ROSC = return of spontaneous circulation; CI = confidence interval Page 23/24 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to dow PedsOHCAGerCARSupplements20230710.docx Page 24/24
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Classifying Patents Based on Their Semantic Content
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RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Bergeaud A, Potiron Y, Raimbault J (2017) Classifying patents based on their semantic content. PLoS ONE 12(4): e0176310. https://doi. org/10.1371/journal.pone.0176310 OPEN ACCESS Citation: Bergeaud A, Potiron Y, Raimbault J (2017) Classifying patents based on their semantic content. PLoS ONE 12(4): e0176310. https://doi. org/10.1371/journal.pone.0176310 Editor: Zhong-Ke Gao, Tianjin University, CHINA Received: December 26, 2016 Accepted: April 7, 2017 Published: April 26, 2017 Copyright: © 2017 Bergeaud 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. Antonin Bergeaud1☯, Yoann Potiron2☯, Juste Raimbault3,4☯* Antonin Bergeaud1☯, Yoann Potiron2☯, Juste Raimbault3,4☯* 1 Paris School of Economics - EHESS and Bank of France, Paris, France, 2 Faculty of Business and Commerce, Keio University, Tokyo, Japan, 3 UMR CNRS 8504 Ge´ographie-cite´s, Universite´ Paris VII, Paris, France, 4 UMR-T 9403 IFSTTAR LVMT, Ecole Nationale des Ponts et Chausse´es, Champs-sur-Marne, France ☯These authors contributed equally to this work. * juste.raimbault@polytechnique.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract In this paper, we extend some usual techniques of classification resulting from a large-scale data-mining and network approach. This new technology, which in particular is designed to be suitable to big data, is used to construct an open consolidated database from raw data on 4 million patents taken from the US patent office from 1976 onward. To build the pattern net- work, not only do we look at each patent title, but we also examine their full abstract and extract the relevant keywords accordingly. We refer to this classification as semantic approach in contrast with the more common technological approach which consists in taking the topology when considering US Patent office technological classes. Moreover, we docu- ment that both approaches have highly different topological measures and strong statistical evidence that they feature a different model. This suggests that our method is a useful tool to extract endogenous information. OPEN ACCESS Citation: Bergeaud A, Potiron Y, Raimbault J (2017) Classifying patents based on their semantic content. PLoS ONE 12(4): e0176310. https://doi. org/10.1371/journal.pone.0176310 Editor: Zhong-Ke Gao, Tianjin University, CHINA Received: December 26, 2016 Accepted: April 7, 2017 Published: April 26, 2017 Copyright: © 2017 Bergeaud 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 Citation: Bergeaud A, Potiron Y, Raimbault J (2017) Classifying patents based on their semantic content. PLoS ONE 12(4): e0176310. https://doi. org/10.1371/journal.pone.0176310 Editor: Zhong-Ke Gao, Tianjin University, CHINA Received: December 26, 2016 Accepted: April 7, 2017 Published: April 26, 2017 Copyright: © 2017 Bergeaud 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. Classifying patents based on their semantic content show that the laser/ink-jet printer technology resulted from the recombination of two differ- ent existing technologies. public, commercial, or not-for-profit sectors for this research. public, commercial, or not-for-profit sectors for this research. Consequently, technological classification combined with other features of patents can be a valuable tool for researchers interested in studying technologies throughout history and to pre- dict future innovations by looking at past knowledge and interaction across sectors and tech- nologies. But it is also crucial for firms that face an ever changing demand structure and need to anticipate future technological trends and convergence (see, e.g., [11]) to adapt to the result- ing increase in competition discussed in [12] and to maintain market share. Curiously, and in spite of the large number of studies that analyze interactions across technologies [13], little is known about the underlying “innovation network” (e.g. [14]). Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. In this monograph, we propose an alternative classification based on semantic network analysis from patent abstracts and explore the new information emerging from it. In contrast with the regular technological classification which results from the choice of the patent reviewer, semantic classification is carried automatically based on the content of the patent abstract. Although patent officers are experts in their fields, the relevance of the existing classi- fication is limited by the fact that it is based on the state of technology at the time the patent was granted and cannot anticipate the birth of new fields. To correct for this, the USPTO regu- larly make changes in its classification in order to adapt to technological change (for example, the “nanotechnology” class (977) was established in 2004 and retroactively to all relevant previ- ously granted patents). In contrast we don’t face this issue with the semantic approach. The semantic links can be clues of one technology taking inspiration from another and good pre- dictors of future technology convergence (e.g. [15] study semantic similarities from the whole text of 326 US-patents on phytosterols and show that semantic analysis have a good predicting power of future technology convergence). One can for instance consider the case of the word optic. Introduction Innovation and technological change have been described by many scholars as the main drivers of economic growth as in [1] and [2]. [3] advertised the use of patents as an economic indicator and as a good proxy for innovation. Subsequently, the easier availability of compre- hensive databases on patent details and the increasing number of studies allowing a more efficient use of these data (e.g. [4]) have opened the way to a very wide range of analysis. Most of the statistics derived from the patent databases relied on a few key features: the iden- tity of the inventor, the type and identity of the rights owner, the citations made by the patent to prior art and the technological classes assigned by the patent office post patent’s content review. Combining this information is particularly relevant when trying to capture the diffu- sion of knowledge and the interaction between technological fields as studied in [5]. With methods such as citation dynamics modeling discussed in [6] or co-authorship networks analysis in [7], a large body of the literature such as [8] or [9] has studied patents citation net- work to understand processes driving technological innovation, diffusion and the birth of technological clusters. Finally, [10] look at the dynamics of citations from different classes to Data Availability Statement: All data files are available from the Dataverse Repository, at URLs http://dx.doi.org/10.7910/DVN/BW3ACK and http:// dx.doi.org/10.7910/DVN/ZULMOY. All source code and results are available on the GitHub repository of the project at https://github.com/ JusteRaimbault/PatentsMining. Funding: Antonin Bergeaud received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors for this research. Yoann Potiron received private funding from Keio University. Juste Raimbault received no specific grant from any funding agency in the PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 1 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Background In our analysis, we will consider all utility patents granted in the United States Patent and Trademark Office (USPTO) from 1976 to 2013. A clearer definition of utility patent is given in S1 Text. Also, additional information on how to correctly exploit patent data can be found in [4] and [23]. An existing classification: The USPC system Each USPTO patent is associated with a non-empty set of technological classes and subclasses. There are currently around 440 classes and over 150,000 subclasses constituting the United State Patent Classification (USPC) system. While a technological class corresponds to the tech- nological field covered by the patent, a subclass stands for a specific technology or method used in this invention. A patent can have multiple technological classes, on average in our data a patent has 1.8 different classes and 3.9 pairs of class/subclass. At this stage, two features of this system are worth mentioning: (i) classes and subclasses are not chosen by the inventors of the patent but by the examiner during the granting process based on the content of the patent; (ii) the classification has evolved in time and continues to change in order to adapt to new technologies by creating or editing classes. When a change occurs, the USPTO reviews all the previous patents so as to create a consistent classification. Classifying patents based on their semantic content Furthermore, most of the existing studies rely on a subsample of patent data, whereas we implement it on the full US Patent database from 1976 to 2013. This way, a general structure of technological innovation can be studied. We draw from this application promising qualita- tive stylized facts, such as a qualitative regime shift around the end of the 1990s, and a signifi- cant improvement of citation modularity for the semantic classification when comparing to the technological classification. These thematic conclusions validate our method as a useful tool to extract endogenous information, in a complementary way to the technological classification. Finally, the statistical model introduced in Section Statistical Model seems to indicate that patents tend to cite more similar patents in the semantic network when fitted to data. In partic- ular, this propensity is shown to be significantly bigger than the corresponding propensity for technological classes, and this seems to be consistent over time. On the account of this infor- mation, we believe that patent officers could benefit very much from looking at the semantic network when considering potential citation candidates of a patent in review. The paper is organized as follows. Section Background presents the patent data, the existing classification and provide details about the data collection process. Section Semantic Classifi- cation Construction explains the construction of the semantic classes. Section Potential Results tests their relevance by providing exploratory results. Finally, section Conclusion discusses potential further developments and conclude. More details, including robustness checking, figures and technical derivations can be found in S2, S3 and S4 Text. Until more recently, this word was often associated with technologies such as photogra- phy or eye surgery, while it is now almost exclusively used in a context of semi-transistor design and electro-optic. This semantic shift did not happen by chance but contains informa- tion on the fact that modern electronic extensively uses technologies that were initially devel- oped in optic. Previous research has already proposed to use semantic networks to study technological domains and detect novelty. [16] was one of the first to enhance this approach with the idea of visualizing keywords network illustrated on a small technological domain. The same approach can be used to help companies identifying the state of the art in their field and avoid patent infringement as in [17] and [18]. More closely related to our methodology, [19] develop a method based on patent semantic analysis of patent to vindicate the view that this approach outperform others in the monitoring of technology and in the identification of novelty innova- tion. Semantic analysis has already proven its efficiency in various fields, such as in technology studies (e.g. [20] and [21]) and in political science (e.g. [22]). Building on such previous research, we make several contributions by fulfilling some short- comings of existing studies, such as for example the use of frequency-selected single keywords. First of all, we develop and implement a novel fully-automatized methodology to classify pat- ents according to their semantic abstract content, which is to the best of our knowledge the first of its type. This includes the following refinements for which details can be found in Sec- tion Semantic Classification Construction: (i) use of multi-stems as potential keywords; (ii) fil- tering of keywords based on a second-order (co-occurrences) relevance measure and on an external independent measure (technological dispersion); (iii) multi-objective optimization of semantic network modularity and size. The use of all this techniques in the context of semantic classification is new and essential from a practical perspective. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 2 / 22 Data collection and basic description Each patent contains an abstract and a core text which describe the invention. To see what a patent looks like in practice, one can refer to the USPTO patent full-text database http://patft. uspto.gov/netahtml/PTO/index.html or to Google patent which publishes USPTO patents in pdf format at https://patents.google.com. Although including the full core texts would be natu- ral and probably very useful in a systematic text-mining approach as done in [26], they are too long to be included and thus we consider only the abstracts for the analysis. Indeed, the seman- tic analysis counts more than 4 million patents, with corresponding abstracts with an average length of 120.8 words (and a standard deviation of 62.4), a size that is already challenging in terms of computational burden and data size. In addition, abstracts are aimed at synthesizing purpose and content of patents and must therefore be a relevant object of study (see [27]). The USPTO defines a guidance stating that an abstract should be “a summary of the disclosure as contained in the description, the claims, and any drawings; the summary shall indicate the technical field to which the invention pertains and shall be drafted in a way which allows the clear understanding of the technical problem, the gist of the solution of that problem through the invention, and the principal use or uses of the invention” (PCT Rule 8). We construct from raw data a unified database. Data is collected from USPTO patent red- book bulk downloads, that provides as raw data (specific dat or xml formats) full patent information, starting from 1976. Detailed procedure of data collection, parsing and consolida- tion are available in S2 Text. The latest dump of the database in Mongodb format is available at http://dx.doi.org/10.7910/DVN/BW3ACK. Collection and homogenization of the database into a directly usable database with basic information and abstracts was an important task as USPTO raw data formats are involved and change frequently. We count 4,666,365 utility patents with an abstract granted from 1976 to 2013. A very small number of patents have a missing abstract, these are patents that have been withdrawn and we do not consider them in the analysis. The number of patents granted each year increases from around 70,000 in 1976 to about 278,000 in 2013. When distributed by the year of application, the picture is slightly different. Classifying patents based on their semantic content information of all citations made by each patent (backward citations) and all citations received by each patent as of the end of 2013 (forward citations). We can thus build a complete network of citations that we will use later on in the analysis. Turning to the structure of the lag between the citing and the cited patent in terms of appli- cation date, we see that the mean of this lag is 8.5 years and the median is 7 years. This distribu- tion is highly skewed, the 95th percentile is 21 years. We also report 164,000 citations with a negative time lag. This is due to the fact that some citations can be added during the examina- tion process and some patents require more time to be granted than others. In what follows, we choose to restrict attention to pairs of citations with a lag no larger than 5 years. We impose this restriction for two reasons. First, the number of citations received peaks 4–5 years after application. Second, the structure of the citation lag is necessarily biased by the truncation of our sample: the more recent patents mechanically receive less citations than the older ones. As we are restricting to citations received no later than 5 years after the application date, this effect will only affect patents with an application date after 2007. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 A bibliographical network between patents: Citations As with scientific publications, patents must give reference to all the previous patents which correspond to related prior art. They therefore indicate the past knowledge which relates to the patented invention. Yet, contrary to scientific citations, they also have an important legal role as they are used to delimit the scope of the property rights awarded by the patent. One can consult [24] for more details about this. Failing to refer to prior art can lead to the invalidation of the patent (e.g. [25]). Another crucial difference is that the majority of the citations are actu- ally chosen by the examiners and not by the inventors themselves. From the USPTO, we gather PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 3 / 22 Classifying patents based on their semantic content following application, 10% of patents receive only one citation and 1% of them receive more than 100 citations. A within class citation is defined as a citation between two patents sharing at least one common technological class. Following this definition, 84% of the citations are within class citations. 14% of the citations are between two patents that share the exact same set of technological classes. Towards a complementary classification Potentialities of text-mining techniques as an alternative way to analyze and classify patents are documented in [26]. The author’s main argument, in support of an automatic classification tool for patent, is to reduce the considerable amount of human effort needed to classify all the applications. The work conducted in the field of natural language processing and/or text analy- sis has been developed in order to improve search performance in patent databases, build tech- nology map or investigate the potential infringement risks prior to developing a new technology (see [28] for a review). Text-mining of patent documents is also widely used as a tool to build networks which carry additional information to the simplistic bibliographic con- nections model as argued in [16]. As far as the authors know, the use of text-mining as a way to build a global classification of patents remains however largely unexplored. One notable exception can be found in [15] where semantic-based classification is shown to outperform the standard classification in predicting the convergence of technologies even in small samples. Semantic analysis reveals itself to be more flexible and more quickly adaptable to the appari- tion of new clusters of technologies. Indeed, as argued in [15], before two distinct technologies start to clearly converge, one should expect similar words to be used in patents from both technologies. Finally, a semantic classification where patents are gathered based on the fact that they share similar significant keywords has the advantage of including a network feature that can- not be found in the USPC case, namely that each patent is associated with a vector of probabil- ity to belong to each of the semantic classes (more details on this feature can be found in Section 1). Using co-occurrence of keywords, it is then possible to construct a network of pat- ents and to study the influence of some key topological features. As reviewed previously, the use of co-occurrences is the usual way to construct a semantic network. Other hybrid tech- nique such as bipartite semantic/authors networks, do not have the nice feature of relying solely on endogenous semantic information contained in data. Semantic classification construction In this section, we describe methods and empirical analysis leading to the construction of semantic network and the corresponding classification. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Data collection and basic description The number of patents steadily increase from 1976 to 2000 and remains constant around 200,000 per year from 2000 to 2007. Restricting our sample to patent with application date ranging from 1976 to 2007, we are left with 3,949,615 patents. These pat- ents cite 38,756,292 other patents with the empirical lag distribution that has been extensively analyzed in [4]. Conditioned on being cited at least once, a patent receives on average 13.5 cita- tions within a five-year window. 270,877 patents receive no citation during the next five years PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 4 / 22 Keywords relevance estimation Relevance definition. Following the heuristic in [29], we estimate relevance score in order to filter multi-stem. The choice of the total number of keywords to be extracted, which we shall denote Kw, is important, too small a value would yield similar network structures but including less information whereas very large values tend to include too many irrelevant key- words. We choose to set this parameter to Kw = 100,000. We first consider the filtration of k  Kw (with k = 4) to keep a large set of potential keywords but still have a reasonable number of co-occurrences to be computed. This step has only very marginal effects on the nature of the final keywords but is necessary for computational purposes. The filtration is done on the unit- hood ui, defined for keyword i as ui = fi  log(1 + li) where fi is the multi-stem’s number of appa- ritions over the whole corpus and li its length in words. A second filtration of Kw keywords is done on the termhood ti, where the formal definition can be found in Eq (1). It is computed as a chi-squared score on the distribution of the stem’s co-occurrences and then compared to a uniform distribution within the whole corpus. Intuitively, uniformly distributed terms will be identified as plain language and they are thus not relevant for the classification. More precisely, we compute the co-occurrence matrix (Mij), where Mij is defined as the number of patents where stems i and j appear together. The termhood score ti is defined as ti ¼ X j6¼i Mij P kMik P kMjk  2 P kMik P kMjk : ð1Þ ð1Þ Moving window estimation. The previous scores are estimated on a moving window with fixed time length following the idea that the present relevance is given by the most recent context and thus that the influence vanishes when going further into the past. Consequently, the co-occurrence matrix is chosen to be constructed at year t restricting to patent which applied during the time window [t −T0; t]. Note that the causal property of the window is cru- cial as the future cannot play any role in the current state of keywords and patents. This way, we will obtain semantic classes which are exploitable on a T0 time span. Keywords extraction Let P be the set of patents, we first assign to a patent p 2 P a set of potentially significant key- words K(p) from its text AðpÞ (that corresponds to the concatenation of its own title and abstract). K(p) are extracted through a similar procedure as the one detailed in [29]: 1. Text parsing and Tokenization: we transform raw texts into a set of words and sentences, reading it (parsing) and splitting it into elementary entities (words organized in sentences). 2. Part-of-speech tagging: attribution of a grammatical function to each of the tokens defined previously. 5 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Classifying patents based on their semantic content 3. Stem extraction: families of words are generally derived from a unique root called stem (for example compute, computer, computation all yield the same stem comput) that we extract from tokens. At this point the abstract text is reduced to a set of stems and their grammatical functions. 4. Multi-stems construction: these are the basic semantic units used in further analysis. They are constructed as groups of successive stems in a sentence which satisfies a simple gram- matical function rule. The length of the group is between 1 and 3 and its elements are either nouns, attributive verbs or adjectives. We choose to extract the semantics from such nomi- nal groups in view of the technical nature of texts, which is not likely to contain subtle nuances in combinations of verbs and nominal groups. Text processing operations are implemented in python in order to use built-in functions nltk library [30] for most of above operations. This library supports most of state-of-the-art natural language processing operations. Source code is openly available on the repository of the project at https://github.com/JusteRaimbault/PatentsMining. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Construction of the semantic network We keep the set of most relevant keywords KW and obtain their co-occurrence matrix as defined in Section 1. This matrix can be directly interpreted as the weighted adjacency matrix of the semantic network. At this stage, the topology of raw networks does not allow the extrac- tion of clear communities. This is partly due to the presence of hubs that correspond to fre- quent terms common to many fields (e.g. method, apparat) which are wrongly filtered as relevant. We therefore introduce an additional measure to correct the network topology: the concentration of keywords across technological classes, defined as: ctechðsÞ ¼ X NðtecÞ j¼1 kjðsÞ 2 P ikiðsÞ 2 ; where kj(s) is the number of occurrences of the sth keyword in each of the jth technological class taken from one of the N(tec) USPC classes. The higher ctech, the more specific to a techno- logical class the node is. For example, the terms semiconductoris widely used in electron- ics and does not contain any significant information in this field. We use a threshold parameter, defined as θc, and keep nodes with ctech(s) > θc. Likewise, edges with low weights correspond to rare co-occurrences and are considered to be noise. To account for this we define the threshold parameter for edges θw, and we filter edges with a weight below θw, follow- ing the rationale that two keywords are not linked “by chance” if they appear simultaneously a minimal number of time. To control for size effect, we normalize by taking yw ¼ yð0Þ w  NP where NP is the number of patents in the corpus (NP ¼ jPj). yð0Þ w is thus a varying parameter interpreted as a noise threshold per patent. Communities are then extracted using a standard modularity maximization procedure as described in [31] to which we add the two constraints captured by θw and θc, namely that edges must have a weight greater than θw and nodes a con- centration greater than θc. At this stage, both parameters θc and yð0Þ w are unconstrained and their choice is not straightforward. Indeed, many optimization objectives are possible, such as the modularity, network size or number of communities. We find that modularity is maxi- mized at a roughly stable value of θw across different θc for each year, corresponding to a stable yð0Þ w across years, which leads us to choose yð0Þ w ¼ 4:1  105. Classifying patents based on their semantic content choice of maximum time lag for citations made in Section 1. Accordingly, the sensitivity analy- sis for T0 = 2 can be found in Appendix S3 Text. choice of maximum time lag for citations made in Section 1. Accordingly, the sensitivity analy- sis for T0 = 2 can be found in Appendix S3 Text. Construction of the semantic network There is a priori no reason to choose any specific point among the dif- ferent optimums. Consequently, we have tried the analysis with all the candidate values for θc and found that the results are the most reasonable when taking θc = 0.06 (see Fig 1). We show in Fig 2 an example of semantic network visualization. Construction of the semantic network Then for the choice of θc, different candidates points lie on a Pareto front for the bi-objective optimization on number of commu- nities and network size. There is a priori no reason to choose any specific point among the dif- ferent optimums. Consequently, we have tried the analysis with all the candidate values for θc and found that the results are the most reasonable when taking θc = 0.06 (see Fig 1). We show in Fig 2 an example of semantic network visualization. where kj(s) is the number of occurrences of the sth keyword in each of the jth technological class taken from one of the N(tec) USPC classes. The higher ctech, the more specific to a techno- logical class the node is. For example, the terms semiconductoris widely used in electron- ics and does not contain any significant information in this field. We use a threshold parameter, defined as θc, and keep nodes with ctech(s) > θc. Likewise, edges with low weights correspond to rare co-occurrences and are considered to be noise. To account for this we define the threshold parameter for edges θw, and we filter edges with a weight below θw, follow- ing the rationale that two keywords are not linked “by chance” if they appear simultaneously a minimal number of time. To control for size effect, we normalize by taking yw ¼ yð0Þ w  NP where NP is the number of patents in the corpus (NP ¼ jPj). yð0Þ w is thus a varying parameter interpreted as a noise threshold per patent. Communities are then extracted using a standard modularity maximization procedure as described in [31] to which we add the two constraints captured by θw and θc, namely that edges must have a weight greater than θw and nodes a con- centration greater than θc. At this stage, both parameters θc and yð0Þ w are unconstrained and their choice is not straightforward. Indeed, many optimization objectives are possible, such as the modularity, network size or number of communities. We find that modularity is maxi- mized at a roughly stable value of θw across different θc for each year, corresponding to a stable yð0Þ w across years, which leads us to choose yð0Þ w ¼ 4:1  105. Then for the choice of θc, different candidates points lie on a Pareto front for the bi-objective optimization on number of commu- nities and network size. Keywords relevance estimation For example, this enables us to compute the modularity of classes in the citation network as in section 1. In the following, we take T0 = 4 (which corresponds to a five year window) consistently with the PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 6 / 22 Characteristics of semantic classes For each year t, we define as N ðsemÞ t the number of semantic classes which have been computed by clustering keywords from patents appeared during the period [t −T0, t] (we recall that we have chosen T0 = 4). Each semantic class k ¼ 1; . . . ; N ðsemÞ t is characterized by a set of key- words K(k, t) which is a subset of KW selected as described in previous sections. The cardinal of K(k, t) distribution across each semantic class k is highly skewed with a few semantic classes containing over 1,000 keywords, most of them with roughly the same number of keywords. In contrast, there are also many semantic classes with only two keywords. There are around PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 7 / 22 Classifying patents based on their semantic content Fig 1. Sensitivity analysis of network community structure to filtering parameters. We consider a specific window 2000–2004 and the obtained plots are typical. (Left panel) We plot the number of communities as a function of the edge threshold parameter θw for different values of the node threshold parameter θc. The maximum is roughly stable across θc (dashed red line). (Right panel) To choose θc, we do a Pareto optimization on communities and network size: the compromise point (red overline) on the Pareto front (purple overline: possible choices after having fixed yð0Þ w ; blue level gives modularity) corresponds to θc = 0.06. https://doi.org/10.1371/journal.pone.0176310.g001 Fig 1. Sensitivity analysis of network community structure to filtering parameters. We consider a specific window 2000–2004 and the obtained plots are typical. (Left panel) We plot the number of communities as a function of the edge threshold parameter θw for different values of the node threshold parameter θc. The maximum is roughly stable across θc (dashed red line). (Right panel) To choose θc, we do a Pareto optimization on communities and network size: the compromise point (red overline) on the Pareto front (purple overline: possible choices after having fixed yð0Þ w ; blue level gives modularity) corresponds to θc = 0.06. https://doi.org/10.1371/journal.pone.0176310.g001 https://doi.org/10.1371/journal.pone.0176310.g001 30 keywords by semantic class on average and the median is 2 for any t. Fig 3 shows that the average number of keywords is relatively stable from 1976 to 1992 and then picks around 1996 prior to going down. Title of semantic classes. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Characteristics of semantic classes USPC technological classes are defined by a title and a highly accurate definition which help retrieve patents easily. The title can be a single word (e.g.: class 101: “Printing”) or more complex (e.g.: class 218: “High-voltage switches with arc preventing or extinguishing devices”). As our goal is to release a comprehensive database in which each patent is associated with a set of semantic classes, it is necessary to give an insight on what these classes represent by associating a short description or a title as in [26]. In our case, such description is taken as a subset of keywords taken from K(k, t). For the vast majority of seman- tic classes that have less than 5 keywords, we decide to keep all of theses keywords as a descrip- tion. For the remaining classes which feature around 50 keywords on average, we rely on the topological properties of the semantic network. [32] suggest to retain only the most frequently used terms in K(k, t). Another possibility is to select 5 keywords based on their network cen- trality with the idea that very central keywords are the best candidates to describe the overall idea captured by a community. For example, the largest semantic class in 2003–2007 is charac- terized by the keywords: Support Packet; Tree Network; Network Wide; Voic Stream; Code Symbol Reader. Size of technological and semantic classes. We consider a specific window of observa- tions (for example 2000–2004), and we define Z the number of patents which appeared during that time window. For each patent i = 1,   , Z we associate a vector of probability where each component p ðsemÞ ij 2 ½0; 1Š, with j = 1,   , N(sem) and where X NðsemÞ j¼1 p ðsemÞ ij ¼ 1 X NðsemÞ j¼1 p ðsemÞ ij ¼ 1 (when there is no room for confusion, we drop the subscript t in N ðsemÞ t ). On average across all time windows, a patent is associated to 1.8 semantic classes with a positive probability. Next 8 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Classifying patents based on their semantic content Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters θc = 0.06 and yw ¼ yð0Þ w  NP ¼ 4:5e5  9:1e5. Characteristics of semantic classes For- mally, we define the number of technological classes as N(tec) (which is not time dependent contrary to the semantic case) and for j = 1,   , N(tec) the corresponding matrix of probability is defined as USPTO and consists in dividing equally the patents between all the classes they belong to. For- mally, we define the number of technological classes as N(tec) (which is not time dependent contrary to the semantic case) and for j = 1,   , N(tec) the corresponding matrix of probability is defined as p ðtecÞ ij ¼ Bij X NðtecÞ k¼1 Bik ; p ðtecÞ ij ¼ Bij X NðtecÞ k¼1 Bik ; where Bij equals 1 if the ith patent belongs to the jth technological class and 0 if not. When there is no room for confusion, we will drop the exponent part and write only pij when refer- ring to either the technological or semantic matrix. Empirically, we find that both classes exhibit a similar hierarchical structure in the sense of a power-law type of distribution of class sizes as shown in Fig 4. This feature is important, it suggests that a classification based on the text content of patents has some separating power in the sense that it does not divide up all the patents in one or two communities. where Bij equals 1 if the ith patent belongs to the jth technological class and 0 if not. When there is no room for confusion, we will drop the exponent part and write only pij when refer- ring to either the technological or semantic matrix. Empirically, we find that both classes exhibit a similar hierarchical structure in the sense of a power-law type of distribution of class sizes as shown in Fig 4. This feature is important, it suggests that a classification based on the text content of patents has some separating power in the sense that it does not divide up all the patents in one or two communities. Characteristics of semantic classes The corresponding file in a vector format (.svg), that can be zoomed and explored, is available as S1 File. https://doi.org/10.1371/journal.pone.0176310.g002 Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters θc = 0.06 and yw ¼ yð0Þ w  NP ¼ 4:5e5  9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available as S1 File Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters θc = 0.06 and yw ¼ yð0Þ w  NP ¼ 4:5e5  9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available as S1 File. Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters θc = 0.06 and yw ¼ yð0Þ w  NP ¼ 4:5e5  9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available as S1 File. https://doi.org/10.1371/journal.pone.0176310.g002 Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters θc = 0.06 and yw ¼ yð0Þ w  NP ¼ 4:5e5  9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available as S1 File. https://doi.org/10.1371/journal.pone.0176310.g002 we define the size of a semantic class as we define the size of a semantic class as we define the size of a semantic class as S ðsemÞ j ¼ X Z i¼1 p ðsemÞ ij : Correspondingly, we aim to provide a consistent definition for technological classes. For that purpose, we follow the so-called “fractional count” method, which was introduced by the PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 9 / 22 Classifying patents based on their semantic content Fig 3. This figure plots the average number of keywords by semantic class for each time window [t −4; t] from t = 1980 to t = 2007. https://doi.org/10.1371/journal.pone.0176310.g003 Fig 3. This figure plots the average number of keywords by semantic class for each time window [t −4; t] from t = 1980 to t = 2007. https://doi.org/10.1371/journal.pone.0176310.g003 USPTO and consists in dividing equally the patents between all the classes they belong to. Potential refinements of the method Our semantic classification method could be refined by combining it with other techniques such as Latent Dirichlet Allocation which is a widely used topic detection method (e.g. [33]), already used on patent data as in [34] where it provides a measure of idea novelty and the counter-intuitive stylized facts that breakthrough invention are likely to come out of local search in a field rather than distant technological recombination. Using this approach should first help further evaluate the robustness of our qualitative conclusions (external validation). Also, depending on the level of orthogonality with our classification, it can potentially bring an PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 10 / 22 Classifying patents based on their semantic content Fig 4. Sizes of classes. Yearly from t = 1980 to t = 2007, we plot the size of semantic classes (left-side) and technological classes (right- side) for the corresponding time window [t −4, t], from the biggest to the smallest. The formal definition of size can be found in Section Characteristics of Semantic Classes. Each color corresponds to one specific year. Yearly semantic classes and technological classes present a similar hierarchical structure which confirms the comparability of the two classifications. This feature is crucial for the statistical analysis in Section Statistical Model. Over time, curves are translated and levels of hierarchy stays roughly constant. https://doi.org/10.1371/journal.pone.0176310.g004 Fig 4. Sizes of classes. Yearly from t = 1980 to t = 2007, we plot the size of semantic classes (left-side) and technological classes (right- side) for the corresponding time window [t −4, t], from the biggest to the smallest. The formal definition of size can be found in Section Characteristics of Semantic Classes. Each color corresponds to one specific year. Yearly semantic classes and technological classes present a similar hierarchical structure which confirms the comparability of the two classifications. This feature is crucial for the statistical analysis in Section Statistical Model. Over time, curves are translated and levels of hierarchy stays roughly constant. https://doi.org/10.1371/journal.pone.0176310.g004 https://doi.org/10.1371/journal.pone.0176310.g004 additional feature to characterize patents, in the spirit of multi-modeling techniques where neighbor models are combined to take advantage of each point of view on a system. Our use of network analysis can also be extended using newly developed techniques of hyper-network analysis. Potential refinements of the method Indeed, patents and keywords can for example be nodes of a bipartite network, or patents be links of an hyper-network, in the sense of multiple layers with different classification links and citation links. The combination of citation network modeling by Sto- chastic Block Modeling with topic modeling was studied for scientific papers by [35], outper- forming previous link prediction algorithms. [36] provide a method to compare macroscopic structures of the different layers in a multilayer network that could be applied as a refinement of the overlap, modularity and statistical modeling studied in this paper. Furthermore, is has recently been shown that measures of multilayer network projections induce a significant loss of information compared to the generalized corresponding measure [37], which confirms the relevance of such development that we left for further research. An other potential research development would be to further exploit the temporal structure of our dataset. Indeed, large progress have recently been made in complex network analysis of time-series data (see [38] for a review). For example, [39] develops a method to construct mul- tiscale network from time series, which could in our case be a solution to identify structures in patents trajectories at different levels, and be an alternative to the single scale modularity anal- ysis we use. Classifying patents based on their semantic content several measures derived from this semantic classification at the patent level: Diversity, Origi- nality, Generality (Section Patent Level Measures) and Overlapping (Section Classes overlaps). We then show that the two classifications show highly different topological measures and strong statistical evidence that they feature a different model (Sections Citation Modularity and Statistical Model). Results In this section, we present some key features of our resulting semantic classification showing both complementary and differences with the technological classification. We first present 11 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Classifying patents based on their semantic content around 1996. This can be seen whether or not we include patents with zero diversity. The diversity of complicated patents stabilizes after a constant decrease, and the overall diversity begins to strongly decrease. This means that on the one hand the number of single class patents begins to increase and on the other hand complicated patents do not change in diversity. It can be interpreted as a change in the regime of specialization, the new regime being caused by more single-class patents. More commonly used in the literature are the measures of originality and generality. These measures follow the same idea than the above-defined diversity in quantifying the diversity of classes (whether technological or semantic) associated with a patent. But instead of looking at the patent’s classes, they consider the classes of the patents that are cited or citing. Formally, the originality Oi and the generality Gi of a patent i are defined as O ðzÞ i ¼ 1 X NðzÞ j¼1 X i02Ii pi0j X NðzÞ k¼1 X i02Ii pi0k 0 B B B B B @ 1 C C C C C A 2 and G ðzÞ i ¼ 1 X NðzÞ j¼1 X i02~I i pi0j X NðzÞ k¼1 X i02~I i pi0k 0 B B B B B @ 1 C C C C C A 2 ; O ðzÞ i ¼ 1 X NðzÞ j¼1 X i02Ii pi0j X NðzÞ k¼1 X i02Ii pi0k 0 B B B B B @ 1 C C C C C A 2 and G ðzÞ i ¼ 1 X NðzÞ j¼1 X i02~I i pi0j X NðzÞ k¼1 X i02~I i pi0k 0 B B B B B @ 1 C C C C C A 2 ; O ðzÞ i ¼ 1 X NðzÞ j¼1 X i02Ii pi0j X NðzÞ k¼1 X i02Ii pi0k 0 B B B B B @ 1 C C C C C A 2 and G ðzÞ i ¼ 1 X NðzÞ j¼1 X i02~I i pi0j X NðzÞ k¼1 X i02~I i pi0k 0 B B B B B @ 1 C C C C C A 2 ; where z 2 {tec, sem}, Ii denotes the set of patents that are cited by the ith patent within a five year window (i.e. if the ith patent appears at year t, then we consider patents on [t −T0, t]) when considering the originality and ~I i the set of patents that cite patent i after less than five years (i.e. we consider patents on [t, t+T0]) in the case of generality. Note that the measure of generality is forward looking in the sense that G ðzÞ i used information that will only be available 5 years after patent applications. Both measures are lower on average based on semantic classi- fication than on technological classification. Fig 6 plots the mean value of O ðsemÞ i , O ðtecÞ i , G ðsemÞ i and G ðtecÞ i . where z 2 {tec, sem}, Ii denotes the set of patents that are cited by the ith patent within a five year window (i.e. if the ith patent appears at year t, then we consider patents on [t −T0, t]) when considering the originality and ~I i the set of patents that cite patent i after less than five years (i.e. we consider patents on [t, t+T0]) in the case of generality. Note that the measure of generality is forward looking in the sense that G ðzÞ i used information that will only be available 5 years after patent applications. Both measures are lower on average based on semantic classi- fication than on technological classification. Fig 6 plots the mean value of O ðsemÞ i , O ðtecÞ i , G ðsemÞ i and G ðtecÞ i . Patent level measures Given a classification system (technological or semantic classes), and the associated probabili- ties pij for each patent i to belong to class j (that were defined in Section Characteristics of Semantic Classes), one can define a patent-level diversity measure as one minus the Herfindhal concentration index on pij by D ðzÞ i ¼ 1 X NðzÞ j¼1 p2 ij; with z 2 ftec; semg: We show in Fig 5 the distribution over time of semantic and technological diversity with the corresponding mean time-series. This is carried with two different settings, namely includ- ing/not including patents with zero diversity (i.e. single class patents). We call other patents “complicated patents” in the following. First of all, the presence of mass in small probabilities for semantic but not technological diversity confirms that the semantic classification contains patent spread over a larger number of classes. More interestingly, a general decrease of diver- sity for complicated patents, both for semantic and technological classification systems, can be interpreted as an increase in invention specialization. This is a well-known stylized fact as doc- umented in [40]. Furthermore, a qualitative regime shift on semantic classification occurs Fig 5. Patent level diversities. Distributions of diversities (Left column) and corresponding mean time-series (Right column) for t = 1980 to t = 2007 (with the corresponding time window [t −4, t]). The first row includes all classified patents, whereas the second row includes only patents with more than one class (i.e. patents with diversity greater than 0). https://doi org/10 1371/journal pone 0176310 g005 Fig 5. Patent level diversities. Distributions of diversities (Left column) and corresponding mean time-series (Right column) for t = 1980 to t = 2007 (with the corresponding time window [t −4, t]). The first row includes all classified patents, whereas the second row includes only patents with more than one class (i.e. patents with diversity greater than 0). PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 12 / 22 Classes overlaps A proximity measure between two classes can be defined by their overlap in terms of patents. Such measures could for example be used to construct a metrics between semantic classes. Intuitively, highly overlapping classes are very close in terms of technological content and one can use them to measure distance between two firms in terms of technology as done in [41]. Formally, recalling the definition of (pij) as the probability for the ith patent to belong to the Fig 6. Patent level originality (left hand side) and generality (right hand side) for t = 1980 to t = 2007 (with the corresponding time window [t −4, t]) as defined in subsection Patent Level Measures. https://doi org/10 1371/journal pone 0176310 g006 Fig 6. Patent level originality (left hand side) and generality (right hand side) for t = 1980 to t = 2007 (with the corresponding time window [t −4, t]) as defined in subsection Patent Level Measures. Fig 6. Patent level originality (left hand side) and generality (right hand side) for t = 1980 to t = 2007 (with the corresponding time window [t −4, t]) as defined in subsection Patent Level Measures. https://doi.org/10.1371/journal.pone.0176310.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 13 / 22 Classifying patents based on their semantic content jth class and NP as the number of patents it writes jth class and NP as the number of patents it writes Overlapjk ¼ 1 NP  X NP i¼1 pijpik: ð2Þ ð2Þ The overlap is normalized by patent count to account for the effect of corpus size: by con- vention, we assume the overlap to be maximal when there is only one class in the corpus. A corresponding relative overlap is computed as a set similarity measure in the number of pat- ents common to two classes A and B, given by oðA; BÞ ¼ 2  jA\Bj jAjþjBj. Intra-classification overlaps. The study of distributions of overlaps inside each classifica- tion, i.e. between technological classes and between semantic classes separately, reveals the structural difference between the two classification methods, suggesting their complementary nature. Their evolution in time can furthermore give insights into trends of specialization. We show in Fig 7 distributions and mean time-series of overlaps for the two classifications. The technological classification globally always follow a decreasing trend, corresponding to more and more isolated classes, i.e. specialized inventions, confirming the stylized fact obtained in previous subsection. Classes overlaps For semantic classes, the dynamic is somehow more intriguing and sup- ports the story of a qualitative regime shift suggested before. Although globally decreasing as technological overlap, normalized (resp. relative) mean overlap exhibits a peak (clearer for normalized overlap) culminating in 1996 (resp. 1999). Looking at normalized overlaps, classifi- cation structure was somewhat stable until 1990, then strongly increased to peak in 1996 and then decrease at a similar pace up to now. Technologies began to share more and more until a breakpoint when increasing isolation became the rule again. An evolutionary perspective on technological innovation [42] could shed light on possible interpretations of this regime shift: as species evolve, the fitness landscape first would have been locally favorable to cross- Fig 7. Intra-classification overlaps. (Left column) Distribution of overlaps Oij for all i 6¼ j (zero values are removed because of the log- scale). Right column) Corresponding mean time-series. (First row) Normalized overlaps. (Second row) Relative overlaps. https://doi.org/10.1371/journal.pone.0176310.g007 Fig 7. Intra-classification overlaps. (Left column) Distribution of overlaps Oij for all i 6¼ j (zero values are removed because of the log- scale). Right column) Corresponding mean time-series. (First row) Normalized overlaps. (Second row) Relative overlaps. https://doi.org/10.1371/journal.pone.0176310.g007 Fig 7. Intra-classification overlaps. (Left column) Distribution of overlaps Oij for all i 6¼ j (zero values are removed because of the log- scale). Right column) Corresponding mean time-series. (First row) Normalized overlaps. (Second row) Relative overlaps. https://doi.org/10.1371/journal.pone.0176310.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 14 / 22 Classifying patents based on their semantic content Fig 8. Distribution of relative overlaps between classifications. (Left) Distribution of overlaps at all time steps; (Right) Corresponding mean time-series. The decreasing trend starting around 1996 confirms a qualitative regime shift in that period. https://doi.org/10.1371/journal.pone.0176310.g008 Fig 8. Distribution of relative overlaps between classifications. (Left) Distribution of overlaps at all time steps; (Right) Corresponding mean time-series. The decreasing trend starting around 1996 confirms a qualitative regime shift in that period. https://doi.org/10.1371/journal.pone.0176310.g008 https://doi.org/10.1371/journal.pone.0176310.g008 insemination, until each fitness reaches a threshold above which auto-specialization becomes the optimal path. It is very comparable to the establishment of an ecological niche [43], the strong interdependency originating here during the mutual insemination resulting in a highly path-dependent final situation. Inter-classification overlaps. Overlaps between classifications are defined as in (Classes overlaps), but with j standing for the jth technological class and k for the kth semantic class: pij are technological probabilities and pik semantic probabilities. Classes overlaps They describe the relative corre- spondence between the two classifications and are a good indicator to spot relative changes, as shown in Fig 8. Mean inter-classification overlap clearly exhibits two linear trends, the first one being constant from 1980 to 1996, followed by a constant decrease. Although difficult to interpret directly, this stylized fact clearly unveils a change in the nature of inventions, or at least in the relation between content of inventions and technological classification. As the tip- ping point is at the same time as the ones observed in the previous section and since the two statistics are different, it is unlikely that this is a mere coincidence. Thus, these observations could be markers of a hidden underlying structural changes in processes. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 Classifying patents based on their semantic content Fig 9. Temporal evolution of semantic and technological modularities of the citation network. (Left) Simple directed modularity, computed with patent main classes (main technological class and semantic class with larger probability). (Right) Multi-class modularity, computed following [44]. Fig 9. Temporal evolution of semantic and technological modularities of the citation network. (Left) Simple directed modularity, computed with patent main classes (main technological class and semantic class with larger probability). (Right) Multi-class modularity, computed following [44]. https://doi.org/10.1371/journal.pone.0176310.g009 https://doi.org/10.1371/journal.pone.0176310.g009 (i.e. the number of citations made by the ith patent to others and the number of citations received by the ith patent). Qd can be defined for each of the two classification systems: z 2 {tec, sem}. If z = tec, ci is defined as the main patent class, which is taken as the first class whereas if z = sem, ci is the class with the largest probability. Multi-class modularity in turns is given by QðzÞ ov ¼ 1 NP X NðzÞ c¼1 X 1i;jNP Fðpic; pjcÞAij bout i;c kout i bin j;ckin j NP " # ; where bout i;c ¼ 1 NP X j Fðpic; pjcÞ and bin j;c ¼ 1 NP X i Fðpic; pjcÞ: We take F(pic, pjc) = pic  pjc as suggested in [44]. Modularity is an aggregated measure of how the network deviates from a null model where links would be randomly made according to node degree. In other words it captures the propensity for links to be inside the classes. Over- lapping modularity naturally extends simple modularity by taking into account the fact that nodes can belong simultaneously to many classes. We document in Fig 9 both simple and multi-class modularities over time. For simple modularity, Q ðtecÞ d is low and stable across the years whereas Q ðsemÞ d is slightly greater and increasing. These values are however low and sug- gest that single classes are not sufficient to capture citation homophily. Multi-class modulari- ties tell a different story. First of all, both classification modularities have a clear increasing trend, meaning that they become more and more adequate with citation network. The speciali- zations revealed by both patent level diversities and classes overlap is a candidate explanation for this growing modularities. Secondly, semantic modularity dominates technological modu- larity by an order of magnitude (e.g. 0.0094 for technological against 0.0853 for semantic in 2007) at each time. This discrepancy has a strong qualitative significance. Our semantic classi- fication fits better the citation network when using multiple classes. As technologies can be seen as a combination of different components as shown by [5], this heterogeneous nature is most likely better taken into account by our multi-class semantic classification. https://doi.org/10.1371/journal.pone.0176310.g009 Citation modularity An exogenous source of information on relevance of classifications is the citation network described in Section A bibliographical network between patents: citations. The correspon- dence between citation links and classes should provide a measure of accuracy of classifica- tions, in the sense of an external validation since it is well-known that citation homophily is expected to be quite high (see, e.g, [14]). This section studies empirically modularities of the citation network regarding the different classifications. To corroborate the obtained results, we propose to look at a more rigorous framework in Section Statistical Model. Modularity is a simple measure of how communities in a network are well clustered (see [31] for the accurate definition). Although initially designed for single-class classifications, this measure can be extended to the case where nodes can belong to several classes at the same time, in our case with different probabilities as introduced in [44]. The simple directed modularity is given in our case by Q ðzÞ d ¼ 1 NP X 1i;jNP Aij kin i kout j NP   dðci; cjÞ; with Aij the citation adjacency matrix (i.e. Aij = 1 if there is a citation from the ith patent to the jth patent, and Aij = 0 if not), kin i ¼ jIij (resp. kout i ¼ j~I ij) in-degree (resp. out-degree) of patents with Aij the citation adjacency matrix (i.e. Aij = 1 if there is a citation from the ith patent to the jth patent, and Aij = 0 if not), kin i ¼ jIij (resp. kout i ¼ j~I ij) in-degree (resp. out-degree) of patents PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 15 / 22 Classifying patents based on their semantic content obtained in Section Citation Modularity. The mere difference between this approach and the citation modularity approach lies in the choice of the underlying model, and the according quantities of interest. In addition for the semantic approach, we want to see if when restricting to patents with higher probabilities to belong to a class, we obtain better results. To do that, we choose to look at within class citations proportion (for both technological and semantic approaches). We provide two obvious reasons why we choose this. First, the citations are com- monly used as a proxy for performance as mentioned in Section Citation Modularity. Second, this choice is “statistically fair” in the sense that both approaches have focused on various goals and not on maximizing directly the within class proportion. Nonetheless, the within class pro- obtained in Section Citation Modularity. The mere difference between this approach and the citation modularity approach lies in the choice of the underlying model, and the according quantities of interest. In addition for the semantic approach, we want to see if when restricting to patents with higher probabilities to belong to a class, we obtain better results. To do that, we choose to look at within class citations proportion (for both technological and semantic approaches). We provide two obvious reasons why we choose this. First, the citations are com- monly used as a proxy for performance as mentioned in Section Citation Modularity. Second, this choice is “statistically fair” in the sense that both approaches have focused on various goals and not on maximizing directly the within class proportion. Nonetheless, the within class pro- portion is too sensitive to the distribution of the shape of classes. For example, a dataset where patents for each class account for 10% of the total number of patents will mechanically have a better within class proportion than if each class accounts for only 1%. Consequently, an ade- quate statistical model, which treats datasets fairly regardless of their distribution in classes, is needed. This effort ressembles to the previous study of citation modularity, but is complemen- tary since the model presented here can be understood as an elementary model of citation net- work growth. Furthermore, the parameters fitted here can have a direct interpretation as a citation probability. We need to introduce and recall some notations. We consider a specific window of observa- tions [t −T0, t], and we define Z the number of patents which appeared during that time win- dow. We let t1,   , tZ their corresponding appearance date by chronological order, which for simplicity are assumed to be such that t1 <    < tZ. For each patent i = 1,   , Z we consider Ci the number of distinctive couples {cited patent, cited patent’s class} made by the ith patent (for instance if the ith patent has only made one citation and that the cited patent is associated with three classes, then Ci = 3). Let z 2 {tec, sem}, we define N ðzÞ i the number of patents associated to at least one of the ith classes at time ti −1. For l = 1,   , Ci we consider the variables Bl, i, which equal 1 if the cited patent’s class is also common to the ith patent. We assume that Bl, i are inde- pendent of each other and conditioned on the past follow Bernoulli variables B min 1; N ðzÞ i i 1 þ yðzÞ ( ) ! ; where the parameter 0  θ(z)  1 indicates the propensity for any patent to cite patents of its own technological or semantic class. When θ(z) = 0, the probability of citing patents from its own class is simply N ðzÞ i ði 1Þ 1, which corresponds to the observed proportion of patents which belong to at least one of the ith patent’s classes. Thus this corresponds to the estimated probability of citing one patent if we assume that the probability of citing any patent k = 1,   , i −1 is uniformly distributed, which could be a reasonable assumption if classes were assigned randomly and independently from patent abstract contents. Conversely if θ(z) = 1, we are in the case of a model where there are 100% of within class citations. A reasonable choice of θ(z) lies between those two extreme values. Finally, we assume that the number of distinctive cou- ples Ci are a sequence of independent and identically distributed random variables following the discrete distribution C, and also independent from the other quantities. ( ) We estimate θ(z) via maximum likelihood, and obtain the corresponding maximum likeli- hood estimator (MLE) ^yðzÞ. Statistical model In this section, we develop a statistical model aimed at quantifying performance of both tech- nological and semantic classification systems. In particular, we aim at corroborating findings PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 16 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 https://doi.org/10.1371/journal.pone.0176310.t001 The likelihood function, along with the standard deviation expres- sion and details about the test, can be found in S4 Text. The fitted values, standard errors and p-values corresponding to the statistical test θ(sem) = θ(tec) (with corresponding alternative hypothesis θ(sem) > θ(tec)) on non-overlapping blocks from the period 1980–2007 are reported on Table 1. Note that the estimation included patents up until 2010 in the period 2006–2007 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310 April 26, 2017 17 / 22 Classifying patents based on their semantic content and not the patents from 1980 in the period 1980–1985 for homogeneity in size with other periods. This doesn’t affect the significativity of the results. Semantic values are reported for four different chosen thresholds p−= .04, .06, .08, .1. It means that we restricted to the couples (ith patent, jth class) such that pij  p−. The choice of considering non-overlapping blocks (instead of overlapping blocks) is merely statistical. Ultimately, our interest is in the significance of the test over the whole period 1980– 2007. Thus, we want to compute a global p-value. This can be done considering the local p-val- ues (by local, we mean for instance computed on the period 2001–2005) assuming indepen- dence between them. This assumption is reasonable only if the blocks are non-overlapping. All of this can be found in S4 Text. Finally, note that from a statistical perspective, including over- lapping blocks wouldn’t yield more information. The values reported in Table 1 are overwhelmingly against the null hypothesis. The global estimates of θ(sem) are significantly bigger than the estimate of θ(tec) for all the considered thresholds. Although the corresponding p-values (which are also very close to 0) are not reported, it is also quite clear that the bigger the threshold, the higher the corresponding θ(sem) is estimated. This is consistently seen for any period, and significant for the global period. This seems to indicate that when restricting to the couples (patent, class) with high semantic proba- bility, the propension to cite patents from its own class θ(sem) is increasing. We believe that this might provide extra information to patent officers when making their choice of citations. Indeed, they could look first to patents which belong to the same semantic class, especially when patents have high probability semantic values. Note that the introduced model can be seen as a simple model of citations network growth conditional to a classification, which can be expressed as a stochastic block model (e.g. [45], [46]). The parameters are estimated computing the corresponding MLE. In view of [47], this can be thought as equivalent to maximizing modularity measures. Classifying patents based on their semantic content Table 1. Estimated values of θ(tec) and θ(sem) and corresponding standard errors obtained from a Maxi- mum Likelihood estimator as presented in section Statistical Model. Approach Estimated Value st. er. p-value 1980–1985 period technological .664 .008 semantic p−= .04 .741 .047 .053 semantic p−= .06 .799 .081 .049 semantic p−= .08 .828 .126 .097 semantic p−= .10 .834 .166 .153 1986–1990 period technological .634 .007 semantic p−= .04 .703 .022 .001 semantic p−= .06 .768 .040 .0004 semantic p−= .08 .804 .069 .007 semantic p−= .10 .832 .114 .041 1991–1995 period technological .619 .006 semantic p−= .04 .655 .009 .0004 semantic p−= .06 .713 .017 9e-08 semantic p−= .08 .731 .025 7e-06 semantic p−= .10 .750 .037 9e-06 1996–2000 period technological .551 .003 semantic p−= .04 .585 .002 0 semantic p−= .06 .638 .004 0 semantic p−= .08 .660 .006 0 semantic p−= .10 .686 .008 0 2001–2005 period technological .567 .003 semantic p−= .04 .621 .004 0 semantic p−= .06 .676 .007 0 semantic p−= .08 .701 .010 0 semantic p−= .10 .710 .013 0 2006–2007 period technological .600 .007 semantic p−= .04 .683 .016 1e-06 semantic p−= .06 .732 .025 2e-07 semantic p−= .08 .760 .036 6e-06 semantic p−= .10 .782 .048 9e-05 1980–2007 global period technological .606 .002 semantic p−= .04 .665 .009 8e-11 semantic p−= .06 .721 .017 9e-12 semantic p−= .08 .747 .025 9e-09 semantic p−= .10 .782 .035 3e-07 https://doi.org/10.1371/journal.pone.0176310.t001 Table 1. Estimated values of θ(tec) and θ(sem) and corresponding standard errors obtained from a Maxi- mum Likelihood estimator as presented in section Statistical Model. 18 / 22 Classifying patents based on their semantic content Regarding firm innovative strategy, a second extension could be to study trajectories of firms in the two networks: technological and semantic. Merging these information with data on the market value of firms can give a lot of insight about the more efficient innovative strate- gies, about the importance of technology convergence or about acquisition of small innovative firms. It will also allow to observe innovation pattern over a firm life cycle and how this differ across technology field. A third extension would be to use dig further into the history of innovation. USPTO patent data have been digitized from the first patent in July 1790. However, not all of them contain a text that is directly exploitable. We consider that the quality of patent’s images is good enough to rely on Optical Character Recognition techniques to retrieve plain text from at least 1920. With such data, we would be able to extend our analysis further back in time and to study how technological progress occurs and combines in time. [50] conduct a similar work by looking at recombination and apparition of technological subclasses. Using the fact that communities are constructed yearly, one can construct a measure of proximity between two successive classes. This could give clear view on how technologies converged over the year and when others became obsolete and replaced by new methods. S2 Text. Data collection procedure. Detailed description of data collection. (PDF) S2 Text. Data collection procedure. Detailed description of data collection. (PDF) S3 Text. Network sensitivity analysis. Extended figures for Network Sensitivity Analysis. (PDF) S3 Text. Network sensitivity analysis. Extended figures for Network Sensitivity Analysis. (PDF) S4 Text. Statistical definitions and derivations. Extended definitions and derivations for the statistical model. (PDF) S4 Text. Statistical definitions and derivations. Extended definitions and derivations for the statistical model. (PDF) S1 File. Semantic network visualization. Vector file of the semantic network (Fig 2). (SVG) Supporting information S1 Text. Definition of utility patent. Describes with more details the definition of patents and context. (PDF) Conclusion The main contribution of this study was twofold. First we have defined how we built a network of patents based on a classification that uses semantic information from abstracts. We have shown that this classification share some similarities with the traditional technological classifi- cation, but also have distinct features. Second, we provide researchers with materials resulting from our analysis, which includes: (i) a database linking each patent with its set of semantic classes and the associated probabilities; (ii) a list of these semantic classes with a description based on the most relevant keywords; (iii) a list of patent with their topological properties in the semantic network (centrality, frequency, degree, etc.). The availability of this data suggests new avenues for further research. Linking our dataset with existing open ones can lead to vari- ous powerful developments. For example, using it together with the disambiguated inventor database provided by [48] could be a way to study semantic profiles of inventors, or of cities as inventor addresses are provided. The investigation of spatial diffusion of innovation between cities, which is a key component of Pumain’s Evolutive Urban Theory [49], would be made possible. 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Pressure-Assisted Development and Characterization of Al-Fe Interface for Bimetallic Composite Castings: An Experimental and Statistical Investigation for a Low-Pressure Regime
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Pressure Assisted Development and Characterization of Al-Fe Interface for bi-metallic Composite Castings: An Experimental and Statistical Investigation for Low Pressure Regime Tayyiba Rashid  University of Engineering & Technology Lahore Muhammad Qaiser Saleem  University of Engineering & Technology Lahore Nadeem Ahmad Mufti  University of Engineering & Technology Lahore Noman Asif  University of Engineering & Technology Lahore Kashif Ishfaq  University of Engineering & Technology Lahore Maham Naqvi  (  mn446@njit.edu ) New Jersey Institute of Technology Tayyiba Rashid  University of Engineering & Technology Lahore Muhammad Qaiser Saleem  University of Engineering & Technology Lahore Nadeem Ahmad Mufti  University of Engineering & Technology Lahore Noman Asif  University of Engineering & Technology Lahore Kashif Ishfaq  University of Engineering & Technology Lahore Maham Naqvi  (  mn446@njit.edu ) New Jersey Institute of Technology Pressure assisted development and characterization of Al-Fe interface for bi-metallic composite castings: An experimental and statistical investigation for low pressure regime Tayyiba Rashid1,2, Muhammad Qaiser Saleem2, Nadeem Ahmad Mufti2, Noman Asif2, Kashif Ishfaq2, Maham Naqvi3* Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engineering, University of ngineering & Technology Lahore, 54890, Pakistan Institute of Quality & Technology Management, University the Punjab Lahore, 54000, Pakistan Research Article Posted Date: April 8th, 2021 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 Metals on October 23rd, 2021. See the published version at https://doi.org/10.3390/met11111687. Keywords: Al-Fe interface, composite castings, interfacial properties, pressure-assisted casting, cooling rate, grey relational analysis g g gy Department of Mechanical & Industrial Engineering, New Jersey Institute of Technology, Newark NJ 071 SA g g gy 3 Department of Mechanical & Industrial Engineering, New Jersey Institute of Technology, Newark NJ 07102, USA 2 Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engineering, University of Engineering & Technology Lahore, 54890, Pakistan Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engineering Engineering & Technology Lahore, 54890, Pakistan Q y gy g y j 2 Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engin 1. Introduction: The continual strive for high strength cast components has garnered a renewed research interest in the development of metal reinforced castings (alternatively termed as dissimilar metal castings or metallic composite castings) with reportages of promising results [1–3]. In this context, a mix metal structure of steel and aluminum alloy poses itself to be a viable tradeoff between cost and strength. Interestingly, the studies on the so-called 'die soldering' phenomenon involving iron and aluminum alloys already indicate towards the affinity existing between these two materials [4, 5]. Aluminum's high affinity for Iron causes a strong reaction when Al melt comes into contact with ferrous material. This reaction produces iron- aluminum-silicon intermetallic compounds, which cause cast metal to stick to it. Various studies in this regard have primarily focused on exploring the factors that are responsible for promoting interface development between the two materials. The soldering is found to be highly dependent on temperature, pre-heat temperature, die surface conditions (topography/coatings) [5] and the chemistry of alloys [6]. A review of some of the available studies in this regard justifies that a strong rationale exists for developing Iron reinforced aluminum composites via casting route. Han et al. [7] studied the soldering for both pure Aluminum as well as Aluminum alloy 380 when fabricated inside a steel mold. In this work, a soldering critical temperature is suggested at which Iron (Fe) begins to react with Aluminum (Al) and results in the formation of an Al- rich liquid phase and solid intermetallic compounds. When the temperature at the die surface is higher than this critical temperature, the aluminum-rich phase is liquid and joins the die with the casting during the subsequent solidification. They purported that the critical temperature for soldering is either the solidus temperature or the coherency temperature between the two metals. In another research, Han et al.[8] reported liquid fraction of the solidifying metal (Al) to be influencing soldering tendency and mentioned surface roughness and placement angle of the steel die to be important. A higher liquid fraction, rougher surface and placement at a certain anglewere reported to be favoring the soldering phenomenon. Kim et al. [4], in their study, reported on the effect of Si concentration (in Al alloy) on the formation of Al-Si-Fe intermetallic layer on different grades of steel (H13 and 1045 steel). They concluded a higher percentage of Si to be resulting in increased thickness of the intermetallic layer. Abstract Review of the available literature indicates that development of metal reinforced castings present intriguing prospects but carry inherent challenges owing to differences in thermal coefficients, chemical affinities, diffusion issues and varying nature of intermetallic compounds. It is supported that pressure application during solidification may favorably influence the dynamics of the aforementioned issues, nevertheless, not only certain limitations have been cited but also some pressure and process regimes have not been found to be investigated and optimized. This work employs the pressure-assisted approach for bi- metallic steel reinforced aluminum composite castings at low-pressure regime and thoroughly investigates the role of three process parameters namely pouring temperature (800°C-900°C), pressure (10-20 bars) and holding time (10-20 sec) for producing sound interfaces. Taguchi L9 orthogonal array has been employed as DOE while dominant factors have been determined via ANOVA and Grey relational analysis multi-objective optimization technique. Supplementary analysis through optical micrographs, SEM and EDS has been relied upon to quantify interfacial layer thicknesses and to study microstructural and compositional aspects of the interface. Nano-indentation tests under static and dynamic loading have also been performed for mechanical strength characterization. It has been found that uniform interfaces with verifiable diffusion are obtainable with pouring temperature being the most influential parameter (PCR 92.84%) in this pressure regime. Optimum parameters determined from the work, yield ~328% thicker interface layer, 19.42% better nano-hardness and 19.10% improved cooling rate when compared to the process conditions with least parametric levels. Keywords: Al-Fe interface, composite castings, interfacial properties, pressure-assisted casting, cooling rate, grey relational analysis Keywords: Al-Fe interface, composite castings, interfacial properties, pressure-assisted casting, cooling rate, grey relational analysis Corresponding Author: Maham Naqvi; mn446@njit.edu 1 1 1. Introduction: They were able to achieve 28µm thick interface with 9% wt Si while keeping the dipping temperature constant at 680ºC. It is important to note that the aforementioned studies and most of the other available literature such as [9–12] have been 2 2 limited to hot dipping and aluminizing methodologies to produce the interfaces; these methods may not be representative of the state-of-the-art procedures employed for producing commercial castings. Work may also be found that employs contemporary methods (namely gravity die casting, high pressure die casting, diffusion-treatment and sand casting) to produce commercial castings for various material combinations including Al-Steel [13–17]. Incidentally, researchers have highlighted some issues in multi material castings such as lower diffusion of one element into the other and non-uniform thickness of the interface layer [15] thus necessitating a thorough investigation for Al-Steel interfaces produced through specific casting processes; this should enable the production of sound multi-material castings to meet industrial demands. Additionally, aspects like cooling rate of the casting and mechanical characterization of the developed interface are found to have received relatively less attention in reported studies. Correspondingly, the dynamics of interface development in a real life scenario with the role of process-specific parameters may need further attention. For the context, the few studies that have used mainstream casting routes while developing the interfaces may be cited not only to mention some positive aspects but to also highlight some process specific issues as reported. Viala et al. [16] used the gravity die molding method and reported on the interfacial chemistry of aluminum and steel. By achieving a continuous metallurgical bond between steel and aluminum, they successfully replaced a 165g cogwheel shaped insert with a 105g simple one. This helped in significant weight reduction of the cast component. In another study, Bhagat et al. [17] employed squeeze casting process for producing aluminum alloy-stainless steel metal composites while experimenting with various fiber volume fractions when stainless steel was used as a mesh in aluminum alloy matrix. They demonstrated the potential effectiveness of squeeze casting process for producing such composites while mentioning 850°C metal pouring temperature, 550°C die pre-heating temperature, and 110 MPa pressure to be suitable parameters. Nevertheless, their work highlighted some issues as encountered. 1. Introduction: Although 40% volume fraction was reported to be resulting in higher tensile strength of the composite in comparison to that reported for aluminum alloy (without reinforcement) but the composites were still reported to have lower strength (in general) to what had been predicted by the rule of mixtures. They attributed it to the generation of a weak interface as a result of 'more than desirable' reaction between fiber and matrix materials in the interface. They also cited limitations in generating uniform interface thickness and observance of large scatter in the values. The need for squeeze casting process parameters optimization was also cited to overcome the issues faced in interface development. Understandably, the higher-pressure 3 3 values employed in their work were those that are typical of a squeeze casting process and follow-up work of any sort that employed pressure assistance in lower value regime(s) for Al- Fe interface hasn’t been found in the available literature as yet. The recent work of Kashif et al. [18] also made use of the squeeze pressure but for producing Al overcast joints; since their work targeted only one type of material (Al) therefore the differences in thermal coefficients and affinity issues were supposedly neither much concerning issues nor were they thoroughly studied. Nevertheless, their work indicates the renewed interest in light of the potential feasibility of pressure-assisted approach for the multi-material components. The potential of the pressure driven casting process and its specific issues as highlighted for previous work on aluminum steel interfaces requires that a lower pressure regime may be investigated to evaluate if the so-called 'more than desirable' reactions may be avoided and better interfaces are developed. Additionally, the limitations as stated for the previous studies on the Al-Fe interfaces, call for a structured parametric investigation for evaluating an extended range of interface properties while correlating the results with important process dynamics, such as thermal/cooling aspects and pressure ratings. Further useful insight and potential benefits could be revealed when study of this sort is supplemented with superior optimization algorithms such as Grey relational analysis (GRA) which is a multi-response optimization technique used to identify the best quality characteristics through the determination of the optimum conditions of input parameters [19]. This work employs pressure-assisted casting route for producing bi-metallic steel reinforced aluminum composite castings. 1. Introduction: A low-pressure regime has been employed and the role of three process parameters, namely pressure, pouring temperature and time for application of pressure (holding time) has been thoroughly investigated for producing sound interfaces. An Al-Si alloy has been used with mild steel reinforcement. Interfacial layer thicknesses have been quantified alongwith the study of its microstructural, compositional and nano-hardness aspects. The evaluation of nano-hardness is given consideration, realizing that in view of the intermetallics formed, interface may be better characterized by nano-scale hardness parameter than microhardness. Discussions are supplemented with analysis of cooling rates as calculated for the experiments to better understand and explain the ensuing process dynamics. Multi-objective parametric optimization (through GRA) has been done for the role of input factors for the targeted outputs. 4 4 2.1 Experimental details A cylindrical shaped Al-5% Si alloy specimen (Փ60 x L=70 mm) reinforced by three equally spaced vertical mild steel rods placed at 120° apart (when viewed in cross-section) was cast using an 80-ton vertical hydraulic press. Instead of using a mesh structure, rods were employed for reinforcement so as to focus on interface development at different experimental conditions while preventing any potential adversities arising from complicated flow patterns (had mesh been used). Six K-type thermocouples interfaced with the data acquisition system4 were inserted into pre-defined locations to capture thermal profiles along radius and height. The thermocouples were connected to a Data Logger5 and ultimately linked to a computer system. Prior to placement in die (made of D2), the mild steel rods (Փ5 x L= 50 mm) were pickled with 90% hydrochloric acid solution followed by immersion in iso-propyle alcohol to clean rust (if present) on the surface as cleaning treatment used by others [20, 21]. For the pouring, melt was prepared in pit furnace. The die was pre-heated to 500°C using an external heating system; upon pouring, pressure was applied and maintained for a specified time (holding time). The bimetal casting was allowed to cool naturally at room temperature afterwards. Three settings of each input parameters, namely pouring temperature, applied pressure and holding time were used for experimentation. The values of input parameters were chosen on the basis of trial experiments conducted prior to actual experimentation. Taguchi L9 orthogonal array design was used as DOE. A total of nine experiments were performed. Fig. 1 provides the schematic of the specimen as placed in the die providing essential details such as locations of thermocouple placements and size of the specimen produced etc. The actual images for experimental setup & die are given in Fig. 2. Table 1 presents the details of variables and constants. For post-experimentation analysis, samples for microstructure, SEM analysis and mechanical characterization were extracted from the castings. Fig. 3 shows the schematic (3a & 3b) and actual cast component (3c) with the locations marked from where specimens for post-experimental analysis were taken. 2.1 Experimental details Table 1 Design of experiment Variables Level 1 Level 2 Level 3 Pouring temperature (°C) 800 850 900 Applied pressure (Bar) 10 15 20 Holding time (Sec) 10 15 20 5 5 Constants Reinforcement type ( RoundMS rod) Melt Mass 1.1kg Matrix Aluminum-5% Silicon Die Temperature (500°C) Fig.1 Schematic of the die Fig.2 Experimental setup Fig.3 a & b) Schematics of the part showing the length & location of the pins c) actual cast component Constants Matrix Aluminum-5% Silicon Melt Mass 1.1kg Die Temperature (500°C) Fig.1 Schematic of the die Fig.2 Experimental setup Fig.3 a & b) Schematics of the part showing the length & location of the pins c) actual cast component Fig.1 Schematic of the die Fig.1 Schematic of the die Fig.2 Experimental setup Fig.2 Experimental setup Fig.2 Experimental setup Fig.3 a & b) Schematics of the part showing the length & location of the pins c) actual cast component 6 Fig.4 Research methodology Fig.4 Research methodology Optical micrographs6 were taken at a magnification of 500X to examine the microstructure and to evaluate the thickness of the diffusion layer. Interface SEM7 images were taken at a magnification of 3000X for in-depth examination of compositional and microstructural details. Samples for microstructure and SEM were prepared using standard metallographic procedures. For mechanical characterization nanoindentation testing8 was done for the interface. The loading and unloading rate of 200mN/min and the acquisition rate of 10Hz were utilized. A record of penetration depth was made over a complete loading cycle with a maximum load of 100mN using depth sensing indentation. Three samples were tested in each testing condition to ensure repeatability and the average is reported herein. The complete research methodology is highlighted in Fig. 4. Fig.4 Research methodology Optical micrographs6 were taken at a magnification of 500X to examine the microstructure and to evaluate the thickness of the diffusion layer. Interface SEM7 images were taken at a magnification of 3000X for in-depth examination of compositional and microstructural details. Samples for microstructure and SEM were prepared using standard metallographic procedures. For mechanical characterization nanoindentation testing8 was done for the interface. The loading and unloading rate of 200mN/min and the acquisition rate of 10Hz were utilized. A record of penetration depth was made over a complete loading cycle with a maximum load of 100mN using depth sensing indentation. 8 NHTX S/N: 01-2569 nano-indentation machine, Austria p y 7 Inspect S-50 of Thermo Fisher Scientific USA 6 Leica DMI 500 microscopic machine, Germany 2.2.1 Data pre-processing GRA begins with the grey relational generationthrough the normalization of data gathered through experimentation. For GRA, data pre-processing is needed as there may be a difference of range and units in different data sequences or the scatter range of the sequence is large. Therefore, in data pre-processing, the original sequence is transferred into a comparable sequence. The range for normalization is kept between 0.00 to 1.00. The best normalized results must be equal to 1.00 [23]. Equation 1 is used for this purpose. 𝑥𝑖(𝑘) = 𝑦𝑖(𝑘) −𝑚𝑖𝑛𝑦𝑖(𝑘) 𝑚𝑎𝑥𝑦𝑖(𝑘) −𝑚𝑖𝑛𝑦𝑖(𝑘) (1) (1) where 𝑥𝑖(𝑘) is the normalized value for kth observation after data pre-processing, 𝑦𝑖(𝑘) is the original sequence while 𝑚𝑖𝑛 𝑦𝑖(𝑘) and 𝑚𝑎𝑥 𝑦𝑖(𝑘) are the smallest and largest experimental values of particular responses from the design matrix, respectively. i is the data number (observations) which in our design matrix is from 1 to 9. 2.2 Statistical analysis Statistical analysis was carried out for the obtained results using statistical software Minitab 19.1. The Analysis of Variance (ANOVA) was performed at a confidence level of 95% in order to find out the significant input parameters and their percentage contribution (PCR) for selected responses. Additionally, Grey Relational Analysis (GRA) was employed for multi- objective optimization of casting parameters. This analysis is a suitable multi-step method [22] used to measure the degree of approximation (among sequences) through Grey Relational Grade (GRG).The main objectives takenfor this work were the maximization of Interface thickness (IT), nano-level hardness (H) and the cooling rate (Rc). Correspondingly, "larger-the-better" was taken as the criteria. 2.1 Experimental details Three samples were tested in each testing condition to ensure repeatability and the average is reported herein. The complete research methodology is highlighted in Fig. 4. 7 7 3 Results, analysis and discussions The experimentation results for the 9 experiment runs are provided in Table 2. The succeeding sections contain the detailed analysis and discussions. 2.2.2 Gray relational coefficient and gray relational grade After the data normalization, grey relational coefficients (GRC) are calculated using normalized values to highlight the relation between desired/ideal and actual normalized results. The GRC highlights the relationship between desirable and real experimental normalized results. The GRG is computed by averaging the GRC. This GRG value gives a single grade for response variables [24]. Equation 2 is used for GRC calculations. GRG values range from 0 to 1. µ𝑖(k) = δ𝑚𝑖𝑛+ mδ𝑚𝑎𝑥 δ0𝑖(k) + 𝑚δ𝑚𝑎𝑥) (2) (2) 8 8 δ0𝑖(k) = ‖𝑥0(𝑘) −𝑥𝑖(𝑘)‖ (3) (3) (3) µ𝑖(k) is the grey relational coefficient, m is the identification/distinguishing coefficient that is usually set to 0.5 [25]. δ𝑚𝑖𝑛 and δ𝑚𝑎𝑥 are the minimum and maximum deviations of each response variable. δ0𝑖(k) is called deviation sequence which is calculated by using Equation 3. This determines the deviation of normalized data from the ideal value of 1.00. On the other hand, Equation 4 is used for GRG calculation by taking the average of GRCs. 𝐺𝑅𝐺𝑖= 1 𝑛∑𝑤𝑖 𝑛 𝑘=1 µ𝑖 ∑𝑤𝑖=1 𝑛 𝑘=1 (4) (4) GRG values range from 0 to 1. The highest value of GRG infers the ultimate combination of eminent casting process characteristics. GRG values range from 0 to 1. The highest value of GRG infers the ultimate combination of eminent casting process characteristics. 3.1.1 Microscopic and compositional analysis The thickness of the interface where materials diffuse into each other has been measured for all the experiments via micrographs. The representative micrographs for all the nine experiment conditions are presented in Fig. 5 for comparison purposes. As can be seen from the micrographs, interface thickness increases as the higher values of parameters (pressure and pouring temperature) are used. An average interface thickness of ~42microns achieved at higher values of above mentioned parameters is around 328% more than ~9.8microns obtained at the least values of process parameters employed herein. In essence, the improved interface thickness values are indicative of the increased diffusion as temperature and pressure values increase. Table 2 Experimentation results Exp. No Pouring Temperature (°C) Applied Pressure (bar) Holding Time (Sec) IT (µm) H (GPa) Rc (°C/sec) 1 800 10 10 9.8 9.01 1.78 2 800 15 15 11.0 9.25 1.94 3 800 20 20 13.8 9.58 2.09 Table 2 Experimentation results 9 4 850 10 15 18.9 9.96 1.86 5 850 15 20 28.5 10.03 1.98 6 850 20 10 30.7 10.14 2.09 7 900 10 20 38.0 10.27 1.93 8 900 15 10 40.9 10.45 2.07 9 900 20 15 42.0 10.76 2.12 Fig.5 Microstructures of the interface region of bimetals made at different temperature and pressure 10bar, 10sec 15bar, 15sec 20bar, 20sec Pouring Temp. (800°C) 10bar, 15sec 15bar, 20sec 20bar, 10sec Pouring Temp. (850°C) 10bar, 20sec 15bar, 10sec 20bar, 15sec Pouring Temp. (900°C) 4 850 10 15 18.9 9.96 1.86 5 850 15 20 28.5 10.03 1.98 6 850 20 10 30.7 10.14 2.09 7 900 10 20 38.0 10.27 1.93 8 900 15 10 40.9 10.45 2.07 9 900 20 15 42.0 10.76 2.12 Fig.5 Microstructures of the interface region of bimetals made at different temperature and pressure 10bar, 10sec 15bar, 15sec 20bar, 20sec Pouring Temp. (800°C) 10bar, 15sec 15bar, 20sec 20bar, 10sec Pouring Temp. (850°C) 10bar, 20sec 15bar, 10sec 20bar, 15sec Pouring Temp. (900°C) 20bar, 10sec 20bar, 10sec Pouring Temp. (850°C) 10bar, 20sec 15bar, 10sec 20bar, 15sec 20bar, 15sec 10bar, 20sec 15bar, 10sec Pouring Temp. (900°C) Pouring Temp. (900°C) Fig.5 Microstructures of the interface region of bimetals made at different temperature and pressure These results wherein higher temperature has been seen to cause more interface thickness in general, are effectively supported by Fick’s law for diffusion [26] when reviewed in conjunction with the diffusivity relation. 3.1.1 Microscopic and compositional analysis It can thus be shown that the diffusionis a function of temperature. At higher temperatures, atoms/molecules gain more mobility in liquids as well as solids. A higher initial concentration of a particular element may cause its diffusion into other solid. Fick’s first law has expressed this diffusional flow mathematically as shown in Equation 5. in Equation 5. 10 𝑱𝒙= −𝑫𝛅𝐂 𝛅𝐗 (5) 𝛅𝐂 (5) 𝑱𝒙= −𝑫𝛅𝐂 𝛅𝐗 Where Jx is the flow rate of the diffusing species in the direction X based on the gradient ( 𝛅𝐂 𝛅𝐗). Here D is termed as the diffusion coefficient (also known as diffusivity). The following expression (Equation 6) is used to calculate diffusivity D. Where Jx is the flow rate of the diffusing species in the direction X based on the gradient ( 𝛅𝐂 𝛅𝐗). Here D is termed as the diffusion coefficient (also known as diffusivity). The following expression (Equation 6) is used to calculate diffusivity D. 𝐷= 𝐷𝑜𝑒−𝑄 𝑅𝑇 ⁄ (6) 𝐷 𝐷𝑜𝑒 ( ) Do, is the pre-exponential coefficient, Q is the activation energy per mole of diffusing species, and R is the universal gas constant. The T is the absolute temperature. This equation shows that the Diffusion coefficient is based on temperature and as the reciprocal of temperature (1/T) decreases, diffusion increases. The increase in temperature triggers diffusion to progress more swiftly, resulting in thicker diffusion layers at the interface. Correspondingly, a higher temperature is seen to result in better values. In terms of pressure, the results arein general conformance to the other available literature [27, 28], which shows the role of pressure to be favorable as well. Here too, for a pouring temperature of 900°C, the thickness at 20 bar is around 10% higher compared to the one obtained at 10 bar even when a relatively lesser holding time has been employed. An additional and visible advantage of the pressure-assisted process is the generation of a nearly uniform interface for each case as can be seen in Fig. 5. More specifically, when compared with other reported work such as by Chen et al. [29] who employed nearly 200 times higher pressure in compound forging process to fabricate Al-Steel bimetal, it is observed that the achieved interface here is much thicker than the already reported work. In their work the interface thickness of 7.9µm was achieved when the initial temperature of the steel ball was kept at 900°C & the applied pressure was 503MPa. Incidentally, the average interface thickness achieved in current study ranges from 9.8µm to 42.0µm which is much higher that of achieved by the compound forging. in Equation 5. The possible reason for higher interface obtained herein is that, a higher pressure employed in the reported work would increase rate of heat transfer which would result in quicker heat loss from the components, thus not allowing much time for the constituents to diffuse; this is not the case herein where the lesser values of pressure would inevitably be resulting into relatively lesser rate of heat loss which is expected to give reasonable time for the constituents to diffuse up to a certain distance. Additionally, the forging process is a solid state process for which the rate of diffusion would be lesser than the current process where aluminum alloy has been poured in liquid state. Furthermore when compared with reported work [30] on compound casting wherein the Furthermore, when compared with reported work [30] on compound casting wherein the applied force is presumably much lesser than the pressure employed in the current work, 11 much lower interface thicknesses has been observed. The maximum interface thickness of around 30µm is reported for Al-steel bimetallic which is ~28.57% less than what has been achieved with the pressure assisted approach herein. Even for the case of other systems such as Al-Cu, it is deduced that very high pressure may not necessarily be resulting in the very thick interfaces. Liu et al. [31] while producing bi- metal component (Al-Cu) through squeeze casting process, observed increase in the thickness of the reaction layer as the temperature was increased (due to the higher diffusion at elevated temperature) however, increase in squeeze pressure resulted in decreasing trend of the overall thickness. The highest value obtained for thickness was about 80µm at 30MPa pressure which further decreased when pressure was increased to 110MPa. As stated earlier, the higher rate of heat transfer during solidification associated with higher pressure values is the most likely reason. Above analysis indicates the importance of the lower pressure regime in producing an appropriate level of interface thickness enabled by the balance between pressure and available time for diffusion to occur. Fig. 6 gives the summary of results for interface thickness in graphical form. Fig.6 Interface thickness obtained for 9 experimental conditions Fig.6 Interface thickness obtained for 9 experimental conditions To confirm the diffusion of materials at the interface and to compare the effects of input parameters on interface constituents, an EDS analysis was performed. Fig. 3.1.2 Quantification for the role of parameters for interface thickness In order to understand the role of different parameters, the main effect plots for interface thickness are provided in Fig. 8. The results are supplemented by analysis of variance (ANOVA) (Table 3) for their percentage contribution (PCR) to the outputs. Here, the pouring temperature comes out to be the only significant parameter at 95% confidence level with a percentage contribution of 92.84%. As can be seen from the plots, both pressure and pouring temperature positively correlate with the thickness whereas holding time in the investigated regime does not seem to be much influential. in Equation 5. 7 presents the elemental profile plots/line scan for all the parametric conditions. Here, the EDS scans of the interface validate the presence of primary constituents of the two metals involved in this composite. This line scan also highlights the percentage variation of Al, Si and Fe at the interface region. Within the interface, the concentration of primary element diminishes as we move to the opposite side of the base metal/alloy to which that constituent belongs. Another interesting observation is that more Al diffuses into the interface in comparison to steel that could be attributed to the smaller size of Al atoms as compared to the Fe atoms [26]. It is 12 pertinent to specify that the percentage of constituents is also influenced by the experimental conditions. Higher pressure and temperature values are seen to favor more aluminum diffusion into the interface than Fe. Correspondingly, at higher values of these parameters, (say experiment 9) the percentage of aluminum increases with the decrease in percentage of Fe. The possible reason for this is the larger dissemination of molten aluminum into steel at a higher temperature and pressure resulting in a rather compact and uniform interface thickness. 3.1.3 Nano-hardness The nano-hardness values obtained for all 9 experiments have been shown in Fig. 9. It can be seen that maximum hardness (10.76 GPa) is obtained at the interface in experiment 9 which is in line with the results obtained for interface thickness. If compared to Experiment 1 conditions, where we have least values of all processing parameters, an improvement of 19.42% is highlighted. Another important aspect is that, the range of hardness achieved at interface is higher than the hardness of individual Al Alloy (1.50GPa) and steel (2.50GPa). It is not uncommon for the interface to develop higher nano-hardness than the base materials. The work by Jiang et al. [32] where magnesium/steel bimetal was developed, reported higher nano-hardness values (11.1GPa) of the interface layer as compared to the hardness of magnesium (1.1GPa) and steel matrixes (4.2GPa). It is important to mention that, Al melt has a capability to spread homogeneously over the iron insert thus initiating the diffusion of elements (Fe, Al & Si) at the interface. The higher values of hardness for the developed interface may be associated to the presence of intermetallics such as, Fe2Al5,τ10-Al9Fe4Si3 FeAl3, Al12Mg17, Al12Mg17. It is obvious that the types of intermetallic layers produced, and their thicknesses play critical role in obtaining materials with desirable performances. In another work [27] an interaction of solid iron rod with molten Al was investigated. 13 Intermetallic such as FeAl3 and Fe2Al5 were identified through XRD analysis within the interface layer. Also the Fe2Al5 layer thickness was found to be larger than the FeAl3 layer. Additionally, Jian et al. [28] also observed intermetallic such as Fe2Al5, τ10-Al9Fe4Si3, FeAl3, τ5-Al8Fe2Si, τ6-Al4.5FeSi which could be the reason for the higher nano-hardness (10.49GPa) for the interface reported in their wok. Literature survey further reveals that the intermetallics such as Fe2Al5 & FeAl3 formed in the interface layer are brittle [33]. The in general inverse relationship of brittle materials and hardness is already established which explains the higher nano-hardness values within the interface. Vendra and Rabiei [34] investigated the diffusion of iron and Aluminum, and observed the presence of brittle Fe2Al7Si phase. They also conducted nano-hardness test while citing it adequate approach in view of the small size of needle shaped light gray/dark phase which could render regular microhardness (MH) testing method as inadequate for the estimation of their hardness. 3.1.3 Nano-hardness A comprehensive load-displacement curve at the interface developed at all experimental conditions is shown in Fig. 10. It is evident from the graph that with the increase in hardness of the interface, the penetration of the indenter is reduced, indicating the increasing strength of the interface. It can be seen that at the same load conditions, the interface developed at more favorable conditions is able to resist deformation more indicating a harder interface. The least indenter penetration (0.95µm) has been achieved for interface produce at highest level of parameters which is 26.92% less when compared to the maximum penetration (1.3µm) achieved with least parametric levels. 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate 14 Fig.7 SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite (for all experiments) Fig.7 SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite (for all experiments) Fig.8 Main effect plots for interface thickness Fig.8 Main effect plots for interface thickness 15 Table 3 ANOVA for interface thickness Source DF Seq SS Adj SS Adj MS F- Value P- Value Contribution T(°C) 2 1241.31 1241.31 620.654 135.48 0.007 92.84% P(bar) 2 68.55 68.55 34.274 7.48 0.118 5.13% Ht(Sec) 2 18.00 18.00 9.001 1.96 0.337 1.35% Error 2 9.16 9.16 4.581 0.69% Total 8 1337.02 100.00% R-sq: 99.31% R-sq(adj): 97.26% Fig.9 Hardness value obtained for all 9 experiments Table 3 ANOVA for interface thickness Source DF Seq SS Adj SS Adj MS F- Value P- Value Contribution T(°C) 2 1241.31 1241.31 620.654 135.48 0.007 92.84% P(bar) 2 68.55 68.55 34.274 7.48 0.118 5.13% Ht(Sec) 2 18.00 18.00 9.001 1.96 0.337 1.35% Error 2 9.16 9.16 4.581 0.69% Total 8 1337.02 100.00% R-sq: 99.31% R-sq(adj): 97.26% Fig.9 Hardness value obtained for all 9 experiments Fig.9 Hardness value obtained for all 9 experiments Fig.9 Hardness value obtained for all 9 experiments Fig.10 Load vs displacement graph -50 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Force (mN) Displacement (µm) Load Vs Displacement Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 Experiment 8 Experiment 9 Load Vs Displacement Fig.10 Load vs displacement graph 16 Fig.11 Main effects plot for nano-hardness Table 4 ANOVA for nano-hardness Source DF Seq SS Adj SS Adj MS F- Value P- Value Contribution T(°C) 2 2.25398 2.25398 1.12699 116.18 0.009 88.28% P(bar) 2 0.25579 0.25579 0.12789 13.18 0.071 10.02% Ht(Sec) 2 0.02400 0.02400 0.01200 1.24 0.447 0.94% Error 2 0.01940 0.01940 0.00970 0.76% Total 8 2.55317 100.00% R-sq: 99.24 % R-sq(adj): 96.96 % Fig.11 Main effects plot for nano-hardness Fig.11 Main effects plot for nano-hardness Fig.11 Main effects plot for nano-hardness Table 4 ANOVA for nano-hardness Table 4 ANOVA for nano-hardness Source DF Seq SS Adj SS Adj MS F- Value P- Value Contribution T(°C) 2 2.25398 2.25398 1.12699 116.18 0.009 88.28% P(bar) 2 0.25579 0.25579 0.12789 13.18 0.071 10.02% Ht(Sec) 2 0.02400 0.02400 0.01200 1.24 0.447 0.94% Error 2 0.01940 0.01940 0.00970 0.76% Total 8 2.55317 100.00% R-sq: 99.24 % R-sq(adj): 96.96 % The results for nano-hardness are best explained when supplemented by cooling rates calculated for the experiments. 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate Fig. 11 shows the main effect plots for nano level hardness wherein an increasing trend for hardness values with an increased temperature. At higher temperatures, the diffusion of Al into steel is increased and improves the formation of intermetallic, resulting in increased hardness values. The increase in pressure and holding time has also shown a higher hardness value. The ANOVA (Table 4) shows that with a p-value of 0.009, the temperature is the most significant parameter and has the highest PCR value (88.28%). 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate It was observed that experiment 9, wherein pressure and temperature were at their maximum values (of the investigated set), resulted into the highest cooling rate. Refer to Fig. 12 which shows values of different cooling rates calculated for all 9 experiments. The cooling rate obtained for experiment 9 is around 20% higher than experiment 1 where pressure and temperature were at their least. In experiment 9, the higher available temperature gradient has been made possible by the higher pouring temperature along with the applied pressure, which in its own independent capacity has been reported to increase the interfacial heat transfer coefficient during solidification [35–37]. All this results into accelerated heat transfer from the casting. Correspondingly, the microstructure which is reported to be a function of cooling rate [38–40] would also be refined which in turn results in the betterment of mechanical properties. The interdependency of mechanical propertiesand microstructure onthe cooling rate has been found in literature [41]. The same could be inferred here wherein higher values of nano-indentation have been achieved at these conditions. Importantly, as can be seen from Fig. 12, the three highest cooling rates (Experiment 9, 6 and 3) are all obtained when the applied pressure was at the highest values 17 (20bar), not only supporting the given explanation but also hinting towards the role of pressure in developing accelerated cooling rates.If we compare this work with reported literature (where higher values of pressure have been used), with the application of pressure, increase in cooling rate has been reported [40]. This is further confirmed when ANOVA is performed for the cooling rates (Table 5). Here, the pressure is the most contributing parameter with PCR value (81.98%) and is statistically significant at 95% confidence level. It is also important to note that the cooling rates pattern doesn't necessarily match entirely with the pattern obtained for nano-hardness results. For example, the two cooling rates (for experiments 3 and 6) that immediately lag the best cooling rate of experiment 9 do not result in the second and third highest nano-hardness values. This can also be explained; at higher cooling rates a possibly adverse effect could be that a very quick temperature drop may not allow enough time for diffusion to properly take place. 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate Interestingly both of these experiment conditions (3 and 6) though having a higher- pressure value (which is statistically significantparameter for cooling rate) but the corresponding pouring temperatures are lower (800 and850°C). The temperature has been determined to be a statistically significant parameter for interface thickness. Accordingly, at these lower pouring temperatures despite getting relatively higher cooling rates, relatively lesser interface thickness and nano-indentation hardness have been achieved. Experiment 9 has both the parameters at their favorable values so not only that highest cooling rate has been the outcome but also interface thickness and nano-indentation are also at their best. Fig.12 Cooling rate at different experimental conditions Table 5 ANOVA for cooling rate Source DF Seq SS Adj SS Adj MS F-Value P-Value Contribution T(°C) 2 0.015268 0.015268 0.007634 5.38 0.157 14.29% P(bar) 2 0.087589 0.087589 0.043794 30.89 0.031 81.98% 3.4 Multi-objective optimization using GRA The optimization has been carried out for development of sound interface. Table 6 contains the normalizing & deviation sequence, GRC and GRG. The highest grade shows the optimal parametric setting for the process. The highest GRG value is obtained for experiment number 9, where temperature is 900ºC, pressure is 20bar, while holding time is 15sec. Table 6 Normalizing sequence & deviation sequence Table 6 Normalizing sequence & deviation sequence Normalizing Sequence Deviation Sequence Grey Relational Coefficient GRG Exp. No IT H Rc IT H Rc IT H Rc - 1 0.0000 0.0000 0.0000 1.0000 1.0000 1.0000 0.3333 0.3333 0.3333 0.3333 2 0.0373 0.1342 0.4693 0.9627 0.8658 0.5307 0.3418 0.3661 0.4851 0.3977 3 0.1242 0.3251 0.9102 0.8758 0.6749 0.0898 0.3634 0.4256 0.8477 0.5456 4 0.2826 0.5432 0.2202 0.7174 0.4568 0.7798 0.4107 0.5226 0.3907 0.4413 5 0.5807 0.5823 0.5873 0.4193 0.4177 0.4127 0.5439 0.5449 0.5478 0.5455 6 0.6491 0.6438 0.9220 0.3509 0.3562 0.0780 0.5876 0.5840 0.8651 0.6789 7 0.8758 0.7205 0.4372 0.1242 0.2795 0.5628 0.8010 0.6414 0.4704 0.6376 8 0.9658 0.8253 0.8459 0.0342 0.1747 0.1541 0.9360 0.7411 0.7645 0.8139 9 1.0000 1.0000 1.0000 0.0000 0.0000 0.0000 1.0000 1.0000 1.0000 1.0000 18 Ht(Sec) 2 0.001154 0.001154 0.000577 0.41 0.711 1.08% Error 2 0.002836 0.002836 0.001418 2.65% Total 8 0.106845 100.00% R-sq:97.35% R-sq(adj): 89.38% 3.4.1 ANOVA for GRA After ranking, an ANOVA is also performed for GRG values to find the most significant parameter(s). It can be seen from Table 7 that temperature and pressure, with a p-value less than 0.05, are significant for GRG. The percentage contribution for temperature is 88.57%, while for pressure it is 31.07%. The main effect plot for GRG is provided in Fig. 14. The response table (Table 8) shows the levels resulting in the highest GRG values. Temperature and pressure at level 3 while holding time at level 2 shows the highest GRG. GRG for all the experimental conditions along with the rank are shown in Fig. 15. 19 Fig.13 Main effects plots for GRG Table 7 ANOVA for GRG Source DF Seq SS Adj SS Adj MS F-Value P-Value Contribution T(°C) 2 0.238796 0.238796 0.119398 52.78 0.019 66.98% P(bar) 2 0.110781 0.110781 0.055390 24.49 0.039 31.07% Ht(Sec) 2 0.002422 0.002422 0.001211 0.54 0.651 0.68% Error 2 0.004524 0.004524 0.002262 1.27% Total 8 0.356523 100.00% R-sq: 98.73% R-sq(adj): 94.92% Table 8 Response table for GRG Level 1 2 3 Delta Rank T(°C) 0.4255 0.5552 0.8172 0.3916 1 P(bar) 0.4708 0.5857 0.6130 0.2707 2 Ht(Sec) 0.6087 0.6130 0.5762 0.0368 3 Fig.14 Gray relational grade for all experiments Fig.13 Main effects plots for GRG Fig.13 Main effects plots for GRG Fig.13 Main effects plots for GRG Fig.14 Gray relational grade for all experiments 20 3.4.2 Confirmation tests A confirmatory experiment was performed for interface thickness, nano-hardness & cooling rate based on optimization done herein. The response table for means (Table 8), highlights levels at which all three parameters will give optimal results. From this table, the levels with highest mean value are selected thus the optimal parametric levels are a temperature of 900ºC, the pressure of 20 bar, and a holding time of 15sec. Based on this suggested parametric setting, confirmatory experimentation was carried out. The results from the initial experimental matrix and the confirmatory experiment are highlighted in Table 9. The values obtained from the confirmatory experiments validated the optimized levels. The confirmatory run not only validated the reliability of the optimization step (results within 5% of the predicted value) but also repeatability was ensured. Table 9 Confirmation experiment Performance characteristics Optimal values from (design matrix) Predicted values Confirmatory experiment values %age error of confirmatory values IT (µm) 42.0 41.20 43.2 4.85 H(GPa) 10.76 10.76 10.84 0.74 Cooling Rate 2.12 2.14 2.14 - Table 9 Confirmation experiment Table 9 Confirmation experiment The samples from the optimum combination of input values were further tested for nano- cyclic loading conditions with a maximum applied load of 200mN. The purpose of applying cyclic load at the interface was to evaluate the interface developed for deformation under dynamic loading conditions. When cyclic load is applied on the metallic materials in plastic range then the tendency to resist deformation is different as compared to continuous load [42]. It can be seen from Fig. 15 that the sample at the optimum condition restricted penetration much more than the other with the least experimental conditions. Due to the increase in pouring temperature and applied pressure, a uniform interface and associated properties resulted in higher hardness at the interface and correspondingly minimum penetration of the indenter. 21 Fig.15 Load vs displacement graph for cyclic loading 0 50 100 150 200 250 0.00 1.00 2.00 3.00 4.00 Load (mN) Displacement(µm) Load vs Displacement (CL) A(Pouring Temp 900 °C, Squeeze Pressure 20bar, Holding Time 15Sec) B(Pouring Temp 800 °C, Squeeze Pressure 10bar, Holding Time 10Sec) Load vs Displacement (CL) Load vs Displacement (CL) Load vs Displacement (CL) Fig.15 Load vs displacement graph for cyclic loading Not Applicable Not Applicable Acknowledgment This work has been facilitated by University of Engineering & Technology (UET), Lahore, Pakistan as part of the post graduate PhD research work at the Department of Industrial & Manufacturing Engineering (UET) Lahore, Pakistan. 4 Conclusions This study while focusing within the context of multi-material castings, developed steel reinforced Al, bi-metallic composite casting using pressure assisted approach and optimized the process parameters for interface thickness, nano-mechanical properties (nano-hardness) and cooling rate. Taguchi L9 orthogonal array was used for experimentation (with pressure, pouring temperature and holding time as input parameters) supplemented by statistical analysis of ANOVA and GRA. It has been found that  It is possible to generate interfaces of uniform thicknesses within the investigated pressure regime of 10-20 bars. Pouring temperature has come out to be the most significant factor in this regime in general. Micrographs and EDS analysis confirms generation of uniform interface along with diffusion of micro-constituents.  Highest interface thickness was achieved at the experiment conditions of temperature 900ºC, pressure 20bar and holding time 15sec where the interface was ~328% higher than the conditions of 800ºC, 10bar, & 10sec. ANOVA reveals that temperature is the most significant factor with PCR of 92.84% for interface thickness. Pressure has a meager contribution of ~5% for interface thickness.  The nano-indentation tests revealed 19.42% increase in nano-hardness while load displacement graph showed 26.92% reduced indenter penetration at experimental conditions of 900 ºC, 20bar and 15sec when compared to the experimental conditions 22 with least levels (800ºC, 10bar, & 10sec). For this output, temperature was found to be the most significant parameter with PCR of 88.28%.  The results are well supplemented by cooling rate calculations for which pressure is the most significant factor.  The results are well supplemented by cooling rate calculations for which pressure is the most significant factor.  The multi-objective optimization using GRA shows highest grey relational grade (GRG) for experimental condition 9 (temperature 900ºC, pressure 20bar and holding time 20sec). Further, the ANOVA for GRG shows the temperature to be the most significant parameter with a p-value of 0.023. The optimized parameters have been seen to exhibit higher nano-hardness also in dynamic loading regime.  An indirect comparison of the presented work with other published literature indicates generation of sound interfaces at considerably less harsh pressure conditions. Uniform interfaces are developed with comparable nano-hardness and thickness (which in certain cases are found to be even thicker). Authors Contributions Tayyiba Rashid conducted the experiments, performed measurements and analysis. Muhammad Qaiser Saleem helped conceptualize the research plan, supervised the work and assisted in article write-up. Nadeem Ahmad Mufti modified the article structure and contributed to analysis and discussion. Noman Asif and Kashif Ishfaq helped in statistical analysis and data interpretation phases. Maham Naqvi helped in analysis and discussion phases. Competing Interests Competing Interests The authors declare that they have no conflict of interest p g The authors declare that they have no conflict of interest p g The authors declare that they have no conflict of interest Consent to Publish Consent to Publish All authors give consent to publish this manuscript in The International Journal of Advanced Manufacturing Technology Consent to Publish All authors give consent to publish this manuscript in The International Journal of Advanced Manufacturing Technology Availability of data and materials Not Applicable Ethical Approval The research does not involve human participants or animals and the authors warrant that the Ethical Approval The research does not involve human participants or animals and the authors warrant that the 23 paper fulfills the ethical standards of the journal. This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal paper fulfills the ethical standards of the journal. This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal Consent to Participate p The research does not involve human participants or animals and the authors warrant that the paper fulfills the ethical standards of the journal References 1. Rajmohan T, Palanikumar K, Ranganathan S (2013) Evaluation of mechanical and wear properties of hybrid aluminium matrix composites. Trans Nonferrous Met Soc China (English Ed 23:2509–2517. https://doi.org/10.1016/S1003-6326(13)62762-4 2. Alidokht SA, Abdollah-zadeh A, Assadi H (2013) Effect of applied load on the dry sliding wear behaviour and the subsurface deformation on hybrid metal matrix composite. Wear 305:291–298. https://doi.org/10.1016/j.wear.2012.11.043 3. 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References Jiang W, Fan Z, Li G, et al (2016) Effects of hot-dip galvanizing and aluminizing on interfacial microstructures and mechanical properties of aluminum/iron bimetallic composites. J Alloys Compd 688:742–751. https://doi.org/10.1016/j.jallcom.2016.07.085 29. Chen G, Chang X, Liu G, et al (2020) Formation of metallurgical bonding interface in aluminum-steel bimetal parts by thixotropic-core compound forging. J Mater Process Technol 283:116710. https://doi.org/10.1016/j.jmatprotec.2020.116710 29. Chen G, Chang X, Liu G, et al (2020) Formation of metallurgical bonding interface in aluminum-steel bimetal parts by thixotropic-core compound forging. J Mater Process Technol 283:116710. https://doi.org/10.1016/j.jmatprotec.2020.116710 30. Jiang W, Fan Z, Li C (2015) Improved steel/aluminum bonding in bimetallic castings by a compound casting process. J Mater Process Tech 226:25–31. https://doi.org/10.1016/j.jmatprotec.2015.06.032 30. Jiang W, Fan Z, Li C (2015) Improved steel/aluminum bonding in bimetallic castings by a compound casting process. J Mater Process Tech 226:25–31. https://doi.org/10.1016/j.jmatprotec.2015.06.032 26 31. Liu T, Wang Q, Sui Y, et al (2016) An investigation into interface formation and mechanical properties of aluminum-copper bimetal by squeeze casting. Mater Des 89:1137–1146. https://doi.org/10.1016/j.matdes.2015.10.072 32. Jiang W, Jiang H, Li G, et al (2020) Microstructure, Mechanical Properties and Fracture Behavior of Magnesium/Steel Bimetal Using Compound Casting Assisted with Hot-Dip Aluminizing. Met Mater Int. https://doi.org/10.1007/s12540-019-00606- 7 33. Bakke AO, Arnberg L, Løland JO, et al (2020) Formation and evolution of the interfacial structure in al/steel compound castings during solidification and heat treatment. J Alloys Compd 849:156685. https://doi.org/10.1016/j.jallcom.2020.156685 34. Vendra LJ, Rabiei A (2007) A study on aluminum-steel composite metal foam processed by casting. Mater Sci Eng A 465:59–67. https://doi.org/10.1016/j.msea.2007.04.037 35. Wang F, Zhao X, Liu J, et al (2020) Study on the relationship between interfacial heat transfer coefficient and interface pressure in squeeze casting by using microscopic contact model. Int J Therm Sci 152:106300. https://doi.org/10.1016/j.ijthermalsci.2020.106300 36. Wang F, Ma Q, Meng W, Han Z (2017) Experimental study on the heat transfer behavior and contact pressure at the casting-mold interface in squeeze casting of aluminum alloy. Int J Heat Mass Transf 112:1032–1043. https://doi.org/10.1016/j.ijheatmasstransfer.2017.05.051 37. Aweda JO, Adeyemi MB (2009) Experimental determination of heat transfer coefficients during squeeze casting of aluminium. J Mater Process Technol 209:1477– 1483. https://doi.org/10.1016/j.jmatprotec.2008.03.071 38. Hajjari E, Divandari M (2008) An investigation on the microstructure and tensile properties of direct squeeze cast and gravity die cast 2024 wrought Al alloy. Mater Des 29:1685–1689. https://doi.org/10.1016/j.matdes.2008.04.012 39. References Benjunior B, Ahmad AH, Mohd M, Reza MS (2017) Effect of Different Cooling Rates Condition on Thermal Profile and Microstructure of Aluminium 6061. Procedia Eng 184:298–305. https://doi.org/10.1016/j.proeng.2017.04.098 40. Amin KM, Mufti NA (2012) Investigating cooling curve profile and microstructure of a squeeze cast Al-4%Cu alloy. J Mater Process Technol 212:1631–1639. https://doi.org/10.1016/j.jmatprotec.2012.02.017 41. Saleem M, Makhlouf M (2012) Helium-Assisted Sand Casting. Int J Met 6:43–59. 27 27 https://doi.org/10.1007/BF03355538 42. Yang F, Peng ÆL, Okazaki ÆK (2007) Cyclic indentation in aluminum. 4513–4520. https://doi.org/10.1007/s10853-006-0480-2 https://doi.org/10.1007/BF03355538 42. Yang F, Peng ÆL, Okazaki ÆK (2007) Cyclic indentation in aluminum. 4513–4520. https://doi.org/10.1007/s10853-006-0480-2 28 Figures Figure 1 Figure 1 Schematic of the die Figure 2 Experimental setup Figure 3 a & b) Schematics of the part showing the length & location of the pins c) actual cast component Figure 2 Experimental setup Figure 3 a & b) Schematics of the part showing the length & location of the pins c) actual cast component Figure 2 Experimental setup Experimental setup p p a & b) Schematics of the part showing the length & location of the pins c) actual cast component a & b) Schematics of the part showing the length & location of the pins c) actual cast component Figure 4 Figure 4 Research methodology Research methodology Figure 5 Microstructures of the interface region of bimetals made at different temperature and pressure Microstructures of the interface region of bimetals made at different temperature and pressure Figure 6 Interface thickness obtained for 9 experimental conditions Figure 6 Interface thickness obtained for 9 experimental conditions Interface thickness obtained for 9 experimental conditions Figure 7 SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite ( experiments) Figure 7 SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite (for all experiments) SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite (for all experiments) Figure 8 Main effect plots for interface thickness Figure 8 Main effect plots for interface thickness Figure 8 Main effect plots for interface thickness Figure 8 Main effect plots for interface thickness Figure 8 Main effect plots for interface thickness Figure 8 Figure 8 Main effect plots for interface thickness Main effect plots for interface thickness Figure 9 Hardness value obtained for all 9 experiments Hardness value obtained for all 9 experiments Hardness value obtained for all 9 experiments Figure 10 Figure 10 Load vs displacement graph Figure 11 Main effects plot for nano-hardness Load vs displacement graph Figure 11 Main effects plot for nano-hardness Figure 11 Main effects plot for nano-hardness Main effects plot for nano-hardness Figure 12 Cooling rate at different experimental conditions Figure 12 Figure 12 Cooling rate at different experimental conditions Figure 13 Main effects plots for GRG Figure 13 Main effects plots for GRG Figure 13 Main effects plots for GRG Main effects plots for GRG Main effects plots for GRG Figure 14 Gray relational grade for all experiments Figure 15 Figure 15 Figure 15 Load vs displacement graph for cyclic loading Load vs displacement graph for cyclic loading
https://openalex.org/W2324903489
https://actavet.vfu.cz/media/pdf/avb_1998067040207.pdf
English
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Cellular Immune Response In Rainbow Trout (Oncorhynchus mykiss): Flow Cytometric Study
Acta veterinaria Brno
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cc-by
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Address for correspondence: Dr. Stefan Chilmoncz .... k INRA. Unitt! de VirologiC et Immunlogie Molcculaires 78J5:! lou)" en Josas France. ACTA VET. BRNO 1998,67: 207-213 ACTA VET. BRNO 1998,67: 207-213 CHILMONCZYK S .. MONGE D. INRA Unite de Virologie et lmmunologie Moleculaires. Jouy en Josas. France Received September 15.1998 Accepted September 21.1998 Received September 15.1998 Accepted September 21.1998 Abstract Chilmonczyk. S .. D. Monge: Cellular Immune Response in Rainbow Trout (Oncorhynchus mykiss): Flow Cytometric Study. Acta vet. Brno 1998.67: 207-213. Chilmonczyk. S .. D. Monge: Cellular Immune Response in Rainbow Trout (Oncorhynchus mykiss): Flow Cytometric Study. Acta vet. Brno 1998.67: 207-213. Assays devoted to the study of non-specific cellular defense mechanisms of rainbow trout: phagocytosis. oxidative burst and nonspecitic cytotoxicity. were developed. Compared to conventional methodologies flow cytometry provides a rapid. suitable and reproducible method for analysing fish cellular immune functions. In addition to the assessment of the defense mechanisms interesting data are directly provided from the observation of unstained cells. Flow cytometric profiles of cells are representative of the organ from which the leukocytes originated and each lymphoid organ displays a characteristic cytogram pattern. Flow cytometry emerges as a useful tool to study immune reactions and pathogenic processes. Flow cytometr),. rainbow trout. leukocytes. phagocytosis. oxidative burst. nonspecific cytotoxic cell Flow cytometry (FCM) allows a cell by cell qualitative and quantitative analysis of cell functions and cell activities. FCM offers great potential for investigations in the areas of fish haematology, pathology and immunology because multiparameter measurements of single cells are easily and rapidly performed. Immunologists routinely used FCM to characterize phenotypically the leukocyte subpopulations. So far, in fish, only a few number of antibodies directed against surface markers are avalaible. The panel of monoclonal antibodies which could identify different subpopulations offish leukocytes is limited (Deluca et al. 1983; Koumans et al. 1994; S liere ndrec h t et al. 1995) and unfortunately the existing markers display no cross reactivity between fish species. Even though now cytometric immunophenotyping offish cells presents a limited interest, FCM offers real advantages over traditional methods. FCM measurements are made on the individual cell and not on the whole population and morphologic, biochemical and functional characteristics can be analyzed simultaneously. Thus it is possible to correlate the cell function or activity with one subpopulation. y p p While in mammals FCM has been used for long, in fish, only a few such studies have been performed. Recently a battery of assays have been developed to monitor different aspects of the rainbow trout (RBT) cellular immune response (Voccia eta1.1994; Chilmonczyk et al. 1995; 1997). The present paper describes some FCM methods we have developed for the investigation of functional aspects of the RBT cell defense mechanisms. Phone: +33 (Oil 34652476 Fax: +33 lOiI 34652591 E-mail: stephan@biorec.jouy.inra.fr CELLULAR IMMUNE RESPONSE IN RAINBOW TROUT (Oncorhynchus mykiss): FLOW CYTOMETRIC STUDY CHILMONCZYK S .. MONGE D. CHILMONCZYK S .. MONGE D. INRA Unite de Virologie et lmmunologie Moleculaires. Jouy en Josas. France Oxidative burst Oxidative burst activity was quantified using flow cytometry as a measure of intracellular hydrogen peroxide production as previously described (Chilmonczyk et aJ. I 997).The assay depends upon the cell incorporation of2' -7' -dichlorofluore'icin diacetate (DCFH-DA). which is a stable nonfluorescent molecule hvdrolvzed to DCFH by cytosolic enzymes, Then by the action of HoO" produced in leukocytes stimulated with' Phorbol Myristate Acetate (PMA). DCFH is oxidised to the fluoresceni dichlorofluorescein (DCF). Briefly. isolated PKL (1.5 x lO6 Iml) were resuspended in I ml MEM and incubated for 15 min at room temperature with DCFH-DA. Cellular baseline of fluorescence (DCF) was determined at this zero time and PMA (10 ng/ml. final concentration) was immediately added and the level of intracellular fluorescence was measured in unstimulated and PMA stimulated cells by flow cytometry. A typical kinetic experiment included the analysis of lO 000 individual cells analysed from the same tube sample 5. 15,30.45 and 60 min over the reaction period, At each sampling time. a stimulation index was determined as the ratio of fluorescence of PMA stimulated cells to that of unstimulated cells. Nonspecific Cytotoxic Cells (NCC) We have adapted to RBT the method of Chang et al. (1993) using the mouse lymphoma YAC-I cell line (Ki e s s lin g et aJ. 1975) as target cells, The assay is based on the simultaneous expression of:2 fluorescent signals allowing the discrimination between a) unstained effector cells. b) green fluorescent viable target cells c) red plus green fluorescent dead target cells. Target cells are labeled with a green fluorescent dye: 3. 3'- dioctadecyloxacarbocyanin perchlorate (DIO-C 18). a lipophilic carbocyanine membrane dye, Target cells are incubated with RBT leukocytes at a effector - target ratio of 50: 1 for 2h at 20°C in presence of propidium iodide which permeates only through the membrane of dead cells and stains the nucleic acids emiting a red fluorescence. FCM acquisitions are performed on the base of green and red fluorescence parameters displayed by target cells: dead and damaged target cells appear doubly stained. Only the target cells. which are easily distinguished from an abundance of effectors by their distinct green stable fluorescence. are considered in the aquisition of data. Phagocytosis h i agocytos s Phagocytosis a"ays were performed il1l'itro as previously described (Chilmonczyk et aJ. 1995), Briefly. leukocyte suspensions (1.5 x 106 cells/mll\IEM) were incubated for 18 h at 16°C with fluoresceinated (FITC) latex beads (Polysciences). Leukocytes were incubated with FITC-beads in a proportion of I cell for 100 FlTC- beads. Non-ingested beads were separated from the phagocytes by centrifuging (lOa g x lO min) mer a cushion of 3% BSA in PBS. The cell pellet was then resuspended in MEM for flow cytometric measurement. The fluorescence histograms of cell number l'eJ'SUS fluorescence intensity were analysed and phagocytic activity was expressed as the percentage of cells that had ingested at least 3 FlTC-Iatex beads. Oxidative burst Cell subpopulations Cell subpopulations p p The auwtluorescence emitted by unstained li\'ing cells allows the distinction between leukocyte subpopulations according to the siLe (forward light scatter: FSC parameter) and the cytosolic complexity (side light scatter: SSC parameter) of the cells. The autofluorescence scattering signals are sufficient on their own to discriminate cells with different morphology: lymphocytes (101\ FSC I SSC) are accurately differentiated from granulocytes (higher FSC I SSC). Quantification of dead cells \\as determined by adding pmpidium iodide (PI) (Sigma) at a final concentration of 5fjg/ml (Dei tc h et aJ. 198~). Phagocytosis Ph i Fish Leukocytes were collected from healthy (control) or experimentally infected rainbow trout (Oncorhynchus .lIlykiss - RBT). Fish (5- IO g) were subjected to waterborne Viral Haemorrhagic Septicaemia Virus (VHSV) while PKX parasite infections (intraperitoneal injection) were performed on 30 g RBT. Control and infected fish were reared in recirculated filtered water therrnoregulated at IO I °C (VHSV infections) or 161°C (PKX infections). Phone: +33 (Oil 34652476 Fax: +33 lOiI 34652591 E-mail: stephan@biorec.jouy.inra.fr 208 Leukocyte collection y Freshl)' collected blood anticoagu]Qted \\ ith heparin was diluted (1:20) with !\1inimum Essential Medium (MEM). Cells from pronephros. spleen and thymus \\ ere obtained by forcing the tissues through a stainless steel mesh in cold serum-free !\1EM. Then leukocvtes were isolated throu~h a Ficoll cushion. The leukoevte-rich interphase \\ as collected. washed in PBS and resuspended in ME!\1. ~ , Flow cytometric analysis y y Flow cytometric anal)ses were can-ied out using a standard fluorescence activated cell analyser (FACScan. Becton Dickinson I. For each sample I () 000 or 20 nOll individual celb \\ ere recorded using a dot plot combination of low an~le forward scattered (FSC) and ri~ht an21e scattered (SSC) laser li2ht. Data \\'ere analvsed with the program LYSIS]] \ersion 2.0. ~ ~ ~ , Table I Table I Variability occurring in the aspect of the FCM cytograms of pronephros leukocytes. Compared to the regular pattern displayed by control RBT. fish subjected to pathogens (VHSV or PKX) or to a crowding stress presented highly modified cytogram patterns. PN Leukocytes Cytogram pattern from regular modified Control 88 % (128/145) 12 % (171145) VHSV infected 60 % (25/42) 40 % (17142) PKX infected 6 % (6/92) 94 % (86/92) Stressed 41 % (10124) 59 % (14/24) riability occurring in the aspect of the FCM cytograms of pronephros leukocytes. Compared the regular pattern displayed by control RBT. fish subjected to pathogens (VHSV or PKX) or to a crowding stress presented highly modified cytogram patterns. or to a crowding stress presented highly modified cytogram patterns. PN Leukocytes Cytogram pattern from regular modified Control 88 % (128/145) 12 % (171145) VHSV infected 60 % (25/42) 40 % (17142) PKX infected 6 % (6/92) 94 % (86/92) Stressed 41 % (10124) 59 % (14/24) Phagocytosis FCM assessment of phagocytic activity is based on the determination of cells engulfing fluorescent (FITC)-latex beads. This method allows a clear delineation between phagocytic and non-phagocytic cell populations on the basis of fluorescence parameters. p g y p p p Phagocytosis assays were carried out on leukocytes collected from the main RBT lymphoid organs including the thymus. In RBT the pronephros is the main phagocytic organ. A slight but not negligible number of active phagocytes occur in the thymus (Table 2). Our data show that the phagocytosis ability may differ greatly between fish (Table 3). Table 2 Table 2 FCM assessment of phagocytosis in leukocyte populations of 8 RBT fingerlings. % ± SD of cells having ingested 3 or more FITC-latex beads. Pronephros Blood Spleen Thymus 12.2 ±5.1 6.1 ±3.1 6.7± 3.9 1.1 ± 0.5 Table 3 FCM assessment of phagocytosis (% of phagocytes having ingested 3 and more FITC-latex beads) and oxidative burst (increase of mean of fluorescence intensity after PMA activation). Both assays were performed on pronephros cells from the same trout and % of granulocytes was recorded from unstained pronephros leukocyte population. RBT Pronephros Phagocytic Ox. Burst granulo. (%) activity ('7c) (15 mm PMA) I 60.4 20.3 77.8 2 32.9 12.6 52.9 3 51.6 19.1 65.4 4 58.6 15.6 85.1 5 20.4 9.1 13.9 6 37.9 16.1 49.7 Table 2 FCM assessment of phagocytosis in leukocyte populations of 8 RBT fingerlings. Results FCM characterization of leukocyte subpopulations Using the autofluorescence of unstained cells, FCM allows a clear delineation of cell subpopulations. In RBT the flow cytometric profiles of cell populations, designed by a two- parameter cytogram using FSC versus SSC (dot plot representation), are representative of the organ from which the leukocytes originated. Each lymphoid organ displays a characteristic cytogram pattern. Spleen and thymus cell populations are for the most part composed of lymphocytes while pronephros leukocytes display 3 populations (lymphocytes, monocytes and granulocytes). In blood the situation is less obvious, blood 209 leukocytes are generally characterized by 2 major populations (lymphocytes and granulocytes), however, blood cytograms may differ greatly between fish. Depending upon experimental and/or pathological conditions, strong modifications may occur and the aspect of the dotplot profiles diverges from that of the characteristic cytogram representative of each lymphoid organ (Table I). During the acquisition of the data cell viability is easily recorded using propidium iodide which stains specifically the DNA of dead cells. g p p p y Electronic gating allows the selection of populations of interest, or the exclusion from aquisition of unwanted population, cell debris or dead cells. Table I % ± SD of cells having ingested 3 or more FITC-latex beads. Pronephros Blood Spleen Thymus 12.2 ±5.1 6.1 ±3.1 6.7± 3.9 1.1 ± 0.5 Table 2 FCM assessment of phagocytosis in leukocyte populations of 8 RBT fingerlings. % ± SD of cells having ingested 3 or more FITC-latex beads. Table 3 Table 3 FCM assessment of phagocytosis (% of phagocytes having ingested 3 and more FITC-latex and oxidative burst (increase of mean of fluorescence intensity after PMA activation) Both assays were performed on pronephros cells from the same trout and % of granuloc was recorded from unstained pronephros leukocyte population. RBT Pronephros Phagocytic Ox. Burst granulo. (%) activity ('7c) (15 mm PMA) I 60.4 20.3 77.8 2 32.9 12.6 52.9 3 51.6 19.1 65.4 4 58.6 15.6 85.1 5 20.4 9.1 13.9 6 37.9 16.1 49.7 M assessment of phagocytosis (% of phagocytes having ingested 3 and more FITC-latex beads and oxidative burst (increase of mean of fluorescence intensity after PMA activation). oth assays were performed on pronephros cells from the same trout and % of granulocytes was recorded from unstained pronephros leukocyte population. 210 Oxidative burst Nonspecific Cytotoxic Cells % ± SD of target cell lysis occurring in pronephros (PN) and spleen was compared between control and PKX infected trout sampled at different time during the course of the disease. A = beginning of the infection; B = acute phase of the disease; C = recovering fish Control PKX infected RBT (n=12) A (n=8) B(n=10) C (n=8) PN 23.4 21.8 21.2 27.6 ± 11.5 ± 8.8 ±15.1 ± 13.1 Spleen 15.4 9.3 21.4 13.4 ±8.6 ±4.9 ±7.3 ±3.6 Nonspecific Cytotoxic Cells Nonspecific Cytotoxic Cells p y NCC activity was demonstrated in the main lymphoid tissues of RBT (Table 4). Cytotoxic effector cells have been mostly demonstrated in pronephros than in blood and spleen which both displayed similar level of killing activity. A significant activity was also measured in the thymus. The modulation of NCC activity under pathologic conditions was assayed in trout experimentally infected with PKX the parasite causative of the Proliferative Kidney Disease. FCM evaluation ofNCC activity was compared between healthy control trout and PKX parasited fish. Infected trout were sampled at 3 different periods during the course of the disease: a) at the beginning, b) in the acute phase, c) in recovering fish (Table 5). Compared to controls, infected trout displayed neither impairment nor enhancement ofNCC activity. Tables 4 and 5 show that percentage of cytotoxicity may vary considerably between individual fish. This large inter-fish variability causes difficulties in the interpretation of the data. Table 4 FCM assessment ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood and lymphoid organs of 8 RBT. Blood Pronephros Spleen Thymus 12.4 ± 6.2 25.3 ± 7.9 15.8±5.6 11.2 ±6.2 Table 5 Modulation of NCC activity in PKX infected fish. % ± SD of target cell lysis occurring in pronephros (PN) and spleen was compared between control and PKX infected trout sampled at different time during the course of the disease. A = beginning of the infection; B = acute phase of the disease; C = recovering fish Control PKX infected RBT (n=12) A (n=8) B(n=10) C (n=8) PN 23.4 21.8 21.2 27.6 ± 11.5 ± 8.8 ±15.1 ± 13.1 Spleen 15.4 9.3 21.4 13.4 ±8.6 ±4.9 ±7.3 ±3.6 Discussion Immunologists routinely used FCM to characterize phenotypically the leukocyte subpopulations. Unfortunately, in trout hitherto only a few number of antibodies directed Table 4 FCM assessment ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood and lymphoid organs of 8 RBT. Blood Pronephros Spleen Thymus 12.4 ± 6.2 25.3 ± 7.9 15.8±5.6 11.2 ±6.2 Table 4 FCM assessment ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood organs of 8 RBT. Table 4 ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood and lymphoid organs of 8 RBT. Table 5 Table 5 Modulation of NCC activity in PKX infected fish. Oxidative burst The assay allows the estimation of intracellular production of hydrogen peroxide in PMA stimulated cells. The fluorescence measured by the flow cytometer is proportional to the amount of HP2 produced by the cells. A typical experiment includes the kinetic analysis of 104 individual cells at 5, 15 and 45 min post PMA stimulation.The results are expressed as the difference between the mean fluorescence produced by PMA stimulated cells and unstimulated cells. As the assay requires a small amount of cells, comparative analysis of phagocytosis and oxidative burst activity are easily performed. In addition. the data recorded from the analysis of cell functions can be correlated with one cell subpopulation. Table 3 shows that pronephros in which the granulocytes constitute the major fraction of the leukocytes are characterized by a high phagocytic activity and a strong oxidative burst (RBT 1 and 4). Inversely a minor fraction of granulocytes display a lower phagocytosis and a very weak oxidative burst (RBT 5). Nonspecific Cytotoxic Cells Discussion Immunologists routinely used FCM to characterize phenotypically the leukocyte subpopulations. Unfortunately, in trout hitherto only a few number of antibodies directed 211 against surface markers are avalaible (Deluca et al. 1983; Slierendrecht et al. 1995). Because of the lack of phenotypic markers the study of the fish cellular immune response involves the screening of morphological characteristics and functional mechanisms. Such combined analysis is easily performed using a flow cytometer. FCM offers a real advantage over conventional techniques because useful informations related to the leukocyte subpopulations are easily provided by the flow cytometer. In spite of the paucity of cell markers, interesting data are obtained from the direct study of non-labeled cells, on the basis of cell autofluorescence. These morphometric informations are very useful. Thus the heterogeneity occurring in the cytogram profiles is correlated with the health status of the fish. This heterogeneity could represent a significant part of the inter-fish variability occurring in the results. g Phagocytosis has been described to playa major role in fish defense mechanisms. The phagocytic system, mechanisms of phagocytosis and killing activity have been described and comprehensively reviewed (MacArthur et al. 1985; Secombes and Fletcher 1992). In mammals, assessment of phagocytosis by FCM has been used for long (Dun and Tyrer 1981; Bjerknes and Bassoe 1983). y j ) More recently in fish, FCM in vitro phagocytosis assays have been developed to assess the modulation of immune response by pathogens (Chilmonczyk 1995), xenobiotics (Voccia et al. 1994; 1997) or both (Chilmonczyk et al. 1997). It is possible to run a phagocytosis assay with a small number of cells (2x 105 ). This is a real advantage allowing in vitro kinetic studies and sampling of small fish. p g Oxidative mechanisms represent an important part of the intracellular microbicidial activity involving a variety of toxic oxygen species produced by activated phagocytes. The associated activity which results from the activation of NADPH oxidases is known as the oxidative or respiratory burst.The FCM technique we have developed, allows an accurate estimation of the capacity of cells to undergo an oxidative burst in response to a variety of stimuli. Oxidative burst assays are commonly used in immunotoxicology and immunopathology to study the modulations of cell defense mechanisms by pollutants, toxicants or pathogens (Chilmonczyk et al. 1997; Voccia et al. 1994; 1997). As in phagocytosis assays, only a number small of cells is needed to assess the oxidative burst. Bunecna imunitni odpoved' u pstruha duhoveho (Oncorhynchus mykiss): stu die pomocl prutokove cytometrie Pozornost byla venovana vyvoji metod vhodnych pro studium nespecifickych bunecnych obrannych mechanismu u pstruha duhoveho: fagocytoza, aktivita respiracniho vzplanuti a nespecificka cytotoxicita. Pfi srovnani s konvencnimi metod ami je prutokova cytometrie rychlou, vhodnou a reprodukovatelnou metodou pro analyzu rybich bunecnych imunitnich funkci. Vedle hodnoceni obrannych mechanismu jsou prezentovany i udaje z pozorovani nebarvenych bunek. Prutokove cytometricke profily bunekjsou reprezentativni pro organ, z nehoz leukocyty pochazeji. Kazdy Iymfoidni organ ma svuj charakteristicky cytogram. Prutokova cytometrie se ukazuje jako vhodny nastroj ke studiu imunitnich reakci a patologickych procesu. Discussion This allows the possibility of performing both assays in parallel on the same cell sample (Table 3). NCC are considered as the fish equi valent of mammalian natural killer (NK) cells (E van s and J aso-Friedmann 1992). NCC activity has been demonstrated in several fish species including catfish (Evans etal.1984)shark(McKinney etaI.1986),RBT(Hayden and Lall x 1985; Green lee et al. 1991; R i stow et al. 1995) or carp (S u z u m ura et al. 1994). NCC activity was demonstrated in the main lymphoid tissues ofRBT (Table 4). The activity measured in the thymus confirms previous observation (Hayden and Laux 1985). This wide-spread cytotoxic activity suggests that a substantial part of the nonspecific defense mechanisms could be due to NCe. NCC flow cytometric assay has the advantage of being able to detect dead target cells before lysis, thus the incubation time required to detect killing is shorterthan in conventional radioactive assay. Our data show that levels of cytotoxicity varied considerably between individual fish. In this particular case the large inter-fish variability could be due, in part, to non-adequate (because of mammalian origin) target cells. The use of fish target cell should improve the accuracy of the NCC assay. So far, in fish, only a few FCM studies have been performed even though FCM offers great potential for the study of the fish immune cells. The functional assays we have developed to assess phagocytosis, oxidative burst and NCC activity, have proven to be accurate and 212 reliable tests. Compared to conventional methods, the use of FCM presents obvious advantages. FCM is a very sensitive method that allows rapid multiparameter analysis of single cells and evaluation of cell functions. FCM assays require much less cells per analysis and are more rapid than conventional assays, thousands of cells can be analyzed within a few seconds. A large number of fluorescent dyes used in mammalian models are suitable for investigations in fish. Thus flow cytometer emerges as a useful tool to study haematopoietic cells and cellular activity. References BASSOE, C. F .. LAERUM, O. D., SOLBERG. C. 0., HANEBERG, B. 1983: Phagocytosis of bacteria by human leukocytes measured by flow cytometry. Proc. Soc. Exp. BioI. Med. 174: 182-186 BJERKNES. R.. BASSOE, C. F. 1983: Human leukocyte phagocytosis of zymosan particles measured by flow cytometry. Acta. Pathol. Microbiol. Immune Scand. 91: 341-348 CHANG. L.. GUSEWITCH. G. A.. CHRITTON, D. B. W., FOLZ, J. c., LEBECK, L. K., NEHLSEN- CANNARELLA, S. L. 1993: Rapid flow cytometric assay for the assessment of natural killer cell activity. J. Immunol. Methods 166: 45-54 CHILMONCZYK. S., VOCCIA, I.. MONGE. D. 1995: Pathogenesis of viral haemorrhagic septicemia virus: cellular aspects. Vet. Res. 26: 501-511 CHILMONCZYK, S., VOCCIA, I., TARAZONA, J. V., MONGE, D. 1997: Flow cytometric analysis of fish leucocyte populations exposed to pollutants and pathogens: modulatory effects induced by experimental procedures. In: Zelikoff, J. T. (ed.) Ecotoxicology: Responses, Biomarkers and Risk Assessment. OECD by SOS Publications, Fair Haven, N1, pp. 171-184 pp DEITCH, A. D .. LAW, H., DEVERE-WHITE. R. 1982: A stable propidium iodide staining procedure for flow cytometry. J. Histochem. Cytochem. 30: 967-972 DELUCA. D .. WILSON, M .. W ARR, G. W. 1983: Lymphocyte heterogeneity in the trout, Salmo gairdneri. defined with monoclonal antibodies to IgM. Eur. J. Immunol.13: 546-551 DUNN, P. A .. TYRER. H. W. 1981: Quantification of neutrophile phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry. J. Lab. Clin. Med. 98: 374-381 YRER. H. W. 1981: Quantification of neutrophile phagocytosis, using fluorescent latex beads. f i d fl J L b Cli M d 98 374 381 EVANS, D. L.. GRAVES, S. S., COBB, D., DA WE, D. L. 1984: Nonspecific cytotoxic cells of fish Uctalurus punctatus). II. Parametre of target cell lysis and specificity. Dev. Compo Immunol. 8: 303-312 GRAVES, S. S., COBB, D., DA WE, D. L. 1984: Nonspecific cytotoxic cells of fish Uctalurus I. Parametre of target cell lysis and specificity. Dev. Compo Immunol. 8: 303-312 s). II. Parametre of target cell lysis and specificity. Dev. Compo Immunol. 8: 303-312 EVANS. D. L..JASO- FRIEDMANN, L. 1992: Nonspecific cytotoxic cells as effectors of immunity in fish. Annual rev. of Fish Diseases. 2: 109-121 GREENLEE. A. R., BROWN. R. A., RISTOW, S. S. 1991: Nonspecific cytotoxic cells of rainbow trout (Oncorhynchus mykiss) kill Y AC-I targets by both necrotic and apoptotic mechanisms. Dev. Compo Immunol. 15: 153-164 HA YDEN. B. 1.. LAUX, D. C. References 1985: Cell mediated lysis of murine target cells by nonimmune salmonid lymphoid preparations. Dev. Compo Immunol. 9: 627-639 KIESSLING. R .. KLEIN. E., WIGZELL, H. 1975: Cytotoxic cells with specificity for mouse Maloney leukemia cells. Specificity and distribution according to genotype. Eur. J. Immunol. 5: 112-117 213 KOUMANS-VAN DIEPEN, 1. C. E., TAVERNE-THIELE, 1. 1.. VAN RENS, B. T. T. M., ROMBOUT, 1. H. W. M. 1994: Immunocytochemical and flow cytometric analysis of B cells and plasma cells in Carp (Cyprinus carpio L.); an ontogenetic study. Fish Shellfish Immunol. 4: 19-28 p ) g y MACARTHUR, 1. I., FLETCHER, T. C. 1985: Phagocytosis in fish. In: Manning, M. 1., Tatner, M. F. (eds) Fish immunology. Academic Press, London, pp. 29-46 MCKINNEY, E. C., HA YNES,L.. DROESE,A. L. 1986: Macrophage-like effector of spontaneous cytotoxicity from the shark. Dev. Compo Immunol. 10: 497-508 p RISTOW, S. S., GRABOWSKI, L. D., WHEELER, P. A., PRIEUR, D. T., THORGAARD, G. H. 1995: Arlee line of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity. Dev. Compo Immunol. 19: 497-505 . SECOMBES, C. 1., FLETCHER, T. C. 1992: The role ofphagocytes in the protective mechanisms offish. Ann. Rev. Fish Diseases. 2: 53-71 SLlERENDRECHT, W. 1., LORENZEN, N., GLAMAN, 1., KOCH, c., ROMBOUT, 1. H. W. M. 1995: Immunocytochemical analysis of a monoclonal antibody specific for rainbow trout (Oncorhynchus mykiss) granulocytes and thrombocytes. Vet. Immunol. Immunopathol. 46: 349-360 g y y p SUZUMURA, E., KURATA, 0., OKAMOTO, N., IKEDA,Y. 1994: Characteristics of natural killer-like cells in carp. Fish Pathol. 29: 199-203 VOCCIA,I., KRZYSTYNIAK, K., FLlPO, D., FOURNIER, M. 1994. In vitro mercury-related cytotoxicity and functional impainnent of the immune cells of rainbow trout (Oncorhyncus mykiss). Aquat. Toxicol. 29: 37-48 VOCCIA, I., SANCHEZ-DARDON, 1., DUNIER, M., CHILMONCZYK. S., FOURNIER, M. 1997: Contaminants on competence of rainbow trout macrophages. In : Chemically induced alteration in functional development and reproduction of fish, Setac Press, pp. 99-109
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Dietary valine requirement of tambaqui (Colossoma macropomum) with different body weights
Ciência e Agrotecnologia
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Exigência de valina dietética para tambaqui (Colossoma macropomum) com diferente peso corporal Jakeline Veras da Silva1 , Felipe Barbosa Ribeiro2* , Jefferson Costa de Siqueira2 Marcos Antonio Delmondes Bomfim2 , Dáphinne Cardoso Nagib do Nascimento1 , Rafael Si Jakeline Veras da Silva1 , Felipe Barbosa Ribeiro2* , Jefferson Costa de Siqueira2 , Marcos Antonio Delmondes Bomfim2 , Dáphinne Cardoso Nagib do Nascimento1 , Rafael Silva Marchão3 1Universidade Federal do Maranhão/UFMA, Programa de Pós-graduação em Ciência Animal/PPGCA, Chapadinha, MA, Brasil 2Universidade Federal do Maranhão/UFMA, Centro de Ciências Agrárias e Ambientais/CCAA, Chapadinha, MA, Brasil 3Universidade Federal do Vale do São Francisco/UNIVASF, Programa de Pós-Graduação em Ciência Animal, Petrolina, PE, Brasil *Corresponding author: felipebribeiro@yahoo.com Received in January 31, 2022 and approved in May 3, 2022 ABSTRACT Tambaqui (Colossoma macropomum) is one of the highly produced freshwater fish in Brazilian fish farming. However, its production efficiency associated with the refinement of diet formulations is limited due to a lack of information on nutritional requirements for certain essential amino acids such as valine. This study was designed to estimate the valine requirement of tambaqui having different body weights. A total of 720 juveniles were used in the feed trials. The first trial consisted of 360 fish with an average initial weight of 33.28 ±0.19 g, and the second trial comprised 360 fish (121.19 ±1.29 g). The trials consisted of dose–response experiments with five levels of valine (5.0, 7.0, 9.0, 11.0, and 13.0 g/kg diet) and four replicate tanks per treatment. The control diet was obtained by adding L-valine to the diet containing 5.0 g/kg diet (the first level of valine tested) to ensure it reached the diet containing 7.0 g/kg diet, corresponding to the second level tested. Analysis of variance, Dunnett’s test for comparison with the control diet, and regression analysis were performed. The results showed that the valine intake, weight gain, specific growth rate, and feed conversion ratio were significantly improved by the dietary valine (P < 0.05) in both trials. Dietary levels of valine exerted quadratic effects on body protein, body fat, body protein deposition, and nitrogen retention efficiency of tambaqui with 33 g and 121 g as body fat deposition was affected by the dietary valine level only in tambaqui weighing 33 g. Quadratic regression analysis of body protein deposition data revealed the optimum dietary valine requirement of tambaqui (33.0 g–83.0 g) and (121.0 g–277.0 g) as 11.9 g/kg diet and 9.1 g/kg diet, respectively. Index terms: Amazon fish; branched-chain amino acid; essential amino acid; fish nutrition. 2022 | Lavras | Editora UFLA | www.editora.ufla.br | www.scielo.br/cagro All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution BY. Ciência e Agrotecnologia, 46:e002122, 2022 http://dx.doi.org/10.1590/1413-7054202246002122 eISSN 1981-1829 Animal Science and Veterinary 2022 | Lavras | Editora UFLA | www.editora.ufla.br | www.scielo.br/cagro ll h f h l h h d l d d b 2022 | Lavras | Editora UFLA | www.editora.ufla.br | www.scielo.br/cagro INTRODUCTION of valine for other fish species has also been determined (Abidi; Khan, 2004; Castillo; Gatlin, 2018; Dong et al., 2013; Ren et al., 2015; Xiao et al., 2018; Zehra; Khan, 2014; Zhou et al., 2020). However, the optimal valine requirements have not been established for tambaqui, especially requirements at different body weights and using the diet dilution technique (Fisher; Morris, 1970). In addition to being the most expensive component of aquatic feeds, proteins are an important source of essential amino acids (EAAs) and non-essential amino acids (non- EAAs) and play a crucial role in the growth and physiological functions of fish (National Research Council - NRC, 2011). However, fish do not have metabolic requirements for protein, necessitating the need for an adequate balance of EAAs and non-EAAs (Costa et al., 2021; Wilson, 2002). Diets formulated solely based on crude protein content may not meet the needs of all amino acids (AA), especially essential ones, thereby compromising performance and increasing the release of nitrogen compounds into the environment due to over-supplementation of nitrogen (Marchão et al., 2020; Silva et al., 2018). Therefore, considering the lack of information about the valine requirement for the tambaqui diet and the importance of specific nutrition for the species, we estimated the valine requirement for tambaqui with different body weights. RESUMO O tambaqui (Colossoma macropomum) é um dos peixes de água doce mais produzidos na piscicultura brasileira. No entanto, sua eficiência produtiva associada ao refinamento das formulações de dietas é limitada devido à falta de informações sobre as exigências nutricionais de determinados aminoácidos essenciais, como a valina. Objetivou-se estimar a exigência de valina para o tambaqui com diferentes pesos corporais. Um total de 720 juvenis foi utilizado nos experimentos, o primeiro com 360 peixes com peso médio inicial de 33,28 ±0,19 g e o segundo com 360 peixes (121,19 ±1,29 g). Os experimentos foram conduzidos em dose- resposta com cinco níveis de valina (5,0; 7,0; 9,0; 11,0 e 13,0 g/kg de dieta) e quatro repetições por tratamento. A dieta controle foi obtida pela adição de L-valina à dieta contendo 5,0 g/kg de dieta (primeiro nível de valina testado), de modo que atingiu a dieta contendo 7,0 g/kg de dieta, correspondente ao segundo nível testado. Foram realizados análise de variância dos dados, teste de Dunnett’s para comparação com dieta controle e análise de regressão. Os resultados para a ingestão de valina, ganho de peso, taxa de crescimento específico, taxa de conversão alimentar foram significativamente melhorados pela valina dietética (P<0,05) em ambos os experimentos. Os níveis dietéticos de valina exerceram efeitos quadráticos sobre a proteína corporal, gordura corporal, deposição de proteína corporal e eficiência de retenção de nitrogênio do tambaqui com 33 g e 121 g, sendo a deposição de gordura corporal afetada pelo nível de valina somente no tambaqui com 33 g. Com base na análise de regressão quadrática dos dados de deposição de proteína corporal, as exigências dietéticas de valina de tambaqui (33,0 g-83,0 g) e (121,0 g-277,0 g) foram estimadas em 11,9 g/kg da dieta e 9,1 g/kg da dieta, respectivamente. rmos para indexação: Peixe da Amazônia; aminoácido de cadeia ramificada; aminoácido essencial; nutriç 2 SILVA, J. V. et al. Ciência e Agrotecnologia, 46:e002122, 2022 aDry matter soybean meal: 95.20% and corn: 93.90%. Experimental diets Valine is an EAA and potentially limiting in conventional fish feeds (Rodrigues et al., 2020). Oliveira et al. (2021), in their study with pacu fish, reported dietary valine, lysine, and arginine as the most limiting EAAs, and signified the importance of the composition of these AAs during the preparation of diets for this species. In juvenile Nile tilapia, methionine, threonine, and valine are the most limiting dietary EAAs (Diógenes et al., 2016). To formulate the experimental diets, we first analyzed the total AA contents of corn and soybean meal. Next, we converted these into digestible AAs using digestibility coefficients of ingredients (Table 1). The total AA contents of corn and soybean meal used in the experimental diets were obtained using high-performance liquid chromatography (HPLC). Valine majorly participates in the synthesis of body proteins, in addition to performing other important physiological functions, including the repair and growth of muscle tissue and the maintenance of nitrogen balance in the body (Suryawan et al., 2011). In addition, valine has been reported to increase fish growth by increasing digestive and absorption capacity, thus, influencing the balance of intestinal microflora (Dong et al., 2013). Therefore, it is necessary to understand valine requirements according to fish species and their development stages. Table 1: Total and digestible amino acid compositions of corn and soybean meal used in experimental diets (based on natural matter). Amino acid (%) Corna Soybean meala TAAb DAAc TAAb DAAc Lysine 0.27 0.25 2.76 2.61 Methionine 0.12 0.12 0.50 0.48 Methionine + Cystine 0.38 0.22 1.58 0.99 Threonine 0.25 0.25 1.71 1.68 Tryptophan 0.23 0.18 0.59 0.56 Isoleucine 0.24 0.24 2.09 2.03 Valine 0.35 0.35 2.10 2.04 Leucine 0.67 0.66 3.30 3.21 Arginine 0.40 0.39 3.31 3.28 Crude protein 7.61 46.66 aDry matter soybean meal: 95.20% and corn: 93.90%. bTotal amino acids, determined using high-performance liquid chromatography (HPLC) by the CBO laboratory, Valinhos, SP. cDigestible amino acids, calculated based on the digestibility coefficients presented by NASCIMENTO et al. (2020) for tambaqui (Colossoma macropomum). Table 1: Total and digestible amino acid compositions of corn and soybean meal used in experimental diets (based on natural matter). Experimental diets Amino acid (%) Corna Soybean meala TAAb DAAc TAAb DAAc Lysine 0.27 0.25 2.76 2.61 Methionine 0.12 0.12 0.50 0.48 Methionine + Cystine 0.38 0.22 1.58 0.99 Threonine 0.25 0.25 1.71 1.68 Tryptophan 0.23 0.18 0.59 0.56 Isoleucine 0.24 0.24 2.09 2.03 Valine 0.35 0.35 2.10 2.04 Leucine 0.67 0.66 3.30 3.21 Arginine 0.40 0.39 3.31 3.28 Crude protein 7.61 46.66 aDry matter soybean meal: 95 20% and corn: 93 90% The tambaqui (Colossoma macropomum), produced in 25 of the 26 Brazilian states, is considered one of the most important species for Brazilian aquaculture (Instituto Brasileiro de Geografia e Estatística - IBGE, 2019). It is particularly known for the flavor of its meat in the consumer market. The tambaqui is an omnivorous species of freshwater from the Amazon and Orinoco rivers, which feeds on fruits, seeds, and small organisms. It displays desirable characteristics for fish farming and can easily adapt to conditions and rearing systems. However, high nutritional value feeds are required when raised in captivity to maximize production (Woynárovich; Van Anrooy, 2019). Th i f EAA f b i h b aDry matter soybean meal: 95.20% and corn: 93.90%. bTotal amino acids, determined using high-performance liquid chromatography (HPLC) by the CBO laboratory, Valinhos, SP. cDigestible amino acids, calculated based on the digestibility coefficients presented by NASCIMENTO et al. (2020) for tambaqui (Colossoma macropomum). The requirement of EAAs for tambaqui has been studied for lysine (Marchão et al., 2020), methionine plus cysteine (Souza et al., 2019), threonine (Firmo et al., 2018), and tryptophan (Bomfim et al., 2020). The requirement Ciência e Agrotecnologia, 46:e002122, 2022 3 Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights Diets were formulated using the dilution technique to obtain increased levels of digestible valine (Fisher; Morris, 1970). The dilution technique, an alternative technique for formulating experimental diets, was recently used by Nascimento et al. (2020) in a study on determining the optimum dietary EAA profile of Nile tilapia and Marchão et al. (2020) in a study on the requirement for digestible lysine for tambaqui juveniles. This technique mixes two diets, one containing a high content of the tested AA and another protein-free diet (PFD), to obtain a diet with intermediate levels of AAs tested and reduced levels of AAs. Crude protein levels ranged from (110.4 to 287.0 g/kg diet) because the diets were formulated using the dilution technique. Experimental diets In this technique, the AA balance of the treatments remained constant because it is based on the use of a high-protein diet deficient in the evaluated AA. The evaluated AA is diluted in a protein-free isoenergetic diet, resulting in its intermediate levels (NRC, 2011; Siqueira et al., 2013). To confirm valine as the first-limiting nutrient and that the responses obtained were a function of valine and not crude protein in the diet, a sixth diet (control diet) was used. The control diet was obtained by adding synthetic valine (L-valine 98.5%) to the diet containing the first level of digestible valine (5.0 g/kg diet) to reach a concentration of 7.0 g/kg diet, corresponding to the second level tested, as proposed by Nonis and Gous (2008). Initially, a reference diet (RD) was formulated containing a 287.0 g/kg diet of crude protein (CP) and a 13.0 g/kg diet of digestible valine (77% digestible valine: lysine ratio) based on corn and soybean meal (Table 2). Subsequently, this diet was diluted with another PFD based on corn starch, containing the same levels of energy, vitamins, and minerals, to obtain diets with increasing dietary valine concentrations (5.0, 7.0, 9.0, 11.0, and 13.0 g/kg diet) (Table 2). Initially, the ingredients of experimental diets were ground (Trf 60, Trapp), sifted, and mixed (Horizontal Mixer 300 kg, Branorte). To obtain intermediate diets (5.0, 7.0, 9.0, 11.0, and 13.0 g/kg diet), mixtures were diluted (Table 2). Subsequently, they were extruded in an equipment with a 3–5 mm sieve (Extruder model MX 40, Inbramaq, Laboratório de Nutrição e Alimentação de Organismos Aquáticos do Maranhão, Chapadinha, Brazil). Other AAs exceeded the ideal ratios to digestible lysine recommended by the NRC (2011) for Nile tilapia by at least two percentage points to prevent another AA from becoming limiting. Table 2: Diet formulation and chemical composition of the protein-free diet (PFD), reference diet (RD), control diet (CD), and experimental diets (g/kg natural matter bases). Table 2: Diet formulation and chemical composition of the protein-free diet (PFD), reference diet (RD), control diet (CD), and experimental diets (g/kg natural matter bases). Table 2: Diet formulation and chemical composition of the protein-free diet (PFD), reference diet (RD), control diet (CD), and experimental diets (g/kg natural matter bases). Table 2: Diet formulation and chemical composition of the protein free diet (PFD), reference diet (RD), control diet (CD), and experimental diets (g/kg natural matter bases). Ciência e Agrotecnologia, 46:e002122, 2022 Experimental diets Ingredients (g/kg) Digestible valine Levels PFD 5.0 7.0 9.0 11.0 13.0(RD) CD Soybean meal 0.00 206.9 289.7 372.5 455.3 538.1 206.9 Corn 0.00 136.5 191.1 245.7 300.3 354.9 136.5 Corn starch 802.9 494.1 370.5 247.0 123.5 0.00 494.1 Soybean oil 103.2 80.6 71.6 62.5 53.5 44.5 80.6 Cellulose 37.5 23.1 17.3 11.5 5.8 0.00 23.1 Lysine-HCl (78.4%) 0.0 0.9 1.3 1.7 2.0 2.4 0.9 DL-Methionine (99%) 0.0 2.0 2.8 3.6 4.5 5.3 2.0 L-Threonine (98.5%) 0.0 0.9 1.3 1.7 2.1 2.4 0.9 L-Tryptophan (98%) 0.0 0.1 0.1 0.2 0.2 0.3 0.1 L-Valine (99%) 0.0 0.3 0.4 0.6 0.7 0.8 2.3 L-Leucine (99%) 0.0 0.6 0.8 1.0 1.2 1.4 0.6 Calcitic limestone 7.0 7.2 7.3 7.4 7.5 7.6 7.2 Dicalcium phosphate 38.2 35.7 34.7 33.6 32.6 31.6 35.7 Vitamin and mineral premixf 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Vitamin Ce 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Continue... SILVA, J. V. et al. SILVA, J. V. et al. 4 Table 2: Continuation. Ingredients (g/kg) Digestible valine Levels PFD 5.0 7.0 9.0 11.0 13.0(RD) CD Salt 5.5 5.4 5.3 5.2 5.2 5.1 5.4 Antioxidant (BHT) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Dilution (%) D1 D2 D3 D4 D5* ** Protein-free diet 100.00 61.54 46.15 30.77 15.38 0.00 Reference diet 0.00 38.46 53.85 69.23 84.62 100.00 Chemical composition (g/kg in the natural matter) Crude protein 0.00 110.4 154.6 198.7 242.9 287.0 110.4 Digestible energy (kcal kg–1)d 3,000 3,000 3,000 3,000 3,000 3,000 3,000 Crude fibera 34.7 34.7 34.7 34.7 34.7 34.7 34.7 Total Caa 12.0 12.0 12.0 12.0 12.0 12.0 12.0 P availablea 7.0 7.0 7.0 7.0 7.0 7.0 7.0 Total Naa 2.2 2.2 2.2 2.2 2.2 2.2 2.2 Digestible Valineb 0.00 5.0 7.0 9.0 11.0 13.0 7.0 Digestible Lysineb 0.00 6.5 9.0 11.6 14.2 16.8 6.5 Digestible Met. Ciência e Agrotecnologia, 46:e002122, 2022 * Reference diet (13.0 g digestible valine/kg of dry diet). f Vitamin and mineral supplement, amounts supplied per kg diet: Vit. A, 6,000 IU; Vit. D3, 1,000 IU; Vit. E, 60.0 mg; Vit. K3, 12.0 mg; Vit. B1, 24.00 mg; Vit. B2, 24.00 mg; Vit. B6, 24.00 mg; Vit. B12, 24.00 mg; Vit. C, 24.00 mg; folic acid, 6.00 mg; Ca pantothenate, 60.00 mg; biotin, 0.24 mg; choline chloride, 108 g; niacin, 100.00 mg; Fe, 250.00 mg; Cu, 15.0 mg; Mn, 100.00 mg; Zn, 150.00 mg; I, 0.5 mg; Co, 0.05 mg; Se, 0.5 mg. Experimental diets + Cistb 0.00 4.3 6.1 7.8 9.5 11.3 4.3 Digestible Threonineb 0.00 4.7 6.6 8.5 10.4 12.3 4.7 Digestible Tryptophanb 0.00 1.4 1.9 2.5 3.1 3.6 1.4 Digestible Arginineb 0.00 7.3 10.3 13.2 16.1 19.1 7.3 Digestible Leucineb 0.00 8.1 11.3 14.5 17.8 21.0 8.1 Relationships Digestible Met + Cys/Lys (0.63)c 0.00 0.66 0.67 0.67 0.67 0.67 0.66 Digestible Thr/Lys (0.69)c 0.00 0.72 0.73 0.73 0.73 0.73 0.72 Digestible Typ/Lys (0.19)c 0.00 0.21 0.21 0.21 0.21 0.21 0.21 Digestible Arg/Lys (0.75)c 0.00 1.12 1.12 1.12 1.12 1.12 1.12 Digestible Val/Lys (0.94)c 0.00 0.77 0.77 0.77 0.77 0.77 1.07 Digestible Leu/Lys(1.19)c 0.00 1.25 1.25 1.25 1.25 1.25 1.25 Table 2: Continuation. * Reference diet (13.0 g digestible valine/kg of dry diet). ** Control diet = D1 + 0.20% L-valine. a Based on the values proposed by Rostagno et al. (2011).fi a Based on the values proposed by Rostagno et al. (2011).fi b Based on the digestibility coefficients proposed by Nascimento et al. (2020). y d Based on the digestibility coefficients proposed by Furuya et al. (2010). d Based on the digestibility coefficients proposed by Furuya et al. (2010). e Vit. C: ascorbic acid 2-monophosphate calcium salt, 42% active ingredient. f Vitamin and mineral supplement, amounts supplied per kg diet: Vit. A, 6,000 IU; Vit. D3, 1,000 IU; Vit. E, 60.0 mg; Vit. K3, 12.0 mg; Vit. B1, 24.00 mg; Vit. B2, 24.00 mg; Vit. B6, 24.00 mg; Vit. B12, 24.00 mg; Vit. C, 24.00 mg; folic acid, 6.00 mg; Ca pantothenate, 60.00 mg; biotin, 0.24 mg; choline chloride, 108 g; niacin, 100.00 mg; Fe, 250.00 mg; Cu, 15.0 mg; Mn, 100.00 mg; Zn, 150.00 mg; I, 0.5 mg; Co, 0.05 mg; Se, 0.5 mg. Ciência e Agrotecnologia, 46:e002122, 2022 5 Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights The experimental diets were provided daily as six meals (08:00, 10:00, 12:00, 14:00, 16:00, and 18:00 h) until satiation. At each meal, they were supplied in small quantities with successive passes, allowing the maximum intake. (dry) in an oven with forced-air circulation, dried for 72 h at 65 °C, and then ground in a micro mill (IKA A11 basic) and stored in plastic containers at -20 °C until body composition was analyzed. Experimental diets The samples were analyzed for whole-body composition (dry matter, moisture content, crude protein, fat, and ash) according to the (Association of Official Analytical Chemists - AOAC, 2019) standard procedures. • FCR (g− 1) = feed intake (g)/weight gain (g). • FBA = final body ash, IBA = initial body ash, IW = initial weight, FW = final weight. • NRE (%) = [FBN (%) × FW(g)]−[IBN (%) × IW(g)]/ [feed intake (g) × N level in ratio (%)]/100i • NRE (%) = [FBN (%) × FW(g)]−[IBN (%) × IW(g)]/ [feed intake (g) × N level in ratio (%)]/100i Sample collection and analysis • FBN = final body nitrogen, IBN = initial body nitrogen, IW = initial weight, FW = final weight. • FBN = final body nitrogen, IBN = initial body nitrogen, IW = initial weight, FW = final weight. • FBN = final body nitrogen, IBN = initial body nitrogen, IW = initial weight, FW = final weight. At the beginning of the experiment, 20 fish were sampled, euthanized by subjecting them to the benzocaine solution (500 mg L–1), and stored at −20 °C to determine the whole-body composition. At the end of each trial period, fish were not fed for 24 h before sampling. All fish from each experimental unit were individually weighted, euthanized with a benzocaine solution (500 mg L–1), and frozen in a freezer at −20 °C. Subsequently, the fish samples were thawed, and the whole fish was crushed and dehydrated • FCR (g− 1) = feed intake (g)/weight gain (g). • FCR (g− 1) = feed intake (g)/weight gain (g). • FCR (g− 1) = feed intake (g)/weight gain (g). Based on the body composition (moisture, ash, protein, and fat), the daily deposition rates of body protein (BPD), body fat (BFD), and body ash (BAD), as well as the nitrogen retention efficiency (NRE), were calculated according to the equations given below: During the trials, the fish were kept in polyethylene boxes with a capacity of 1000 L each, equipped with individual water supplies, drainage systems, and aeration systems. The water supply of boxes was derived from an artesian well, with a flow rate of 30 L h–1 per box. • BPD (mg day− 1) = {[FBP (%) × FW (mg)] − [IBP (%) × IW (mg)]/100}/experimental period (days)i The temperature of the water was measured daily at 7:30, and 17:30 h using a mercury bulb thermometer graduated from 0 to 50 °C. Controls for pH, the content of dissolved oxygen, and ammonia in the water were measured every 3 days using a pH meter (HI 8424, Hanna), oximeter (HI 9146, Hanna), and a commercial kit (Arcor) for toxic ammonia test, respectively. The temperature of water ranged from 24.7 ±0.6 °C to 27.1 ±0.2 °C (first trial) and 26.4 ±0.7 °C to 27.5 ±0.1 °C (second trial). The pH was kept at 7.02 ±0.3, dissolved oxygen was maintained above 10.5 ±0.4 mg/L, and ammonia nitrogen was kept below 1.0 ppm. • IBP = initial body protein, FBP = final body protein, IW = initial weight, FW = final weight. • BFD (mg day− 1) = {[FBF (%) × FW (mg)]−[IBF (%) × IW (mg)]/100}/experimental period (days)i • BFD (mg day− 1) = {[FBF (%) × FW (mg)]−[IBF (%) × IW (mg)]/100}/experimental period (days) • FBW = final body fat, IBW = initial body fat, IW = initial weight, FW = final weight. • FBW = final body fat, IBW = initial body fat, IW = initial weight, FW = final weight. • BAD (mg day− 1) = {[FBA (%) × FW (mg)]−[IBA (%) × IW (mg)]/100} experimental period (days)i • BAD (mg day− 1) = {[FBA (%) × FW (mg)]−[IBA (%) × IW (mg)]/100} experimental period (days) • FBA = final body ash, IBA = initial body ash, IW = initial weight, FW = final weight. Ciência e Agrotecnologia, 46:e002122, 2022 Ethics Statement, experimental facility, fish, and water quality parameters The study was conducted at the Laboratory of Nutrition and Food for Aquatic Organisms of Maranhão, located at the Center of Agrarian and Environmental Sciences of the Federal University of Maranhão, Chapadinha, in Brazil (03◦ 44′ 33′ ′ S, 43◦ 21′ 21′ ′ W; altitude 105 m) and lasted for 50 days for each trial. It was conducted using experimental procedures approved by the Animal Use Ethics Committee of the Federal University of Maranhão (Protocol N° 23115.031736/2020–83). At the end of each trial, the following performance and feed efficiency indexes were evaluated: feed intake (FI), digestible valine intake (DVI), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR), according to the equations given below: • FI (g) = dry feed consumed during the experimental period • FI (g) = dry feed consumed during the experimental period • DVI (g) = [feed intake (g) × digestible valine level in feed (%)]/100i The study was divided into two trials. In total, 720 tambaqui (C. macropomum) were used, being 360 fish, with an average initial weight of 33.3 ±0.2 g, in the first trial and 360 fish, with an average initial weight of 121.2 ±1.3 g, in the second trial. In each trial, the fish were randomly distributed into 24 experimental units, with 15 fish in each unit. i • WG (g) = final mean weight (g)−initial mean weight (g) • SGR (% day–1) = [In final weight (g) −In initial weight (g)] × 100]/experimental period (days) Statistical analysis The data were submitted to the Shapiro–Wilk and Levene tests to test for normality and homoscedasticity. Once the assumptions met, the data were then submitted to analysis of variance. The variables affected by valine levels were subjected to regression analysis performed Ciência e Agrotecnologia, 46:e002122, 2022 6 SILVA, J. V. et al. using linear and quadratic models. To compare the effects of the CD (1.125%) with other treatments, Dunnett’s test was used. All statistical analyses were performed using the SAS 9.0 software (Statistical Analysis System - SAS, 2002), with a value of P < 0.05 indicating significance. In both trials, the fish raised on the control diet (CD) with 7.0 g/kg valine in the diet showed superior responses to those raised on a diet containing 5.0 g/kg valine in the diet, with emphasis on body protein deposition, feed conversion ratio, and nitrogen retention efficiency. The experimental diets in our study were formulated using the dilution technique (Fisher; Morris, 1970), and despite the variations in protein intake among treatments, the results obtained in animals submitted to the CD confirmed that valine was the first limiting AA in experimental diets. In addition, the results were related only to variations in the valine intake and not to the crude protein content of the diets. Sep-Pooled standard error of the mean. *Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). p *Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). P > F – Significance of the “F” test of the analysis of variance. ( ) Significance of the “F” test of the analysis of variance. l d t d d f th Sep-Pooled standard error of the mean.f P > F – Significance of the “F” test of the analysis of variance. he mean. ffer from the control diet (CD) by the Dunnett test (P < 0.05). y tandard error of the mean. e same column differ from the control diet (CD) by the Dunnett test (P < 0.05). CD – Control diet (D1 + 0.20% L-valine).i he F test of the analysis of variance. ror of the mean. umn differ from the control diet (CD) by the Dunnett test (P < 0.05). he “F” test of the analysis of variance. ror of the mean. i d standard error of the mean. n the same column differ from the control diet (CD) by the Dunnett test (P < 0 05) RESULTS AND DISCUSSION Variables Body weight (g) Digestible valine levels (g/kg diet) FI (g fish –1) DVI (g fish –1) WG (g) SGR (% day–1) FCR (g g–1) 33 g CD 103.74 ±9.18 257.20 ±8.77 47.08 ±1.76 4.21 ±0.02 2,23 ±0.25 5.0 116.31 ±4.17 193.84 ±6.94* 36.57 ±2.50 4.07 ±0.04* 3.21 ±0.14 * 7.0 113.22 ±3.61 264.19 ±8.43 47.13 ±3.23 4.21 ±0.04 2.42 ±0.10 9.0 110.87 ±4.61 332.60 ±13.84* 56.02 ±5.66 4.31 ±0.06 2.02 ±0.13 11.0 108.45 ±2.85 397.63 ±10.44* 59.12 ±0.97 * 4.35 ±0.01 1.84 ±0.02 13.0 114.75 ±5.69 497.26 ±24.66* 63.61 ±1.95* 4.40 ±0.02* 1.80 ±0.06 P>F1 0.7167 <0.0001 0.0003 0.0002 <0.0001 Linear effect 0.5699 <0.0001 <0.0001 <0.0001 <0.0001 Quadratic effect 0.2621 0.3200 0.1636 0.0900 0.0004 121 g SEp 10.84 27.14 6.14 0.072 0.27 CD 224.28 ±2.53 341.11 ±4.62 98.16 ±1.36 4.10 ±0.00 2.40 ±0.15 5.0 220.12 ±1.89 244.58 ±2.10* 79.84 ±2.24 4.03 ±0.01 2.76 ±0.09 7.0 220.24 ±2.04 342.60 ±3.17 106.9 ±15.13 4.09 ±0.05 2.15 ±0.28 9.0 222.14 ±4.61 444.29 ±9.22* 124.84 ±8.51 4.19 ±0.03 1.81 ±0.14 11.0 220.40 ±1.99 538.76 ±4.87* 102.77 ±9.22 4.11 ±0.03 2.19 ±0.18 13.0 220.13 ±0.52 635.92 ±1.49* 91.92 ±3.63 4.08 ±0.01 2.41 ±0.11 P>F1 0.9760 <0.0001 0.0147 0.0316 0.0094 Linear effect 0.9844 <0.0001 0.4442 0.2289 0.2002 Quadratic effect 0.6524 0.6404 0.0017 0.0068 0.0012 SEp 5.14 0.054 14.35 0.054 0.30 CD – Control diet (D1 + 0.20% L-valine).i Ciência e Agrotecnologia, 46:e002122, 2022 Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights 7 reflects lower growth rates, possibly due to the more rapid assimilation of crystalline AAs than AAs present in the diet as a component of protein. The use of the diet dilution technique provides more accurate results with respect to the performance of animals than the supplementation technique because the dilution technique provides constant levels of AAs in all treatments, uses a lower concentration of synthetic AAs, and provides better efficiency in the use of dietary nutrients (Siqueira et al., 2013). This formulating diet approach avoided the need to include large quantities of crystalline AAs in the diet, similar to ours during the supplementation technique. RESULTS AND DISCUSSION No dead fish were observed during the 50 days of each trial. No evidence of outward pathological signs was noted in fish, considering the low levels of dietary valine. The growth performance, body composition, and body deposition are shown in Tables 3, 5, and 7, respectively. Table 3: Mean values ±standard error of feed intake (FI), digestible valine intake (DVI), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) of tambaqui with different body weights as a function of digestible valine levels in the diet. Table 3: Mean values ±standard error of feed intake (FI), digestible valine intake (DVI), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) of tambaqui with different body weights as a function of digestible valine levels in the diet. g g g pi growth rate (SGR), and feed conversion ratio (FCR) of tambaqui with different body weights as a function of digestible valine levels in the diet. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION Therefore, comparisons between digestible valine values reported in the literature and those described in our study are difficult, even among species considered omnivorous. In this study, improvements in weight gain, specific growth rate, feed conversion ratio, body protein, body protein deposition, and nitrogen retention efficiency of tambaqui fed with increasing dietary valine levels indicated the importance of valine for the productive performance of this species and showed its ability to assimilate the valine crystal for growth. Previous reports on feeding trials with different dietary valine levels showed that growth performances in fish were improved by supplementation of dietary valine (Ahmad; Ahmed; Dar, 2021; Dong et al., 2013; Rollin et al., 2006; Xiao et al., 2018). For body composition and body deposition in tambaqui with an initial body weight of 33 g, the increased dietary valine levels influenced the body protein (%), body protein deposition, and nitrogen retention efficiency in a quadratic way (P < 0.05), which increased up to the levels of dietary valine of 10.7, 11.9, and 9.5 g/kg diet, respectively. In addition, body fat and body fat deposition showed a quadratic response (P < 0.05), which decreased until the dietary valine levels of 10.7 and 9.4 g/kg diet, respectively (Tables 5, 6, 7 and 8 and Figure 2). Xiao et al. (2018) conducted a study with juvenile Nile tilapia (initial weight = 6.48 g) and estimated the optimum valine requirement in 11.5 g/kg diet for weight gain, which was higher than the dietary valine values reported in our study for weight gain in fish with an initial weight of 121 g. Although Nile tilapia is also an omnivorous species, fingerlings are expected to have a higher metabolic rate and higher AA requirements than post-juvenile fish. Values ​​higher than those presented in the present study have also been reported by Dong et al. (2013), which estimated the valine requirement in 13.7 g/kg diet for improving the specific growth rate of juvenile Jian carp, characterized by being the first artificial breed Chinese carp, which grows 30% faster than the common carp (Dong; Yuan, 2002). In trial two (with an initial weight of 121 g), the levels of dietary valine influenced body protein (%), body protein deposition, and nitrogen retention efficiency in a quadratic manner (P < 0.05), which increased up to 9.0, 9.1, and 8.2 g/kg diet, respectively. RESULTS AND DISCUSSION (2010) reported that comparisons should not be made between AA requirement values in species with distinct feeding behaviors, except when they are expressed as dietary proteins. Similar to trial one, the tambaqui with an initial body weight of 121 g (trial two) showed an increase in the valine intake with valine supplementation (P < 0.05) and a quadratic reduction in feed conversion rate up to the valine level of 9.3 g/kg diet. However, the weight gain and specific growth rate of trial one differed by showing a quadratic response, which increased up to the levels of dietary valine of 9.2 and 9.4 g/kg diet, respectively, and gradually decreased thereafter (Tables 3 and 4 and Figure 1). The higher level determined to optimize the feed conversion ratio of tambaqui with 33 g as compared to the level determined for tambaqui with 121 g of body weight can be explained by a change in AA requirements during the growth, which decreases throughout an animal’s life due to the reduction in the growth rate (NRC, 2011). The improvement in feed conversion ratio was provided by a higher concentration of valine because no feed intake differences were observed in any of the trials. The optimal valine requirement to maximize the weight gain and specific growth rate of tambaqui with an initial weight of 121 g was recorded in 9.2 and 9.4 g/kg of dry diet, respectively; these values were lower than the valine requirements of tambaqui with an initial weight of 33 g, which showed a linear increase in weight gain and specific growth rate with the supplementation of dietary valine. As different stages of growth have metabolic or physiological requirements for specific AAs, the requirement for AAs may change during life stages, as observed in the present study (Wilson; Poe; Robinson, 1985).i Abidi and Khan (2004) and Xiao et al. (2018) reported an improved feed conversion ratio with an increase in the dietary concentration of valine up to 15.0 and 12.7 g/kg diet, respectively. These values were lower than those mentioned in our study. Several factors, such as fish size, developmental stages, type of diet, dietary protein and energy concentrations, feeding frequency, and environmental conditions, interfere with AA requirements (Abidi; Khan, 2004; Abimorad et al., 2010; Yue et al., 2014; Zehra; Khan, 2014). RESULTS AND DISCUSSION Moreover, according to Cowey (1995), the presence of large amounts of crystalline AAs in fish diets Regarding the tambaqui with a bodyweight of 33 g (trial one), the feed intake was not influenced (P > 0.05) by the increasing valine levels in the diets, whereas the valine intake, weight gain, and specific growth rate increased by an increment in the valine level from 5.0 to 13.0 g/kg diet (P < 0.05). The feed conversion ratio was reduced in a quadratic manner, estimating the optimum valine requirement at 11.7 g/kg diet (Tables 3 and 4 and Figure 1). Table 4: Adjusted regression equations, coefficients of determination and requirement values for the variables, digestible valine intake (DVI), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) of tambaqui with different body weights as a function of digestible valine levels in the diet. Body weight (g) Variable Model Equation R2 P>F Level (g/kg diet) 33 g DVI (g) Linear Ŷ = 37.014× + 3.9789 0.99 <0.0001 - WG (g) Linear Ŷ = 3.3043× + 22.752 0.94 0.0003 - SGR (% day–1) Linear Ŷ = 0.04× + 3.908 0.93 0.0002 - FCR (g g–1) Quadratic Ŷ = 0.030893 × 2 – 0.72657× + 6.0457 0.99 <0.0001 11.7 121 g DVI (g) Linear Ŷ = 48.941× + 0.7547 0.99 <0.0001 - WG (%) Quadratic Ŷ =-2.0702 × 2 + 38.261× – 58.831 0.87 0.00017 9.2 SGR (% day–1) Quadratic Ŷ =-0.00639 × 2 + 0.12061× + 3.5828 0.79 0.0068 9.4 FCR Quadratic Ŷ = 0.042604 × 2 – 0.80044× + 5.6765 0.90 0.0012 9.3 P > F-Significance of the “F” test of the analysis of variance. P > F-Significance of the “F” test of the analysis of variance. P > F-Significance of the “F” test of the analysis of variance. Figure 1: Graphic representation of feed conversion ratio of 33 g (solid line) and 121 g (dotted line) tambaqui fed with diets containing different levels of digestible valine. Figure 1: Graphic representation of feed conversion ratio of 33 g (solid line) and 121 g (dotted line) tambaqui fed with diets containing different levels of digestible valine. 8 SILVA, J. V. et al. maximize weight gain (45.3 g/kg of dietary protein). The data here were also expressed in g/kg of dietary protein because the red sea bream Pagrus major is a strictly carnivorous species and tambaqui is omnivorous. In addition, Abimorad et al. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION For body fat (%), a decrease was observed until the valine level of 10 g/kg of the diet with a subsequent increase in fat and no effect of valine supplementation on body fat deposition (P > 0.05). For dry matter, a linear reduction was observed (P < 0.05) in this trial (Tables 5, 6, 7 and 8 and Figure 2). Rahimnejad and Lee (2013) evaluated the digestible valine requirement of juvenile red sea bream Pagrus major and estimated 9.0 g/kg diet for the maximum weight gain, equivalent to 20.0 g/kg of dietary protein, which was lower than the dietary valine values mentioned in our study to Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights 9 9 Table 5: Mean values ±standard error of dry matter, protein, fat, moisture, and body ash contents of tambaqui with different weights as a function of digestible valine levels in the diet. Table 5: Mean values ±standard error of dry matter, protein, fat, moisture, and body ash contents of tambaqui with different weights as a function of digestible valine levels in the diet. Table 5: Mean values ±standard error of dry matter, protein, fat, moisture, and body ash contents of tambaqui with different weights as a function of digestible valine levels in the diet. RESULTS AND DISCUSSION Variable Body weight (g) Digestible valine levels (g/kg diet) Dry matter (%) Protein (%) Fat (%) Moisture (%) Ash (%) 33 g CD 30.00 ± 0.67 16.33 ± 0.18 10.69 ± 0.15 70.00 ± 0.67 3.23 ± 0.18 5.0 28.13 ± 1.21 12.42 ± 0.32 * 13.02 ± 0.41* 71.87 ± 1.21 2.69 ± 0.16 7.0 30.09 ± 1.27 16.04 ± 0.25 11.20 ± 0.25 69.91 ± 1.27 3.22 ± 0.30 9.0 26.50 ± 1.23 15.05 ± 0.41* 8.83 ± 0.47* 73.50 ± 1.23 2.86 ± 0.13 11.0 28.75 ± 0.27 18.21 ± 0.26* 7.51 ± 0.30 * 71.25 ± 0.27 3.03 ± 0.15 13.0 26.21 ± 0.62 15.92 ± 0.19 9.57 ± 0.13 73.79 ± 0.62 3.22 ± 0.17 P > F1 0.0803 <0.0001 <0.0001 0.8303 0.2598 Linear effect 0.1235 <0.0001 <0.0001 0.1235 0.1681 Quadratic effect 0.4134 <0.0001 <0.0001 0.4134 0.8300 SEp 1.91 0.56 0.62 1.91 0.38 121 g CD 39.95 ± 0.79 16.28 ± 0.46 19.08 ± 0.58 60.05 ± 0.79 4.59 ± 0.06 5.0 40.63 ± 0.82 15.74 ± 0.49 19.93 ± 0.66 59.37 ± 0.82 4.97 ± 0.25 7.0 40.50 ± 0.74 16.81 ± 0.23 18.93 ± 0.49 59.51 ± 0.74 4.75 ± 0.13 9.0 37.60 ± 1.08 19.11 ± 0.52* 13.50 ± 0.50* 62.41 ± 1.08 4.99 ± 0.33 11.0 37.61 ± 0.82 16.96 ± 0.41 15.49 ± 0.55* 62.39 ± 0.82 5.16 ± 0.30 13.0 37.89 ± 1.11 15.73 ± 0.45 17.08 ± 0.64 62.12 ± 1.11 5.08 ± 0.31 P>F1 0.0587 0.0004 <0.0001 0.0587 0.8589 Linear effect 0.0119 0.9300 0.0001 0.0119 0.4649 Quadratic effect 0.2983 <0.0001 <0.0001 0.2983 0.8406 SEp 1.80 0.87 1.14 1.80 0.49 CD – Control diet (D1+0.20% L-valine). P > F – Significance of the “F” test of the analysis of variance. Sep-Pooled standard error of the mean Table 6: Regression equations, coefficients of determination, and requirement values for variables fat, protein, and dry matter in tambaqui with different body weights as a function of the levels of digestible valine in the diet. Table 6: Regression equations, coefficients of determination, and requirement values for variables fat, protein, and dry matter in tambaqui with different body weights as a function of the levels of digestible valine in the diet. P > F-Significance of the “F” test of the analysis of variance. ( ) P > F – Significance of the “F” test of the analysis of variance. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION Body weight (g) Variable Model Equation R2 P>F Level (g/kg diet) 33 g Protein (%) Quadratic Ŷ =-0.1369 × 2 + 2.9239x+1.4031 0.72 <0.0001 10.7 Fat (%) Quadratic Ŷ = 0.15732 × 2 + 3.3613x+26.276 0.91 <0.0001 10.7 121 g Protein (%) Quadratic Ŷ = –0.16194 × 2 +2.9208x+4.9953 0.77 <0.0001 9.0 Fat (%) Quadratic Ŷ = 0.225 × 2 + 5.4067x + 37.519 0.73 <0.0001 10.0 Dry matter (g g–1) Linear Ŷ =-0.4189x + 42.613 0.70 0.0119 - P > F-Significance of the “F” test of the analysis of variance. P > F-Significance of the “F” test of the analysis of variance. Ciência e Agrotecnologia, 46:e002122, 2022 10 SILVA, J. V. et al. Table 7: Mean values ±standard error of body protein (BPD), fat (BFD), and ash (BAD) deposition and nitrogen retention efficiency (NRE) in tambaqui with different body weights as a function of digestible valine levels in the diet. retention efficiency (NRE) in tambaqui with different body weights as a function of digestible valine levels in the diet. RESULTS AND DISCUSSION Variable Body weights (g) Digestible valine BPD BFD BAD NRE levels (g/kg diet) (mg day–1) (mg day–1) (mg day–1) (%) 33 g CD 162.99 ± 6.82 155.68 ± 14.76 26.17 ± 3.41 40.24 ± 1.83 5.0 64.22 ± 7.91* 154.32 ± 7.45 10.19 ± 2.89 22.41 ± 2.39* 7.0 158.05 ± 13.56 153.13 ± 8.18 26.50 ± 7.44 40.46 ± 2.45 9.0 168.70 ± 12.70 128.76 ± 17.37 25.50 ± 5.29 34.34 ± 1.24 11.0 245.37 ± 6.87* 107.03 ± 7.12* 30.77 ± 3.52 41.93 ± 0.77* 13.0 213.96 ± 4.91* 158.80 ± 2.92 37.62 ± 2.27 29.43 ± 1.41* P>F1 <0.0001 0.0090 0.0120 <0.0001 Linear effect <0.0001 0.2511 0.0012 0.0148 Quadratic effect <0.0001 0.0099 0.4810 <0.0001 SEp 18.71 21.62 8.97 3.57 CD 281.66 ± 36.85 395.79 ± 13.06 55.82 ± 9.72 36.35 ± 1.00 121 g 5.0 151.70 ± 15.93* 314.42 ± 35.30 42.64 ± 9.02 28.06 ± 2.84* 7.0 266.90 ± 63.00 343.99 ± 76.32 53.07 ± 21.64 36.06 ± 2.32 9.0 492.54 ± 38.02* 160.66 ± 39.64* 95.43 ± 27.85 50.25 ± 2.33* 11.0 295.07 ± 48.42 193.20 ± 26.84* 78.43 ± 18.38 24.64 ± 1.54* 13.0 193.38 ± 27.90 232.33 ± 38.40 61.70 ± 16.56 13.77 ± 1.98* P>F1 0.0004 0.0626 0.3794 <0.0001 Linear effect 0.4135 0.0592 0.3238 <0.0001 Quadratic effect <0.0001 0.1974 0.1435 <0.0001 Sep 82.25 85.66 36.80 4.18 CD – Control diet (D1 + 0.20% L-valine). P > F – Significance of the “F” test of the analysis of variance. Sep-Pooled standard error of the mean. *Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). CD – Control diet (D1 + 0.20% L-valine).i CD Control diet (D1 0.20% L valine). P > F – Significance of the “F” test of the analysis of variance. gi Sep-Pooled standard error of the mean. f Table 8: Adjusted regression equations, determination coefficients, and requirement values for variables body protein deposition (BPD), body fat deposition (BFD), and body ash deposition (BAD), and nitrogen retention efficiency (NRE) in tambaqui with different body weights as a function of the level of digestible valine in the diet. ( ) P > F – Significance of the “F” test of the analysis of variance. l d d d f h Sep oo ed sta da d e o o t e ea . *Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). gi y Sep-Pooled standard error of the mean. f Significance of the F test of the analysis of variance. ooled standard error of the mean. f ror of the mean. umn differ from the control diet (CD) by the Dunnett test (P < 0.05). “F” test of the analysis of variance. RESULTS AND DISCUSSION Body Weight (g) Variable Model Equation R2 P>F Level (g/ kg diet) 33 g BPD (mg day–1) Quadratic Ŷ =-3.2942 × 2 + 78.634x – 244.47 0.92 <0.0001 11.9 BFD (mg day1) Quadratic Ŷ = 1.9384 × 2 – 36.748x + 298.62 0.50 0.0099 9.4 BAD (mg day–1) Linear Ŷ = 2.957x – 0.49 0.86 0.0012 - NRE (%) Quadratic Ŷ =-0.8465 × 2 + 16.012x – 35.06 0.71 <0.0001 9.5 121 g BPD (mg day–1) Quadratic Ŷ =-15.302 × 2 + 281.01x – 887.31 0.77 <0.0001 9.1 NRE (%) Quadratic Ŷ =-1.3845 × 2 + 22.921x – 52.511 0.79 <0.0001 8.2 P > F-Significance of the “F” test of the analysis of variance. Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights 11 Figure 2: Graphic representation of body protein deposition of tambaqui with 33 g (solid line) and 121 g (dotted line) fed diets containing different levels of digestible valine. Figure 2: Graphic representation of body protein deposition of tambaqui with 33 g (solid line) and 121 g (dotted line) fed diets containing different levels of digestible valine. Regarding body composition, the responses of BP, body protein deposition, and nitrogen retention efficiency to the increased dietary valine levels in tambaqui with an initial body weight of 33 g were a little higher than those reported for tambaqui with an initial weight of 121 g due to different growth rates, which is directly related to different depositions of muscle tissue. The optimum valine requirement for tambaqui (33 g and 121 g) in our study was lower than the dietary valine values reported for juvenile hybrid grouper (15.8 g/kg diet; Zhou et al., 2020), juvenile tilapia (12.7 g/kg diet; Xiao et al., 2018), Indian major carp (15.8 g/kg diet; Abidi; Khan, 2004). Despite differences in valine levels to maximize body protein deposition, studies have shown an improvement in protein deposition with dietary valine supplementation, suggesting that a better dietary AA balance probably prevents the selective catabolism of AAs and consequently increases protein synthesis (Tantikitti; Chimsung, 2001). increased. This result is consistent with previous reports of feeding trials with red sea bream (Rahimnejad; Lee, 2013) and Jian carp (Dong et al., 2013). The body fat deposition for tambaqui weighing 121 g was not affected by increased dietary valine; however, the highest body fat deposition was reported at the two lowest dietary valine levels. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION Only the tambaqui weighing 33 g showed a significant increase in body ash deposition with an increase in the dietary valine level. As previously discussed, a better balance of AAs improves the body’s protein deposition, leading to better use of dietary minerals for the formation of osseous tissues to support muscle tissues (Pianesso et al., 2015). A close functional relationship exists between osseous, and muscle tissues, Bomfim et al. (2020) evaluated the growth curve of tambaqui (C. macropomum) and reported that the maximum development of total length was reached at 243.4 days, whereas the maximum development of body weight was reached at 333.9 days, suggesting an earlier bone development than muscle tissue because that fish weight gain is associated with the accretion of proteins.i In addition, the quadratic nitrogen retention efficiency response to increasing levels of valine in the diet showed that at excessive levels, the percentage of retained nitrogen decreased, consequently increasing the excretion of nitrogen compounds into the environment and contributing to contamination and eutrophication of the aquatic environment (Furuya et al., 2013). The AA requirement for fish has been determined by the level that optimizes performance variables, mainly weight gain (NRC, 2011). However, weight gain consists of increases in proteins, lipids, minerals, and water (NRC, 2011), and there is no metabolic demand for AAs to maintain lipid and mineral reserves (Marchão et al., 2020). In this sense, body protein deposition is the most suitable variable to determine the AA requirement for fish (Bomfim et al., 2020; Lee et al., 2020; Silva et al., 2018; Costa Sousa et al., 2021), especially valine because it is used in greater proportion for protein synthesis (Rodrigues et al., 2018). A better balance of AAs in the diet can contribute to an increase in protein synthesis, with a concomitant decrease in the accumulation of lipid reserves (Tantikitti; Chimsung 2001), as demonstrated in our studies, in which the whole-body fat composition decreased with supplementation of dietary valine, and subsequently Ciência e Agrotecnologia, 46:e002122, 2022 12 SILVA, J. V. et al. AUTHOR CONTRIBUTION COWEY, C. B. Protein and amino acid requirements: A critique of method. Journal of Applied Ichthyology, 11(3-4):199-204, 1995. Conceptual idea: Silva, J.V.; Ribeiro, F.B.; Siqueira, J.C.; Bomfim, M.A.D.; Methodology design: Silva, J.V; Ribeiro, F.B; Siqueira, J.C.; Bomfim, M.A.D.; Marchão, R.S.; Data collect: Silva, J.V; Nascimento, D.C.N.; Marchão, R.S.; Data analysis and interpretation: Siqueira, J.C.; Silva, J.V; Ribeiro, F.B.; Writing and editing: Silva, J.V; Ribeiro, F.B.; Siqueira, J.C.; Nascimento, D.C.N.; Marchão, R.S. DIÓGENES, A. F. et al. Establishing the optimal essential amino acid ratios in juveniles of Nile tilapia (Oreochromis niloticus) by the deletion method. Aquaculture Nutrition, 22(2):435-443, 2016. DONG, M., et al. Growth, body composition, intestinal enzyme activities and microflora of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of dietary valine. Aquaculture Nutrition, 19(1):1-14, 2013. CONCLUSIONS BOMFIM, M. A. D. et al. Digestible tryptophan requirement for tambaqui (Colossoma macropomum) fingerlings. Revista Ciência Agronômica, 51(2):e20196724, 2020. The results of our study demonstrated that dietary valine provides optimal growth and increases the body protein of tambaqui with different body weights. Using a quadratic regression analysis of body protein deposition data, the optimum dietary valine requirement of tambaqui (33.0 g–83.0 g) and (121.0 g–277.0 g) was estimated to be 11.9 g/kg diet and 9.1 g/kg diet, respectively, corresponding to 4.53% of dietary crude protein. These values are within the range of valine requirements of similar fish species reported by the NRC, 2011. CASTILLO, S.; GATLIN, D. M. III. Dietary requirements for leucine, isoleucine and valine (branched-chain amino acids) by juvenile red drum Sciaenops ocellatus. Aquaculture Nutrition, 24:1056-1065, 2018. COSTA SOUSA, M. et al. Lysine requirements of tambatinga (♀Colossoma macropomum × ♂Piaractus brachypomus) fingerlings using different diet formulation techniques. Aquaculture Nutrition, 27(6):1825-1836, 2021. ACKNOWLEDGMENTS DONG, Z. J.; YUAN, X. H. The utilizations of heterosis in common carp in China. Aquaculture Asia, 12:14-15, 2002. The Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) for project financing. This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)-Brazil, Finance code 001. FIRMO, D. S. et al. Threonine to lysine ratio in diets of tambaqui juveniles (Colossoma macropomum). Semina: Ciências Agrárias, 39(5):2169-2180, 2018. FISHER, C.; MORRIS, T. R. The determination of the methionine requirements of laying pullets by a diet diluition technique. British Poultry Science, 11(1):67-82, 1970. REFERENCES ABIDI, S. F.; KHAN, M. A. Dietary valine requirement of Indian major carp, Labeo rohita (Hamilton) fry. Journal of Applied Ichthyology, 20(2):118-122, 2004. FURUYA, W. M. et al. Exigência de lisina digestível para a tilapia do Nilo de 87 a 226 g alimentada com dietas balanceadas para a relação arginina:lisina. Semina: Ciências Agrárias, 34(4):1945-1954, 2013. ABIMORAD, E. G. et al. Dietary digestible lysine requirement and essential amino acid to lysine ratio for pacu (Piaractus mesopotamicus). Aquaculture Nutrition, 16(4):370-377, 2010. FURUYA, W. M. Tabelas brasileiras para nutrição de tilápias. Toledo: GFM, 2010. 100p. AHMAD, I.; AHMED, I.; DAR, N. Dietary valine improved growth, immunity, enzymatic activities and expression ofTOR signaling cascade genes in rainbow trout, Oncorhynchus mykiss fingerlings. Scientifc Reports, 11:22089, 2021. INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA - IBGE. Pecuária, 2020. Available in: <https://cidades.ibge.gov.br/ brasil/ma/pesquisa/18/16459>. Access in: August, 10, 2021. ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS - AOAC. Official methods of analysis. 21.ed. Rockville, MD.: AOAC, 2019. 3390p. LEE, S. et al. 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https://openalex.org/W2898135192
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HIGH-TOOL – a strategic assessment tool for evaluating EU transport policies
Journal of shipping and trade
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© 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. HIGH-TOOL – a strategic assessment tool for evaluating EU transport policies E. Szimba1* , J. Ihrig2, M. Kraft2, K. Mitusch2, M. Chen3, M. Chahim3, J. van Meijeren3, J. Kiel4, B. Mandel5, A. Ulied6, E. Larrea6, G. De Ceuster7, R. Van Grol8, Z. Berki9, A. Székely9 and R. Smith10 * Correspondence: szimba@kit.edu 1Karlsruhe Institute of Technology (KIT), Network Economics, Institute of Economics (ECON), Waldhornstr. 27, Bldg. 01.96, 76131 Karlsruhe, Germany Abstract In this paper the strategic transport policy assessment instrument HIGH-TOOL is presented. The model has been developed for the European Commission, allowing policy-makers to identify the most advantageous transport policies and to strategically evaluate the impacts of transport policies on transport, environment and economy. Full list of author information is available at the end of the article The main innovation of this policy assessment tool lies in the integration of originally independently functioning models – i.e. passenger and freight demand, demography, and vehicle stock models, as well as economic, environmental and safety assessment models. With its traffic zones at the regional level of NUTS-2 and its aggregated view on the transport system, the instrument has a relatively lean structure avoiding runtime problems, without losing the spatial dimension. What distinguishes HIGH-TOOL from all other European transport policy assessment instruments: the model is an open source tool, it is freely available and does not require any commercial software to be run. In combination with its modular structure the HIGH-TOOL model can relatively easily be adjusted to other modelling methodologies or data. It can also comparatively easily be made responsive to “new” policies which are not in the scope of the current model version. Thus the HIGH- TOOL model lays the foundation for further innovations in the assessment of transport policies and mobility concepts. Keywords: Assessment tool, Transport policy, Transport demand modelling, Transport model, Impact assessment, European Union (2018) 3:11 (2018) 3:11 Szimba et al. Journal of Shipping and Trade https://doi.org/10.1186/s41072-018-0037-y Journal of Shipping and Trade Journal of Shipping and Trade Open Access Full list of author information is available at the end of the article Introduction and policy context Decisions on transport policy measures proposed by the European Union (EU) as ad- dressed by the White Paper on Transport (European Commission 2011a) have long-term and important impacts on economy, environment and society. Transport policy measures can lock up capital for decades and cause manifold external effects and indirect effects in many sectors – thus, policy measures may have a tremendous scope, especially if proposed at the European level. Various authors, such as Sieber et al. (2013), Nilsson et al. (2008) or McIntosh et al. (2011), emphasize the increasing importance of impact assessment tools as decision support instruments for policy making, allowing policy-makers to analyze relationships within a complex system and to reach decisions based on quantitative information. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 2 of 30 Page 2 of 30 In this context, the strategic assessment model “HIGH-TOOL” has been devel- oped to compute economic, environmental and social impacts of transport pol- icies, allowing European policy-makers to identify the most advantageous transport policies and to carry out strategic assessments of transport policies. HIGH-TOOL, which stands for “high-level strategic transport model”, addresses EU transport policies in the scope of the European Commission’s White Paper on Transport. The tool’s output indicators are largely aligned with assessment indica- tors of EU policy documents such as the EU’s Impact Assessment Guidelines (European Commission, 2009). The main objective of the HIGH-TOOL project has been the development of an open strategic transport policy assessment instrument for the European Commission, which is largely based on already existing tools, which has a low runtime and which provides a high level of user-friendliness. Within this setting, the HIGH-TOOL model integrates originally independently functioning models. This paper provides an overview of structure, modelling approach and application as- pects of the HIGH-TOOL model. Thus, the paper has a “synopsis” character, providing an overview perspective rather than a description of the modelling or validation ap- proach at a highly detailed level. The paper is structured as follows: Chapter 2 provides an overview of general tool features, structure of the model, and of the methodology behind the individual model- ling entities (modules). Chapter 4 summarizes the testing and validation approach. Chapter 5 addresses the scope of transport policy measures considered, the approach chosen to operationalize these policies, and presents the “hypernet” conception. Modelling philosophy Reflecting one of the key goals of the HIGH-TOOL project – i.e. the development of an assessment instrument that is largely based on already existing tools –, the HIGH-TOOL model integrates originally independently functioning models – i.e. pas- senger and freight demand, demography, and vehicle stock models, as well as eco- nomic, environmental and safety assessment models. This requires that already existing European state-of-the-art transport demand and assessment models are integrated by a common platform and are connected to a common database, relying on a common foundation, such as a common business-as-usual scenario, geographical scope, spatial entities, and demand segmentation. The contents of this chapter are partly based on Szimba et al. (2017), as well as on project reports by Szimba (2016), Mandel et al. (2016), and Van Grol et al. (2016). Introduction and policy context Chap- ter 6 presents a case study. The paper closes with chapter 7, the conclusions. General scope The HIGH-TOOL model has a global scope. However, the main focus is attached to Europe, and particularly to the Member States of the European Union. The spatial scope is the level of NUTS-21 for all EU Member States (EU28), Norway and Switzerland, NUTS-0 for EU neighboring countries, and country bundles for intercon- tinental transport. In total 314 modelling zones are considered. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 3 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 3 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 The tool’s timeline are 5-years steps from 2010 to 2050. The year 2010 is the base year of the HIGH-TOOL model. The HIGH-TOOL baseline or business-as-usual sce- nario is aligned with the EU Reference Scenario 2013 (European Commission 2013). Thus the forecasts of the HIGH-TOOL baseline are largely consistent with those of the EU Reference Scenario 2013. Passenger demand is differentiated by following modes: air, rail, road (passenger car and powered 2-wheelers), and long-distance coach. The urban demand sub-module additionally considers urban bus, urban tram/metro, cycling and walking. The demand differentiation by trip purpose covers business, private, vacation, and commuting trips. The freight transport is modelled by multi-modal transport chains, considering the transport modes air, rail, road, inland waterways, and maritime transport. The demand is considered for NST-2 commodities (52 commodity groups). The vehicle fleet is dis- tinguished by 60 vehicle types and 17 fuel types. The HIGH-TOOL model was largely developed in Java, thus ensuring platform inde- pendence. The User Interface was programmed as a stand-alone online application based on AngularJS and SailsJS, both free and open source software components pro- grammed in JavaScript. The HIGH-TOOL Database that stores input, output data as well as modelling parameters of all modules, is realized as a PostgreSQL database with PostGIS extension (Kiel et al. 2016b). Model structure The HIGH-TOOL model consists of three main elements: Core modules that represent the modelling framework; the Database that facilitates the exchange of data; and the User Interface for application of the model and providing access to the assessment re- sults. The basic structure follows the natural architecture common for most Decision Support Systems (DSS). The core modules are assembled such that a wide spectrum of specific transport topics can be addressed by the model while minimizing interdepend- encies in favor of short model runtime. In consequence, the number of core modules was finally condensed to seven and their internal structure was designed to allow for fast processing avoiding loops, while reflecting essential relations consistently. The core modules of the HIGH-TOOL model are constrained to these design features over de- tailed implementation of complex interdependencies, as the tool is outlined to provide decision support by testing policy scenarios for the European Union on a strategic level before the most promising ones are implemented in more detailed (network-based) models such as TRANSTOOLS or Vaclav. The core modules are as follows: Demography (DEM), Economy & Resources (ECR), Passenger Demand (PAD), Freight Demand (FRD), Vehicle Stock (VES), Environment (ENV), and Safety (SAF). In the center of the model are the passenger and freight de- mand modules (PAD and FRD). Since transport demand is driven (among others) by demographic and economic developments, DEM and ECR provide inputs for the de- mand modules. Based on transport demand, environmental and safety impacts are computed by ENV and SAF. To estimate economic impacts of transport policies, PAD and FRD provide demand data to ECR. As the composition of the vehicle fleets effects both transport costs and emission factors, VES provides inputs to the demand modules PAD and FRD, as well as to ENV. Figure 1 displays the detailed structure of the model, Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Page 4 of 30 Page 4 of 30 also depicting the structure within the core modules. The model structure is explained in detail, including the variables which are exchanged between the modules, by Mandel et al. (2016). The core modules interact sequentially with each other (see section “Module Interaction”). Fig. 1 Detailed structure of the HIGH-TOOL model Fig. 1 Detailed structure of the HIGH-TOOL model also depicting the structure within the core modules. Model structure The model structure is explained in detail, including the variables which are exchanged between the modules, by Mandel et al. (2016). The core modules interact sequentially with each other (see section “Module Interaction”). Demography module The Demography module (DEM) calculates the regional population and labor force in the 28 EU Member States as well as in Norway and Switzerland. UN projections (United Nations 2014) are used for other countries worldwide and are adapted to the geographic zoning system used in HIGH-TOOL. The population and labor force are derived from the Europop assumptions on demo- graphic trends (European Commission 2011b) at country level for the EU 28, Norway and Switzerland. These trends are aligned with the EU Reference Scenario 2013’s as- sumptions concerning fertility rates, life expectancies at birth and net migration Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 5 of 30 Page 5 of 30 (European Commission 2013). Net migration is not modelled explicitly; it is taken exogenously from the EU Reference Scenario. The projected population values from the EU Reference Scenario by country are disaggregated to geographic zones based on historical demographic trends concerning population development. This is done by using a demographic model which calculates future population by zone, based upon fertility rates and life expectancies. The population and labor force values from Europop are then disaggregated based upon the results from the demographic model. This allows consistent results with the Europop population trends. Kiel et al. (2016b) and Van Grol et al. (2016) contain a more detailed speci- fication of the demographic model and the calibrated values of the parameters and coefficients. Regional disaggregation of the population excluding migration is based on the 2010 historical regional distribution. Net migration is regionally distributed using a distribu- tion proxy based on income and employment rate. Labor force is estimated from the labor force percentage defined in the EU Reference Scenario and underlying assumptions. Being calibrated to the EU Reference Scenario 2013 and the projections by the United Nations (UN), the results of the Demography module are aligned with the expected economic development in Europe. However, the demographic develop- ment is an exogenous variable in HIGH-TOOL, i.e. the module is not sensitive to any policy. Figure 2 provides an overview of the structure of the Demography module. The Demography module’s most relevant data interchanges with other modules are population data at regional scale (NUTS-2), differentiated by age and gender cohort, which are input to ECR, VES and PAD. Furthermore, regional labor force data are pro- vided to ECR and VES. Fig. 2 Structure of the Demography module Fig. Demography module 2 Structure of the Demography module Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 6 of 30 Szimba et al. Journal of Shipping and Trade Economy & Resources Module The Economy & Resources module (ECR) comprises two components: Economy and Resources, as well as of the outputs GDP, Trade, Energy, Resources, and Production/ Distribution (see Fig. 3). The Economy & Resources module (ECR) comprises two components: Economy and Resources, as well as of the outputs GDP, Trade, Energy, Resources, and Production/ Distribution (see Fig. 3). The Economy sub-module estimates total output, capital stock and labor use in the economy, for which the general drivers (Gross Domestic Product (GDP), household in- come per capita and population) are – for an initial run without any policies – exogen- ously defined by the EU Reference Scenario 2013 (European Commission 2013). These drivers are disaggregated from country to zone based on ETISplus data (regional GDP, re- gional population, and labor force). The combined component (GDP, Trade, Energy, Re- sources, Production/ Distribution) estimates employment, trade, resource consumption, and purchasing power. The resources component calculates environmental indicators (without combustion) using the EXIOBASE database (Wood et al. 2015) for CO2, NOx, SOx, PM, biomass, fossil fuel use, metal use, mineral use, wood use, and water use. The methodology chosen for the Economy & Resources module is driven by a set of sequential algebraic equations, based on the first-order conditions for utility and profit maximization. The Economic module follows the algebraic framework of a (comput- able) general equilibrium model. The total output per region is generated using a Cobb-Douglas equation using total wages and total value of capital stock by region as inputs. Future trends in terms of growth in Gross Domestic Product and development of sectoral shares in Gross Value Added are obtained from the EU Reference Scenario 2013. The growth paths of economic and environmental indicators (e.g., intermediate inputs, CO2 emissions, water use or wood use) are driven by the total sectoral output trajectory. Moreover, projected demand is calculated as the sum of final demand of Fig. 3 Structure of the Economy & Resources module Fig. 3 Structure of the Economy & Resources module Fig. 3 Structure of the Economy & Resources module Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 7 of 30 Page 7 of 30 households and the intermediate demand of sectors (under the assumption of fixed Leontief technology). For modelling trade flows the following method is applied: For the base year, trade flows by country pairs in the base year are derived from the ETISplus database. Economy & Resources Module ETIS- plus estimates NUTS-2 inter-regional trade using national data from Eurostat statistics with its known limitation such as less detailed or missing data at regional level. Subse- quently, sectoral Gross Value Added data at NUTS-2 level (source: Eurostat) are ap- plied to scale down the country-level trade to the regional level. This creates different trade shares per commodity for each region. In this way the heterogeneity of each re- gion in a country is incorporated, i.e. not all regions export the same product mix. On the import side, the regional GDP share is applied to disaggregate the trade flows. In order to compute the share of intra-regional trade with respect to outgoing inter-regional trade, we combine RHOMOLO data (see Ivanova et al. 2011) on intra-regional trade (i.e. trade between NUTS-2 regions within in a country) with ETIS- plus inter-regional trade (i.e. trade between NUTS-2 regions of different countries) for regions where both data is available. The average commodity share of inter-regional trade is used to estimate the intra-regional trade for the remaining regions. By doing so, we implicitly assume that the freight handling factor (i.e. the frequency of lifts of the tons in the supply chain from the place of production to the place of consumption) for intra-regional trade is at least equal to the inter-regional trade. For intra-regional trade we assume that the share of a com- modity is equal to the average exporting share of a commodity to the different regions. For the assessment of economic impacts of a policy scenario the following effects are captured by the ECR module. The changes in monetary costs of freight transport (i.e. the cost differences between the policy scenario and the baseline), differentiated by mode and commodity, are trans- lated into changes in consumer prices. The consumer prices include both transport and trade margins as well as consumption taxes and subsidies. A decrease in transportation cost improves regional accessibility, which has a positive impact on the sector output at the regional level through provision of better access to labor markets and more varieties of intermediate goods. Next, the projected demand for goods and services in a region are calculated as the sum of the final demand of households and the intermediate demand of sec- tors. Economy & Resources Module We calculate regional intermediate demand as the sum of intermediate de- mand over all the sectors located in the region (under the assumption that intermediate goods and services are used in the production according to fixed Leontief technology). The regional intermediate demand is recalculated using the spending on the purchase of new vehicles in each sector and time period, new transport investment in Research and Development (RTD) and new infrastructure investments (these variables drive direct change in intermediate demand due to transport related investments). Finally, we assume that the household sector is driven by a Cobb-Douglas utility function (see, e.g. Ivanova 2014). Changes in the total costs of passenger transport have an impact on the level of disposable income of the households. Increases in passenger transportation costs reduce the amount of money available for purchasing of other goods and services, affecting also the household sector’s consumption of cars. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 8 of 30 Page 8 of 30 Changes in trade are modelled by the change in regional sectoral output between the baseline trajectory and the policy scenario (i.e. growth of sectoral output is applied to exports). Finally, the (new) Gross Domestic Product is derived at regional level on the basis of the variables calculated above as the sum of the value added of individual sectors plus taxes on final demand. The ECR module’s most relevant input data are regional population and labor force data provided by DEM, transport costs and transport demand by PAD and FRD, as well as vehicle tax revenue and vehicle stock data (purchase costs of new vehicles, vehicle stock by mode and fuel type) by VES. ECR’s main output to other modules are regional GDP per capital values for VES, trade flows in monetary values to FRD, and regional GDP, GVA and employment data for PAD. Vehicle stock module Journal of Shipping and Trade (2018) 3:11 Page 9 of 30 Page 9 of 30 (2018) 3:11 Szimba et al. Journal of Shipping and Trade Fig. 4 Structure of the Vehicle Stock module Fig. 4 Structure of the Vehicle Stock module Vehicle stock module The Vehicle Stock module (VES) converts passenger and freight demand to vehicle fleet size, which is disaggregated to vehicle type and vehicle age cohort for calculation of emissions and energy use. Vehicle types include propulsion and fuel technologies, and the module embraces 61 road and 12 non-road vehicle types. The vehicle age co- horts range from 0 to 29 years. Fleet stock forecasts are provided at country and region for each of the 28 EU Mem- ber States and for each period (5-year intervals) up to 2050. The module also delivers forecasts of average fixed (e. g., purchase costs, vehicle technology related costs) and variable costs (e.g., fuel costs, taxes, maintenance/repair costs, insurances, crew costs, cargo handling costs) for each vehicle type, and total tax revenue per country. Taking into account the transport demand and the vehicle stock in the previous period, as well as the vehicles that survived in current period, the demand for new vehi- cles and the average mileage per vehicle are calculated. The logit and the stock dynamic model inside the Vehicle Stock module use the cal- culated average generalized costs and behavioral aspects to define the shares of the dif- ferent types of new vehicles entering the market as well as their numbers. This calculation produces the detailed existing vehicle stock in the current period. The methodology underlying the Vehicle Stock module is aligned with TRACCS (Papadimitrio et al. 2013) and TREMOVE (De Ceuster et al. 2007). The structure of the module is shown by Fig. 4. There is no explicit distinction between company cars and private cars, but the effect is implicitly included in the decreasing annual mileage with increasing age, as well as by the different annual mileage for different vehicle types (e.g. higher annual mileage for diesel as these are more likely to be company cars). The module’s most important input data are population data by DEM, GDP per capita data by ECR, as well as demand data (vehicle-km, load factors/ car occupancy rates) by PAD and FRD. The VES module’s main outputs to other modules are vehicle tax revenue and vehicle stock data (purchase costs of new vehicles, vehicle stock by mode and fuel type) to ECR, vehicle stock by mode, fuel type and vehicle technology to ENV, as well as generalized fixed and variable costs per vehicle-km (differentiated by mode and fuel type) to PAD and FRD. Szimba et al. Passenger demand module The core Passenger Demand (PAD) module computes the number of passenger trips per year by transport mode for each origin/destination (O/D) relation at NUTS-2 level. It distinguishes the four main transport modes, rail, road, air, coach. PAD largely fol- lows the classical four-step approach in transport demand modelling of generation, dis- tribution, modal split and assignment (Ortúzar and Willumsen 2011). However, instead of the assignment step, the module translates the number of trips into transport per- formance indicators by the conversion step. The methodology of PAD is outlined by Fig. 5. It relies on a rather complex mathem- atical framework encompassing 44 equations that are described in detail by Van Grol et al. (2016). Considerably simplified, the computation of the O/D trip matrices (Tijm) car- ried out by the generation, distribution and the modal split models can be outlined as follows: Tijm ¼ T i  Aij X j Aij  Sijm Tijm ¼ T i  Aij X j Aij  Sijm where: Tijm Number of trips per year from origin i to destination j by transport mode m Ti Generated passenger trips in zone i Aij Accessibility of opportunities in destination j from origin i Sijm Market share of transport mode m of those trips from zone i to zone j. In the first step, the trip demand Ti is generated for each origin based on the popula- tion and specific trip rate factors. These trip rate factors are distinguished by year and Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 10 of 30 Szimba et al. Journal of Shipping and Trade Fig. 5 Structure of the Passenger Demand module Fig. 5 Structure of the Passenger Demand module by country under consideration of four trip purposes and eight age groups. They are computed by a regression approach that takes economic and demographic changes in European countries into account with the regression approach being calibrated for the first year 2010 based on ETISplus data (Szimba et al. 2013). For the forecast years and for the policy scenarios trip rate factors are dependent on changes of the explanatory variables GDP, employment, and income level. PAD is therefore sensitive to economic and demographic changes. In an intermediate step, four indicators are subsequently computed in order to inte- grate the trip distribution and the modal split model. Passenger demand module First, Generalized Travel Times (GTTijm) are computed by the cost functions based on travel impedance indicators, such as travel time, travel distance and level of service. All inter-zonal public transport modes (rail, air, coach) are considered by the GTT calculation as trip chains consisting of access mode, main mode and egress mode. GTTs follow the concept of travel time in which the cost unit refers to minutes rather than to monetary terms. Second, based on GTTs, Expected Minimum Costs (EMCij) and market shares of transport modes (Sijm) are computed by a Nested Logit model following the concept of logsums (De Jong et al. 2007). Third, Deterrence Values (DVij) are calculated by the deterrence model and in the last step, Accessibilities (Aij) are computed based on EMCs and DVs. Finally, Aij are used by the trip distribution model for the computation of the O/D trip matrix, and Sijm by the modal split model in order to divide the matrix by transport mode. The last modelling step is the conversion stage. In this step, transport performance indicators (passenger-kilometers and vehicle-kilometers) are derived, based on the O/D trip matrices, as well as travel distances and occupancy rates from ETISplus. In addition, road trips are split by car and powered two-wheelers, under the assumption of country-specific shares and motorization levels. Page 11 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 The core PAD module is complemented by two additional modules, the urban pas- senger demand, and the intercontinental air passenger sub-module. The urban passenger demand sub-module computes the number of urban trips per year at country level. It distinguishes six transport modes: Car, motorcycle, tram/ metro, bus, cycling, walking. Given that urban trips by car and motorcycle are a subset of the intra-zonal NUTS-2 trip demand modelled by the core PAD module, the urban sub-module is linked to the generation step of the core PAD module in order to ensure the overall consistency. The urban sub-module follows a generic, elasticity-based approach and consists of a generation, a modal split, and a conversion model. In the first step, the number of urban trips is generated for each NUTS-2 region based on “urban share factors” and trip rate factors. Passenger demand module The “urban share factors” contain the share of the urban population which are pre-calculated for each NUTS-2 region on the basis of GIS data. Also the trip rate factors are pre-calculated for each NUTS-2 region. They rely on trip purpose-specific trip rates derived from household surveys. In the second step, the market shares for each urban transport mode is estimated under application of a logit model for the three transport mode categories: private road, public transport and slow modes. Finally, the conversion model disaggregates the three mode categories into the six transport modes and computes aggregated demand indicators at country level. The intercontinental air passenger sub-module uses a regression-based approach to estimate the number of flights respectively demand between European regions and intercontinental destinations based on socio-economic data, regional attractors and network impedances. In general air transportation impedances consider cost structures such as CO2 taxation, national air transport charges or expenditures calculated by air- lines in respect of passenger rights. The split between European and intercontinental air transport mirrors the necessity to deal with the different regional granulation under- lying the assessment model’s purpose: while Europe is in the focus and modeled at re- gional level (NUTS-2), the other regions of the world are considered through a less detailed approach, i.e. 19 aggregated zones such as Far East, USA, Russia, or South-America. In order to avoid biased model results because of a highly different size of assumed traffic cells, air demand modeling has been split. The PAD module’s most important input data are population and labor force data at regional level by DEM, regional GDP, GVA and employment by ECR, as well as the ve- hicle stock and generalized fixed and variable costs per vehicle-km (differentiated by country, mode and fuel type) by VES. The module’s main outputs to other modules are transport demand (vehicle mileage, passenger-kilometers, number of passengers) by mode and region, as well as costs by region and origin/destination relation to ECR, transport demand and vehicle occupancy rates to VES, transport demand (vehicle mileage by O/D, origin and mode, and differ- entiated by urban and non-urban) to ENV and SAF. Figure 5 displays the structural overview of the Passenger Demand module. Freight demand module The Freight Demand (FRD) module uses national and international freight transport chains as a base. FRD consists of four components: trade conversion, distribution, Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 12 of 30 mode split and conversion (see Fig. 6). Together with the Economy & Resources mod- ule, it follows an approach analogue to the classical four-step methodology of gener- ation, distribution, modal split and assignment. The latter is replaced by calculation of performance indicators in the conversion component. The module delivers trade in value by production-consumption location (P/C), which is converted to volumes, applying volume density assumptions per O/D (related to legs in a chain between production and consumption location) and commodity (assumed constant over time) extracted from ETISplus (Szimba et al. 2013). The trade projections are produced by the Economy & Resources module (in Euro, 2010 constant values). The input from the Economy & Resources module is defined by sectors that (apart from transport sectors) match commodity types c. The trade projec- tions are used to calculate growth factors. As such the freight model can be regarded as a marginal model. The FRD applies the value/volume conversion method from NEAC-10 to ETISplus data to estimate the volumes in tons per year (Tt,i,j,c) by com- modity type c and production-consumption relation ij as follows: Tt;i: j:c ¼ aci∋i;cj∋j;c  bci∋i;cj∋j;c  cci∋i;cj∋j;c  V t;i;j;c where: Tit,i,j,c Freight trade flow of commodity c between production and consumption loca- tion i and j in time period t [tonnes] aci,cj,c Value-volume ratio for trade from production country ci to consumption coun- try cj per commodity type c [Euro] aci,cj,c Value-volume ratio for trade from production country ci to consumption coun- try cj per commodity type c [Euro] bci,cj,c Logistics factor for domestic trade when country ci is country c bci,cj,c Logistics factor for domestic trade when country ci is country cj cci,cj,c Logistics factor for intra-zonal trade when zone i is zone j. Fig. 6 Structure of the Freight Demand module Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 13 of 30 Page 13 of 30 Kiel et al. (2016a, 2016b) and Van Grol et al. (2016a) contain a more detailed meth- odology description of the freight demand model and the calibrated values of the model parameters. The distribution of mode chains follows a multinomial logit model and distributes the freight flow across multi-modal transport chains (routed through transshipment points) collected from the ETISplus database. In HIGH-TOOL a mode chain consti- tutes a multimodal routing from production location to consumption location through up to two transshipment points. The ETISplus database provides multimodal freight flows (in tons) per production-consumption location pair ij in the form of mode chains r using at most two transshipment points. The probability (Pt,I,j,c,r) of using a specific route r is calculated as follows: Pt;i;j;c;r ¼ eTCt;i; j;c;r X m∈M eTCt;i; j;c;r in which in which TCt;i;j;c;r ¼ β0 min costt;i;j;c;r TCt;i;j;c;r ¼ β0 min costt;i;j;c;r where: where: Pt,i,j,c,r|m Probability of choosing route r between consumption location i and produc- tion location j for commodity c in time period t give mode m [%] TCt,I,j,c,r Minimal total costs for route r between production location i and consump- tion location j for commodity c at time period t min_costt,i,j,r,c Minimum total cost of route r between consumption location i and pro- duction location j for commodity c in time period t β0 Constant β0 Constant r The route for a production-consumption location pair given the set of mode(s) m used to traverse it. The specific set of modes used in a route allows it to be differentiated from another route going through the same transshipment points. The modal combination with low- est costs for a certain route is used as a proxy for the attractiveness of all routes con- necting the production-consumption location pair ij. Here, the probability that a specific route is selected for a P/C pair ij is calculated by comparing the estimated costs for all available routes. The number of total tons as pro- vided in the previous step is applied on these routes to obtain route flows (Tt,i,j,c,r): Tt;i; j;c;r ¼ Tt;i; j;c;  Pt;i;j;c;r where: Tt,i,j,c,r Freight flow for commodity c from production location i to consumption loca- tion j in time period t using route r [tons] Pt,i,j,c,r Probability choosing route r between production location i to consumption lo- cation j for commodity c in time period t [%] Tt,i,j,c Freight flow for commodity c from production location i to consumption loca- tion j in time period t [tons]. The end product of this step is for each P/C pair ij a list of chains with allocated tons. Van Grol et al. (2016) provides a detailed description of the distribution of chains. The Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 14 of 30 mode split calculation is based on the approach applied in ETISplus and the NEAC-10 model (Newton et al. 2015). The cost functions and utilities in the modal split compo- nent rely on TRANSTOOLS v2. The modal split calculation estimates a modal share for each leg of the chains produced by the distribution step. For each of these legs the total leg costs are the summation of the costs encountered in each country traversed along the leg. Each origin and destination is connected by the transport routes extracted from the ETISplus database. These transport routes are part of transport chains that form a set of up to three legs that connect an origin and destination through up to two transship- ment regions. For each of the legs the modal split calculation is applied. The modal split component considers various cost elements such as fixed costs, variable costs, en- ergy costs, waiting costs, and loading/unloading costs. These are influenced by the VES module that can be affected by policy measures to compute generalized cost per avail- able mode connecting an origin and destination of a leg through a multinomial logit function according to TRANSTOOLS (Burgess et al. 2008; NEA 2007). Van Grol et al. (2016) provides a detailed description of the mode split model. where: The mode split model uses the following logit approach: Pmjcdij ¼ eV mjcij X m∈M eV mjcij Pmjcdij ¼ eV mjcij X m∈M eV mjcij with V mjcij ¼ αcmcmcij þ βcm where: Pm|cij Probability of choosing mode m given commodity group c, origin i and destin- ation j Pm|cij Probability of choosing mode m given commodity group c, origin i and destin- ation j Vm|cij Utility of mode m for commodity group c and O/D relation ij Vm|cij Utility of mode m for commodity group c and O/D relation ij Cmcij Costs for mode m given commodity group c and O/D relation ij αcm Logit parameter for commodity group c and mode m βcm Constant for commodity c and mode m m Set of available modes (road, rail, inland shipping, sea, other). Subsequently based on total generalized costs for routes connecting the trade rela- tion’s origin and destination, demand is distributed across the route’s chains connecting origin and destination through transshipment regions in the route choice component. As the model uses a hyper network connecting the different zones, the route choice is performed at an aggregated level. The changes in route choice are expressed as changes in time and/or distance which are translated into fixed, variable and energy costs. The translation of the chain matrices into unimodal demand matrices is fairly straight forward. Each chain comprises up to a maximum of three legs. Each leg contains an origin and destination. The origin or destination can be the production or consumption location, but they can also be the transshipment points. The chain matrix is split into different leg-matrices by commodity group and mode. This results in mode specific matrices by commodity group that can be used to calculate the ton-kilometers by mode and commodity. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 15 of 30 Page 15 of 30 The translation of the unimodal demand matrices in tons into trip matrices is done by using assumptions on the development of the average load per mode or vehicle and the share of empty trips. This has been calibrated for the base year, in order to achieve consistency with the amount of trips reported by statistics. The conversion step, i.e. where: the calculation of transport demand performance indicators (ton-kilometer and vehicle-kilometer), is conducted both for the origin region and re- lated to the “on the territory” perspective. The latter is calculated by applying the shares of distances of a leg per country, using data from ETISplus. Finally, assumptions on share of only-cargo flights and capacity of air freight trans- port are applied to extract air freight transport by only-cargo aircraft from the total de- mand for air transport. The module’s most important input data are regional trade data (differentiated by commodities) from ECR, and generalized fixed and variable costs per vehicle-km (dif- ferentiated by country, mode and fuel type) from VES. The module’s main outputs to other modules are transport demand by mode and commodity (vehicle mileage, ton-kilometers, tons carried) as well as transport costs by region and origin/destination relation to ECR, transport demand and vehicle occupancy rates to VES, transport demand (vehicle mileage by O/D, mode and commodity) to ENV and vehicle mileage by country and mode on the basis of the “on the territory” principle to SAF. Environment module The Environment (ENV) module calculates wheel-to-tank fuel consumption and emis- sions for each vehicle type. The key variables in this calculation are fuel consumption or fuel intensity, and emission factors or emission index. These factors are divided into technologies that are represented in the module by age cohort or vintage. The module produces estimates of CO2 emissions and five other pollutants: CO, VOC, NOx, SO2 and PM2.5. Fuel consumption and emissions are calculated per origin country and disaggregated to zones based on the share of transport demand in each zone. The module comprises of two parts (see Fig. 7). Firstly, the predicted transport de- mand segmented by country, mode and fuel type is disaggregated by vehicle type and vehicle technology (represented by the vehicle age cohort). Secondly, fuel consumption and emissions are derived and calculated for each mode, vehicle type, fuel, and age co- hort (technology) using the previously disaggregated transport demand, fuel consump- tion and emission factors. Data on fuel consumption and emission factors for all vehicle age cohorts (technol- ogy) are available for the year 2010. For each period in the remaining simulation period (2015–2050), only factors of the new vehicles (vehicles between 0 and 4 years old) are available in the dataset. These factors are modifiable to enable policy simulation, such as introduction of new emission standards in a specific time or simulation period. Fuel consumption and emission factors of older vehicles (vehicles more than 4 years old) are derived from the dataset for the previous period. The ENV module uses vehicle mileage data (differentiated by O/D and mode) from PAD and FRD. The vehicle mileage used by PAD differentiates between urban and Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Page 16 of 30 non-urban transport, while the FRD data differentiate by commodity. Furthermore, VES provides the vehicle fleet by country, mode, fuel type, vehicle type and vehicle technology. There are no data flows from ENV to any other core module. Fig. 7 Structure of the Environment module Fig. 7 Structure of the Environment module non-urban transport, while the FRD data differentiate by commodity. Furthermore, VES provides the vehicle fleet by country, mode, fuel type, vehicle type and vehicle technology. There are no data flows from ENV to any other core module. Safety module The Safety module (SAF) assesses the impact of transport policy measures on safety, and yields predictions of the number of fatalities and injuries, and associated social costs (see Fig. 8). The required input includes historical mobility data from the Data Stock, predicted mobility (from the Passenger and Freight Demand modules), and user input changes to safety risk and safety risk causal factors. Risk is defined as the number of occurrences (fatalities, injuries) per unit of mobility (in vehicle-kilometer or number of trips). The module distinguishes between road and non-road modes, that are subsequently dealt with at different levels of detail. Road safety is treated in the most detail and pre- dicts fatalities as well as serious and minor injuries. Road is further split into car, truck, powered two-wheelers, public transport, bike, and pedestrians. Non-road modes in- clude rail, air, short sea shipping, and inland waterways. The results are computed per country and time period. p p y p For each transport mode, there are two components. The first is the Business-as-Usual (BAU), which calculates safety risks and makes predictions based on risk trend lines (from historical mobility and safety data) and mobility predictions (from the Passenger and Freight Demand modules). The second is the scenario component that adapts the BAU risks according to the anticipated effect of safety measures mod- elled. The effect is derived from changes in accident causal factors (which are the policy inputs) and the elasticities and equations relating these to changes in risk. Safety pre- dictions for the scenario follow from these scenario risks and mobility predictions. Road fatalities, serious and minor injuries are predicted. For the other transport modes, Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 17 of 30 (2018) 3:11 Szimba et al. Journal of Shipping and Trade Fig. 8 Structure of the Safety module the focus is on fatalities. For all modes the social costs are calculated. The general ap- proach of adjusting risk trends based on changes in accident causal factors is based on the European Road Safety Action Program (ERSAP) (Delhaye et al. 2010). The SAF module uses vehicle mileage by country and mode on the basis of the “on the territory” principle from PAD and FRD. Furthermore, passenger demand data (pas- senger-kilometers, number of trips) per country, age group and gender, differentiated by urban and non-urban transport, are used from PAD. Safety module The module does not provide any data to other core modules. Module interaction The core modules interact sequentially with each other. The sequential solution reduces the computation loops, as results for a period t are passed to computations in t + 1. An iterative process would be much more time consuming as the modules would interact, re-compute, store and read data several times until the results for a certain time period become available and the model can move forward to the next time period. The sequence starts with DEM to produce demographic outputs for all forecast years 2015–2050. Subsequently ECR is run, fed by DEM results of time step t and by VES, PAD and FRD outputs of time step t-1. Afterwards VES is activated, on the basis of DEM/ECR (step t), and PAD/FRD (step t-1) outputs. Subsequently, PAD and FRD are run, using results from DEM/ECR/VES, and ECR/VES, respectively. Finally, results by PAD, FRD and VES are delivered for all years to ENV for the computation of the environmental impacts and by PAD and FRD to SAF for the computation of the safety impacts. The tool’s base year is 2010. Thus, the first time step 2015 is partly driven by 2010 results, and 2020 by 2015 results etc. The interaction scheme is shown by Fig. 9. Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Page 18 of 30 Fig. 9 Model execution order Fig. 9 Model execution order Page 19 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Model validation The HIGH-TOOL model was subjected to an extensive validation and testing ap- proach, consisting of: robustness tests to ensure that the model is capable of discover- ing invalid inputs; check of coherence of model outputs with the EU Reference Scenario 2013 (European Commission 2013); comparison of model outputs with the ASTRA-EC model; sensitivity tests to test the plausibility of the modules’ reactions on changes in input variables; and the conduction of case studies (see van Meijeren et al., 2016; Kiel et al. (2016a)). Since the results of the HIGH-TOOL baseline required consistency with the forecasts of the EU Reference Scenario 2013 (EU Ref), significant effort was spent to validate the HIGH-TOOL modules against this reference. The validation approach embraced both the validation of each module as stand-alone module and the validation of the inte- grated tool, in which all modules are interconnected with each other. As an example of validation outcomes, Table 1 compares the modelled rail passenger transport demand for the years 2010, 2020, 2030 and 2050 with the forecasts by the EU Reference Scenario 2013, while Table 2 shows the development of the total freight transport demand. Overall the validation analyses have revealed a high level of consistency between HIGH-TOOL results and the EU Reference Scenario 2013. Any major differences, which occur for a limited number of results, can be well explained, largely by differ- ences in data definition. Moreover, the differences between forecasts by HIGH-TOOL and the EU Reference Scenario are explained by the pattern that no information was accessible concerning how the policy measures underlying the EU Reference Scenario 2013 have been operationalized into the PRIMES model for the generation of the fore- casts. Thus also differences how the policies underlying the EU Reference Scenario 2013 have been operationalized by HIGH-TOOL and PRIMES, explain differences among the results. More details on validation approach and results are provided by van Meijeren et al. (2016). Transport policy measures Operationalization Transport Policy Measures (TPMs) are implemented in HIGH-TOOL by a specific set of policy levers (or input parameters of the HIGH-TOOL model) and associated values. To arrive at the set and the values for the policy levers several sources have been reviewed. These sources differ in the strength of their underpinning and the scalability of parameter values to the spatial level that is considered in HIGH-TOOL. The follow- ing sources have been reviewed (see Van Grol et al., 2016): Table 1 HIGH-TOOL results vs. EU Ref, rail passenger transport demand (million pkm) 2010 HIGH-TOOL 2010 EU Ref 2030 HIGH-TOOL 2030 EU Ref 2050 HIGH-TOOL 2050 EU Ref EU28 417,213 405,505 581,036 606,485 708,525 770,552 EU15 371,151 359,537 499,760 520,224 602,662 656,002 EU13 46,063 45,967 81,276 86,260 105,863 114,551 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 20 of 30 Table 2 HIGH-TOOL results vs. EU Ref, EU28 + 2 total freight transport demand (million tkm) 2010 HIGH-TOOL 2010 EU Ref 2030 HIGH-TOOL 2030 EU Ref 2050 HIGH-TOOL 2050 EU Ref EU28 2,239,665 2,312,415 3,175,986 3,199,693 4,102,472 3,652,618 EU15 1,663,948 1,718,291 2,279,797 2,305,708 2,871,405 2,616,397 EU13 575,717 594,124 896,189 898,386 1,231,067 1,042,883 Table 2 HIGH-TOOL results vs. EU Ref, EU28 + 2 total freight transport demand (million tkm) (1) Large-scale (European) model applications – TPMs that have been modelled in models such as SUMMA, EXPEDITE, and TRANSTOOLS. (1) Large-scale (European) model applications – TPMs that have been modelled in models such as SUMMA, EXPEDITE, and TRANSTOOLS. (2) Examples from practice – TPMs that have already been implemented in practice and are described in the literature. (3) Research publications – TPMs for which useful information was found in research publications. Most of these studies describe model simulations, field tests, surveys, or expert judgement. (4) Qualitative guidelines – TPMs for which a qualitative description of policy impacts is adapted from the ASSIST project. Under consideration of this review, one or more variables of the HIGH-TOOL model have been selected for each TPM, which translate a TPM’s effect on the transport sec- tor. For a majority of the TPMs, mode-specific user cost variables, i.e. elements of the generalized cost functions, have been selected to operationalize a TPM. Detailed information on how the different policy measures are operationalized is pro- vided by Mandel et al. (2016). Transport policy measures Operationalization In this project report each transport policy measure is categorized, and its general impact mechanism is drafted. Furthermore, the report pro- vides a documentation on which specific model variable(s) is (are) altered in order to operationalize a transport policy measure. Policy selection Instead of embracing a dedicated transport supply module, the HIGH-TOOL instrument’s supply parameters can be adjusted by the user either indirectly by the selection of transport policy measures or bundles, or directly by changing specific policy levers. Furthermore, the experienced user may conduct any change in the tool’s database to simulate specific policies. The HIGH-TOOL instrument offers 30 pre-defined Transport Policy Measures (TPM), which can be selected either individually or in combinations. The scope of the pre-defined Transport Policy Measures is shown by Table 3. Policies can be specified by the user in terms of intensity, temporal effectiveness (2015 to 2050) and geographical distribution (countries and regions in Europe). Also combinations of pre-defined TPMs can be applied. All combinations of TPMs have been analyzed in terms of interdependencies (see section “Consideration of policy bundles”). While the majority of the policies have been revealed to be additive, the user is informed by the system on the existence of interdependencies, if interdependent pol- icy combinations are chosen. Furthermore, customized policy packages can be defined using any combination of policy levers. The policy levers are organized per module. The number of levers per module are shown in Table 4. Page 21 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Szimba et al. Policy selection Journal of Shipping and Trade Table 3 Transport Policy Measures covered by HIGH-TOOL Category Single Pre-Defined Transport Policy Measures Efficiency standards and flanking measures Improving local public transport Deployment of efficient vehicles Replacement of inefficient LDVs and buses HDV limitation for urban areas LDV speed limit Diffusion of H2 fuel cell cars Diffusion of electro cars Replacement of inefficient cars Pricing CO2 feebates for road transport CO2 certificate system for road transport Circulation tax for cars Internalization of external costs HDV infrastructure change Urban road charging Research and innovation Intelligent road vehicles Dynamic traffic management for road Intelligent traffic information system for road Road vehicle safety technology protecting other transport users Safety systems for road vehicle users Internal market Acceleration of TEN-T implementation River information system European Rail Traffic Management System Harmonized handling of dangerous goods Harmonization of rail safety Harmonized social rules for truck drivers Opening the internal IWW market Enhance service quality at ports Maritime traffic management system Freight corridor management Single rail vehicle authorization and certification Finally, using the Expert Mode the user can edit input tables and/or the hypernet to control the impedances used in the model. The Expert Mode is an optional feature for advanced editing of the database values before running the model. Consideration of policy bundles When TPMs are combined their dependency is analyzed by reviewing a possible overlap in policy levers. If the sets of policy levers do not overlap, the TPMs are independent and their result additive. If the sets of policy levers do overlap, special care is needed in the implementation of policy lever values because these TPMs may not be completely independent. Depending on the nature of the involved TPMs, the combined effect of their policy levers might be as follows: Page 22 of 30 Page 22 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Table 4 Number of policy levers per module for the Customised Policy package interface Module Number of individual levers Economy and Resources 3 Vehicle Stock 430 Passenger Demand 100 Freight Demand 79 Environment 127 Safety 60 (1) Reduced – when TPMs are conflicting and cancel each other out; (2) Maximized – the effect of the combined TPMs equals the effect of the strongest TPM; (3) Limited – combining the TPM has some added effect, but this added effect is limited, and (4) Maximum – although the TPMs share the same policy levers they are independent and thus additive. (4) Maximum – although the TPMs share the same policy levers they are independent and thus additive. There are no pairs of TPMs that fall into the first group, while pairs in the last group can be freely combined by adding up their policy lever values. However, six groups of TPMs have been identified that share the same policy levers and are considered to be partly or largely overlapping. For the shared policy levers of these groups it has been defined how to model them simultaneously (see Van Grol et al., 2016). The hypernet approach With its requirement to provide assessment results quickly and at a strategic level, the HIGH-TOOL model is not a network-based transport model such as TRANSTOOLS. Nevertheless, the passenger and freight demand modules are based on transportation impedances at the level of NUTS-2 O/D relations. The impedances for the HIGH-TOOL base year 2010 are founded on ETISplus impedances, while the baseline’s impedances for the forecast years have been computed by the network-based transport model Vaclav for the infrastructure configuration underlying the EU Reference Sce- nario, and aligned with the impedances for the year 2010. This approach allows the modelling of infrastructure policies by modifying mode-specific travel impedances generically for transport in the whole EU, selected countries or selected NUTS-2 regions, which however does not allow the consideration of network effects of infrastructure policies. Therefore, in order to allow the consideration of network effects in the strategic as- sessment of transport infrastructure policies, a “hypernet” approach was developed. The hypernet approach allows the change of travel impedances on a virtual link be- tween two NUTS-2 regions. After running a shortest-path algorithm new impedance matrices are obtained, thus covering the network dimension of the infrastructure pol- icy. The hypernet is an optional submodule covering road and rail and is linked to the passenger demand module. For the creation of the hypernet, regions have been connected in the hypernet if: ei- ther the zones are bordering each other and have an ETISplus network link that Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 23 of 30 connects both zones, or they are connected by a ferry link in the ETISplus network. A representative NUTS-3 zone, i.e. the NUTS-3 zone containing the largest city/ capital city within the NUTS-2 zone, was chosen for each NUTS-2 zone. Each hypernet node is allocated an access/ egress impedance to model travel impedances to access the inter-regional hypernet links. The intra-zonal impedances are based on inter-NUTS-3 impedance values from ETISplus and can be influenced by policies. Also the inter-zonal impedances are based on the ETISplus impedance matrices. Future travel impedances up to the model’s forecast horizon 2050 were calculated from enhanced ETISplus networks, including all infrastructure projects planned on the TEN-T core and comprehensive networks (see European Union, 2013). The hypernet approach Figure 10 displays the constructed hyper-network links for road (ETISplus road net- work (red), Zone centroids (green) and constructed HIGH-TOOL road hypernet (blue) for the base year 2010). Case study description This case study examines the application of the hypernet facility of the HIGH-TOOL instrument. The assumption is made that rail passenger travel times will further de- crease by 10% along the “Magistrale” corridor Paris–Strasbourg–Karlsruhe–Munich– Vienna–Bratislava (see Fig. 11). The travel time decrease is assumed to be on top of the time savings due to TEN-T/CEF policies already in the baseline scenario. Thus, the in- vestment assumptions do not refer to concrete rail infrastructure projects, but are hypothetical. The infrastructure improvements are assumed to become effective in the year 2030. Further details on this case study are provided by Kiel et al. (2016b). Fig. 10 Scope of the hypernet (road) (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 24 of 30 Fig. 11 Map showing the hypernet rail links of the Magistrale corridor (blue), other hypernet rail links (red) and NUTS-2 zone centroids (green) Fig. 11 Map showing the hypernet rail links of the Magistrale corridor (blue), other hypernet rail links (red) and NUTS-2 zone centroids (green) Model results The model predicts an increase in rail passenger demand while the demand of Ac- knowledgements modes (road – i.e. private passenger cars –, coach and air) is expected to decrease (see Table 5). The results do not only reveal a mode shift effect, but also re- veal that the increase in rail passenger-kilometers exceeds the loss of passenger-kilometers by competing mode. Thus, the assumed improvement of rail level-of-service is expected to generate induced traffic that explains almost half of the additional rail traffic demand. This pattern is largely in line with the observations made for the French high-speed rail line Paris-Lyon (Vickerman 1996) and can be explained by direct and indirect effects, such as changes in travelers’ mobility behavior and the emerging of new activities due to the reduced travel time (Cascetta and Coppola 2013). The percentage changes in relation to the total passenger transport demand are rela- tively limited, which is explained by the limited geographical scope of the measures, as well as by the pattern that the assumed infrastructure improvements relate to inter-zonal passenger transport flows at the level of NUTS-2, which represent only a small share of the overall market. In 2050, the rail passenger demand is expected to increase by 0.02% for EU28 + 2, representing 4.4 million passenger-kilometers per day (1.6 Gpkm per year). The results of our rather hypothetical case study are largely in line with the findings of the “Magistrale corridor study” predicting an increase of about 14 million rail passenger trips per year due to upgrading this railway corridor (IWW et al. 2001). Regarding impacts on demand by mode of transport for 2050 at country level, the strongest impacts in absolute terms are expected for Germany, followed by France, Table 5 Case study – Total passenger-kilometres 2010–2050, by modes (in million), EU28 + 2, difference Scenario-Baseline 2030 2035 2040 2045 2050 Air −45 −55 −53 −55 −55 Rail 1306 1569 1534 1575 1608 Coach −35 −42 −43 −45 −47 Road −605 −743 −713 −734 −742 Table 5 Case study – Total passenger-kilometres 2010–2050, by modes (in million), EU28 + 2, diff S i B li Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 25 of 30 Page 25 of 30 Austria and Slovakia (see Fig. 12). These countries are the key beneficiaries of the as- sumed infrastructure investments. Model results Due to the network effects, which are covered by the hypernet approach, also the demand structures of other countries, which are not directly concerned by the investments – such as the Czech Republic, Italy and Romania – reveal slight impacts in favor of rail. The modal shift from road and air to rail leads to a decrease in fuel consumption, CO2 emissions and the emission of air pollutants (see Table 6). Furthermore, the modal shift results in a slight reduction in the number of road accidents (see Table 7). Finally, the HIGH-TOOL model predicts moderate economic impacts (see Table 8): the de- crease in rail passenger travel times results in savings in generalized costs and, thus, improves regional accessibility, which has a positive impact on Gross Value Added through provision of better access to labor markets and lower costs of intermediate goods (second order effects). The economic impacts do not embrace any first order ef- fects (direct or multiplier effects of the infrastructure investments), as the underlying investment amount has not been specified for the purposes of the case study. Conclusions The HIGH-TOOL model is an open source instrument, is both publicly and freely available and does not require any commercial software products to be run. These fea- tures clearly distinguish the HIGH-TOOL model from any other European transport demand model or policy assessment instrument. The model’s openness ensures thor- ough transparency of computations, and allows the experienced user to modify calcula- tion methodologies, data or model parameters. To develop the HIGH-TOOL model, originally independently functioning models have been integrated on a common platform. A key enabler of the development work in HIGH-TOOL was the European reference database ETISplus, covering a large share of data sets relevant for the models which were integrated in HIGH-TOOL. In this re- spect, HIGH-TOOL can be regarded as a logical consequence of the European Union’s strategy to provide not only publicly available data for transport policy and modelling – as accomplished by EU-funded projects such as ETIS-BASE and ETISplus –, but also to establish a publicly available open source tool for strategic policy assessment. Fig. 12 Impact on passenger transport demand by transport mode per country (year 2030, changes in millions pkm) Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 26 of 30 Table 6 Case study – Impact on emissions and fuel consumption p.a. (in tons), EU28 + 2, difference Scenario-Baseline Table 6 Case study – Impact on emissions and fuel consumption p.a. (in tons), EU28 + 2, difference Scenario-Baseline Year Fuel consumption CO2 NOx PM SO2 2030 −9963 −35,105 −54 −3 −1 2035 −9411 −33,449 −60 −3 0 2040 −9956 −34,320 −62 −3 0 2045 −10,253 −35,044 −65 −3 −1 2050 −8934 −31,284 −62 −3 −1 Also the existence of the EU Reference Scenario, which outlines long-term projec- tions until 2050 and covers aspects such as transport demand, energy consumption and vehicle fleet, was a substantial support for the calibration of the HIGH-TOOL model. On the other hand, it is becoming a broadly accepted view that there will be large structural changes in the (near) future in many economic sectors, also within transport and mobility (see e.g., Chen et al. 2016; Brynjolfsson and McAfee 2014; Blasi et al. 2013). The National Research Council of the USA (2010) states: “All forecasting meth- odologies depend to some degree on the inspection of historical data. Conclusions (million €), EU28 + 2, difference Scenario-Baseline Year Primary Secondary Tertiary 2030 0 0 0 2035 0.1 6.5 3.7 2040 0.1 7.0 4.1 2045 0.2 7.3 4.4 2050 0.1 7.4 4.6 Table 8 Case study – Impact on Gross Value Added by sector p.a. (million €), EU28 + 2, difference Scenario-Baseline application and improve the spatial representation of infrastructure-related policies. In- creasing the regional level of detail of traffic cells and connecting the HIGH-TOOL model with a network-based model however, imply unfavourable impacts on model run time, which needs to be avoided as far as possible by smart data handling and process- ing methodology. application and improve the spatial representation of infrastructure-related policies. In- creasing the regional level of detail of traffic cells and connecting the HIGH-TOOL model with a network-based model however, imply unfavourable impacts on model run time, which needs to be avoided as far as possible by smart data handling and process- ing methodology. In combination with an enhanced approach to model network effects of transport policies, a dedicated transport supply module could be implemented to consolidate the representation of the supply side (including the operationalization of policies) in one entity. Moreover, the increasing relevance of Sharing Economy concepts in transport (e.g., car/bike sharing; ride sharing) calls for a more sophisticated and explicit consideration of these schemes by transport demand and policy assessment tools. Further mega trends in transport are electrification and autonomous driving. The electrification of the road transport sector indicates the requirement to connect transport demand and energy modelling more closely, in order to obtain a better understanding of interdependencies between these sectors and to explore the po- tential of electrification of transport for decentral energy supply concepts (e.g., by using electric vehicles as mobile energy storage). Autonomous driving will result in tremendous impacts on the transport sector by enhancing access to mobility, im- prove safety, and add the potential to alleviate congestion, reduce travel time and reduce environmental impacts (see e.g., Harper et al. 2016; Fagnant and Kockelman 2014; Hars 2014). Though it remains unclear how much of the potential benefits will actually be exploited, since vehicle mileage is expected to increase (Szimba and Orschiedt 2017). Conclusions Supported by further research on travel behaviour, traffic engin- eering and the scope of business models expected from autonomous driving, an extended version of the HIGH-TOOL model could be used to estimate the impacts of autonomous driving at European scale. Finally, the consideration of future modes of transport such as drones or the Hyper- loop concept may provide avenues for model enhancement. With its high degree of openness and its modular structure, the HIGH-TOOL model provides the basis for an efficient further development of the model in the indicated di- rections and beyond, and thus lays the foundation for further innovations in the realm of assessment of transport policies and mobility concepts. Conclusions However, exclu- sive reliance on historical data inevitably leads to an overemphasis on evolutionary innovation and leaves the user vulnerable to surprise from rapid or nonlinear develop- ments. […] A methodology that can forecast disruptive technologies must overcome the evolutionary bias and be capable of identifying unprecedented change”. Also the currently instable political and geo-political situation in many world regions, wars and migration cause a high level of uncertainty in terms of future develop- ment of demographic, social, societal and socio-economic patterns which substan- tially influence transport demand. However, the official national and EU forecasts currently tend to suffer from a lack of adapted methodology to be able to antici- pate for the future in which direct and indirect impacts of disruptive technologies will play a major role. Thus, further research is needed to develop a generally ac- cepted, trusted, transparent and repeatable approach that does not solely rely on historical developments, but which allows to deal with breaks in trends and derived developments. The current version of the HIGH-TOOL model offers various possibilities for further developments: for instance, enhancing the zoning system of transport demand model- ling from the spatial level of NUTS-2 to NUTS-3 will significantly reduce the share of intra-zonal transport demand and increase the accuracy of transport demand model- ling. Also, a closer link to a network-based model, which goes beyond the currently im- plemented hypernet approach for passenger transport, will enhance the tool’s scope of Table 7 Case study – Impact on road accidents p.a. (number of injured persons), EU28 + 2, difference Scenario-Baseline Table 7 Case study – Impact on road accidents p.a. (number of injured persons), EU28 + 2, difference Scenario-Baseline Year Serious injuries Slight injuries 2030 −9 −67 2035 −5 −77 2040 −9 −69 2045 −11 −82 2050 −8 −73 Table 7 Case study – Impact on road accidents p.a. (number of injured persons), EU28 + 2, difference Scenario-Baseline Year Serious injuries Slight injuries 2030 −9 −67 2035 −5 −77 2040 −9 −69 2045 −11 −82 2050 −8 −73 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 27 of 30 Page 27 of 30 Table 8 Case study – Impact on Gross Value Added by sector p.a. 1NUTS: Nomenclature of Units for Territorial Statistics Competing interests The authors declare that they have no competing interests. Funding The research has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no° 321624. Declaration I confirm that I have read SpringerOpen’s guidance on competing interests and have included a statement indicating that none of the authors have any competing interests in the manuscript. Abbreviations AngularJS: A client framework to build web applications in JavaScript; BAU: Business as usual; CEF: Connecting Europe Facility; CO2: Carbon Dioxide; DEM: Demography module of the HIGH-TOOL model; DV: Deterrence Value; ECR: Economy & Resources module of the HIGH-TOOL model; EMC: Expected Minimum Cost; ENV: Environment module of the HIGH-TOOL model; ERSAP: European Road Safety Action Program; EU Ref: EU Reference Scenario 2013; EU: European Union; EU28 + 2: 28 Member States of the European Union plus Norway and Switzerland; EU28: 28 Member States of the European Union; Eurostat: Statistical Office of the European Union; FRD: Freight Demand module of the HIGH-TOOL model; GDP: Gross Domestic Product; Gpkm: Giga passenger-kilometer; GVA: Gross Value Added; HDV: Heavy Duty Vehicle; LDV: Light Duty Vehicle; NOx: Nitric oxide and nitrogen dioxide; NST: Eurostat’s Standard Goods Classification for Transport Statistics; NUTS: Nomenclature of Territorial Units for Statistics; A number assigned to it defines the level of granularity, such as 0 for the country level; O/D: Origin/ Destination; P/C: Production/ Consumption; PAD: Passenger Demand module of the HIGH-TOOL model; PostGIS: An open source software program that adds support for geographic objects to the PostgreSQL object-relational database; PostgreSQL: An object- relational database; RTD: Research and Development; SAF: Safety module of the HIGH-TOOL model; Sails.js: A server framework to build Node.js applications; SOx: Sulfur oxide; TEN-T: Trans-European Networks for Transport; TPM: Transport Policy Measures Acknowledgements Th h ld lik The authors would like to thank Hettie Boonman, Tina Laparidou and Cristian Lorenzo Martínez for their dedication for contributing to the development of the tool. The authors are also grateful to the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. Endnotes 1NUTS: Nomenclature of Units for Territorial Statistics Page 28 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Availability of data and materials y The (co-)author(s) declare that the tool presented in this paper (and the involved data) is publicly available and can be requested from the HIGH-TOOL homepage (www.high-tool.eu). Authors’ contributions All authors have contributed to the conception and design of the presented model, the development of the tool and involved data collection, analysis and interpretation. Under coordination by the corresponding author, all authors have been involved in the drafting of the article, and the critical revision of the paper. All authors have approved the version to be published. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 15 January 2018 Accepted: 14 October 2018 Received: 15 January 2018 Accepted: 14 October 2018 Received: 15 January 2018 Accepted: 14 October 2018 Author details 1 1Karlsruhe Institute of Technology (KIT), Network Economics, Institute of Economics (ECON), Waldhornstr. 27, Bldg. 01.96, 76131 Karlsruhe, Germany. 2Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. 3Netherlands Organisation for Applied Scientific Research TNO, The Hague, The Netherlands. 4Panteia, Zoetermeer, The Netherlands. 5MKmetric Gesellschaft für Systemplanung mbH, Karlsruhe, Germany. 6MCRIT S.L., Barcelona, Spain. 7Transport & Mobility Leuven NV (TML), Leuven, Belgium. 8Significance, The Hague, The Netherlands. 9Főmterv, Budapest, Hungary. 10E-mergo, Delft, The Netherlands. 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Panteia, Zoetermeer Nilsson M, Jordan A, Turnpenny J, Hertin J, Nykvist B, Russel D (2008) The use and non-use of policy appraisal tools in public policy making: an analysis of three European countries and the European Union. Policy Science 41:335–355. https://doi. org/10.1007/s11077-008-9071-1 g Ortúzar JD, Willumsen LG (2011) Modelling transport, 4th edn. John Wiley & Sons Ltd, Chichester Papadimitrio G, Ntziachristos L, Wüthrich P, Notter B, Keller M, Fridell E, Winnes H, Styhre L, Sjödin A (2013) Transport data collection supporting the quantitative analysis of measures relating to transport and climate change Project acronym: TRACCS. References Blasi J, Freeman R, Kruse D (2013) The citizens share, reducing inequality in the 21st century. Yale University Press, New Haven & London Brynjolfsson E, McAfee A (2014) The second machine age. W.W. Norton & Company, New York Burgess A, Chen T, Snelder M, Schneekloth N, Korzhenevych A, Szimba E, Kraft M, Krail M, Nielsen O, Hansen C, Martino A, Fiorello D, Christidis R (2008) Final report TRANS-TOOLS. In: deliverable D6. Funded by the 6th framework RTD Programme, Delft. Available under http://www.transport-research.info/sites/default/files/project/documents/20100304_ 172116_94411_TRANSTOOLS%20-%20Final%20Report.pdf Accessed 08 May 2018. Cascetta E, Coppola P (2013) High speed rail (HSR) induced demand models. Procedia - Soc Behav Sci 111:147– Chen M, Bodea G, Huijboom N (2016) Anticipating EU transport sector governance. Paper presented at FORMForum 201 Brussels De Ceuster G, van Herbruggen B, Ivanova O, Carlier K, Martino A, Fiorello D (2007) TREMOVE: service contract for the further development and application of the transport and environmental TREMOVE model. Transport and Mobility Leuven. De Ceuster G, van Herbruggen B, Ivanova O, Carlier K, Martino A, Fiorello D (2007) TREMOVE: service contract for the further development and application of the transport and environmental TREMOVE model. Transport and Mobility Leuven. Page 29 of 30 Page 29 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Available under http://www.tmleuven.be/methode/tremove/Final_Report_TREMOVE_9July2007c.pdf Accessed 08 May 2018 De Jong G, Daly AJ, Pieters M, van der Hoorn T (2007) The logsum of an evaluation measure: review of the literature and new results. Transp Res A 41:874–889 p Delhaye E, Akkermans L, De Ceuster G, Vanhove F, Bosetti S (2010) Lot 2 Impact Assessments and Evaluations in the field of transport. The preparation of the European Road Safety Action Program 2011–2020. Not publicly available European Commission (2009) Impact Assessment Guidelines SEC(2009) 92. Available under. htt European Commission (2009) Impact Assessment Guidelines SEC(2009) 92. Available under. ht regulation/impact/commission guidelines/docs/iag 2009 en.pdf. Accessed 8 May 2018 pact/commission_guidelines/docs/iag_2009_en.pdf. Accessed 8 May 2 regulation/impact/commission_guidelines/docs/iag_2009_en.pdf. Acc y European Commission (2011a) Roadmap to a Single European Transport Area - Towards a competitive and resource efficient transport system COM(2011) 144 final. Available under http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX: 52011DC0144&from=EN. Accessed 08 May 2018. European Commission (2011b) The 2012 Ageing Report: Underlying Assumptions and Projection Methodologies. Eur Econ 4(2011). https://doi.org/10.2765/15373 European Commission (2013) EU energy, transport and GHG emissions trends to 2050. Reference scenario 2013. Available under https://ec.europa.eu/transport/sites/transport/files/media/publications/doc/trends-to-2050-update-2013.pdf Accessed 08 May 2018. 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Transp Res Procedia 25:2843–2860. https://doi.org/10.1016/j.trpro.2017.05.262 structure of the tool and key features. Transp Res Procedia 25:2843 2860. https://doi.org/10.1016/j.trpro.2017.05.262 Szimba E, Orschiedt Y (2017) How beneficial is fully automated driving in urban areas from a socio-economic point of view? Proceedings of the conference Future City 2017: urban sustainable development and mobility. University of Transport and Communications, Hanoi Van Grol R, De Bok M, De Jong G, Van Eck G, Ihrig J, Kraft M, Szimba E, Mandel B, Ivanova O, Corthout R, Purwanto J, Smith R, Laparidou K, Helder E, Grebe S, Székely A (2016b) Elasticities and Equations of the HIGH-TOOL model (Final Version). In: HIGH-TOOL Deliverable D4.3. References Project co-funded by the European Commission under the 7th Framework Programme, Karlsruhe Available under http://www.high-tool.eu/public_content/downloads/HT_D_4_3_v1_2.pdf Accessed 08 May 2018 Van Grol R, De Bok M, De Jong G, Van Eck G, Ihrig J, Kraft M, Szimba E, Mandel B, Ivanova O, Corthout R, Purwanto J, Smith R, Laparidou K, Helder E, Grebe S, Székely A (2016b) Elasticities and Equations of the HIGH-TOOL model (Final Version). In: HIGH-TOOL Deliverable D4.3. Project co-funded by the European Commission under the 7th Framework Programme, Karlsruhe Available under http://www.high-tool.eu/public_content/downloads/HT_D_4_3_v1_2.pdf Accessed 08 May 2018 van Meijeren J, Davydenko I, Chahim M, Szimba E, Kraft M, Ihrig J, Smith R, Laparidou T, Purwanto J, Corthout R (2016) Validation by Coherence Checks, HIGH-TOOL Deliverable D8.1, project co-funded by the European Commission under the 7th Framework Programme, Karlsruhe g Vickerman R (1996) High-speed rail in Europe: experience and issues for future development. Ann Reg Sci 31(1):21–38. https://doi.org/10.1007/s001680050037 Wood R, Stadler K, Bulavskaya T, Lutter S, Giljum S, de Koning A, Kuenen J, Schütz H, Acosta-Fernández J, Usubiaga A, Simas M, Ivanova O, Weinzettel J, Schmidt JH, Merciai S, Tukker A (2015) Global sustainability accounting-developing EXIOBASE for multi-regional footprint analysis. Sustainability 7(1):138–163. https://doi.org/10.3390/su7010138 Wood R, Stadler K, Bulavskaya T, Lutter S, Giljum S, de Koning A, Kuenen J, Schütz H, Acosta-Fernández J, Usubiaga A, Simas M, Ivanova O, Weinzettel J, Schmidt JH, Merciai S, Tukker A (2015) Global sustainability accounting-developing EXIOBASE for multi-regional footprint analysis. Sustainability 7(1):138–163. https://doi.org/10.3390/su7010138
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De novo sequencing of the transcriptome reveals regulators of the floral transition in Fargesia macclureana (Poaceae)
BMC genomics
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De novo sequencing of the transcriptome reveals regulators of the floral transition in Fargesia macclureana (Poaceae) Ying Li  International Center for Bamboo and Rattan https://orcid.org/0000 Chunxia Zhang  Nanjing Forestry University Kebin Yang  International Center for Bamboo and Rattan Jingjing Shi  International Center for Bamboo and Rattan Yulong Ding  Nanjing Forestry University Zhimin Gao  (  gaozhimin@icbr.ac.cn ) https://orcid.org/0000-0003-4464-7159 Ying Li  International Center for Bamboo and Rattan h Chunxia Zhang  Nanjing Forestry University Kebin Yang  International Center for Bamboo and Rattan Jingjing Shi  International Center for Bamboo and Rattan Yulong Ding  Nanjing Forestry University Zhimin Gao  (  gaozhimin@icbr.ac.cn ) https://orcid.org/0000-0003-4464-7159 Research article Keywords: Transcriptome, Floral transition, Bamboo, Qinghai–Tibet Plateau Posted Date: January 3rd, 2020 DOI: https://doi.org/10.21203/rs.2.10521/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on December 30th, 2019. See the published version at https://doi.org/10.1186/s12864-019-6418-2. Page 1/27 Abstract Background: Fargesia macclureana (Poaceae) is a woody bamboo species found on the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3,800 m above sea level. It rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. To date, little is known regarding the molecular mechanism of bamboo flowering, and no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we report the first de novo transcriptome sequence for F. macclureana to investigate the putative mechanisms underlying the flowering time control used by F. macclureana to adapt to its environment. Results: Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, assembled into 99,056 unigenes. A comprehensive analysis of the broadly, specifically and differentially expressed unigenes (BEUs, SEUs and DEUs) indicated that they were mostly involved in metabolism and signal transduction, as well as DNA repair and plant-pathogen interactions, which may be of adaptive importance. In addition, comparison analysis between non-flowering and flowering tissues revealed that expressions of FmFT and FmHd3a, two putative F. macclureana orthologs, were differently regulated in NF- vs F- leaves, and carbohydrate metabolism and signal transduction were two major KEGG pathways that DEUs were enriched in. Finally, we detected 9,296 simple sequence repeats (SSRs) that may be useful for further molecular marker-assisted breeding. Conclusions: F. macclureana may have evolved specific reproductive strategies for flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. Our findings will provide new insights to future investigations into the mechanisms of flowering time control and adaptive evolution in plants growing at high altitudes. Background The flowering time is of crucial importance to ensure the reproductive success of flowering plants. Previous results have indicated that the floral transition is orchestrated by several parallel and interactive genetic pathways that are regulated by a variety of environmental and endogenous signals [1]. Many key genes and regulatory networks have been identified in herbaceous annual plants such as Arabidopsis [2, 3], rice [4], gourds [5], potato [6] and sorghum [7]. However, much less is known about such regulation in perennial plants. Despite the increasing attention on perennial dicotyledonous woody plants such as poplar [8, 9], eucalyptus [10] and citrus [11] species, to date, the molecular mechanism underlying floral regulation in monocotyledonous woody plants remains elusive. Furthermore, previous studies investigated flowering mainly by artificially altering the external signals (e.g. photoperiod and light intensity) and did not assess the impact of the original environment on the adaptive evolution of species-specific reproductive strategies. Bamboo plants are an important group in the Bambusoideae subfamily of the monocotyledonous Poaceae. They exhibit a wide degree of variation in the timing (1-120 years) and nature (sporadic vs. gregarious) of flowering among species [12]. Sporadic flowering involves flowering in only a few isolated clumps, which set little or no seed and usually remain alive afterward [13]. In contrast, gregarious flowering involves all individuals of a species regardless of age and/or location within and among the populations at the same time, which is usually followed by death and seed setting [14]. And the simultaneous death of many individuals triggers serious ecological consequences, including changes in the population dynamics of neighboring plants, differences in soil properties, various effects on endangered animals that depend on bamboo [15], and the knock-on effects on human economies in many parts of the world [16]. Therefore, dissecting the regulators that Page 2/27 Page 2/27 control the unique life history of bamboo may be of use for plant ecology and human society. However, to date, little is known regarding the molecular mechanisms of bamboo flowering, in part because of the sporadic occurrence of these flowering episodes and the long intervals between events. control the unique life history of bamboo may be of use for plant ecology and human society. However, to date, little is known regarding the molecular mechanisms of bamboo flowering, in part because of the sporadic occurrence of these flowering episodes and the long intervals between events. Background Many genes have been identified as regulators of reproductive development in different bamboo species, including the MADS-box transcription factors [17-19], CONSTANS (CO) [20] and FLOWERING LOCUS T (FT) [21], among others. In addition, studies of sequenced transcriptomes have identified microRNAs related to floral development [22-24]. However, samples collected in these analyses were limited to mature spikelets or to different spikelets at different development stages. Thus, it is likely that dynamic changes in genes occurring at different development stages may be missing. In addition, the specific response of particular tissues to internal and external cues and how plants integrate these signals to regulate different phases of reproductive development (including the floral transition, florigen transport, and floral organ specification) has not yet been elucidated in bamboo. Furthermore, no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we took advantage of an unexpected flowering event in highland arrow bamboo, Fargesia macclureana [25], and performed the first de novo transcriptome analysis. This transcriptome includes data from six different tissues collected at different development stages, including inflorescences in the initial and peak flower stage (I- and P- spikelets), branchlets, and leaves from both flowering and non-flowering bamboo plants (F/NF-branchlets and F/NF -leaves). F. macclureana is a woody bamboo species found in areas 2,000 ~ 3,800 m above sea level on the Qinghai–Tibet Plateau (QTP) (Fig. 1), which is the highest and largest plateau in the world. The growth environment of the QTP is characterized by low temperature and low oxygen availability, reduced pathogen incidence, and intense radiation [26]. F. macclureana rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. Our goal is to use the transcriptomic data to gain a deeper understanding of the mechanisms underlying the control of flowering time and the adaptation of F. macclureana to the complex extreme conditions of the QTP. On one hand, we expect to detect regulatory hubs involved in the flowering mechanisms. On the other hand, we aim to discover signs of the adaptive evolutionary changes in F. macclureana in response to the harsh environmental conditions in the QTP, which may, in turn, provide a broader insight into the adaptive mechanisms for plants that grow at high altitudes. De novo transcriptome assembly yielded 99,056 unigenes Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, including 471,537,304 clean reads in 18 unique samples (Additional file 1: Table S1). The average Q20 (sequencing error rate less than 1%) and Q30 (sequencing error rate less than 0.1%) percentages were 95.64% and 89.95% respectively. The GC content of all samples ranged from 53.78% to 55.86%, with an average of 54.81%. Sample data were assembled into 289,122 transcript scaffolds, with an N50 and average length of 1,765 bp and 1,183 bp, respectively. The final de novo assembly included 99,056 unigenes, with an N50 and average length of 1,587 bp and 926 bp, respectively. Among these unigenes, 71.02% (70,354) were shorter than 1,000 bp and 12.06% (11,950) were longer than 2,000 bp (Table 1). Page 3/27 Page 3/27 Page 3/27 Page 3/27 Most unigenes were functionally annotated and classified A total of 47,306 unigenes were annotated (Additional file 2: Table S2). Of these, 45,516 (96.22%) unigenes were found to encode products that showed significant similarity to characterized proteins in the non-redundant protein sequence database (Nr) at an E-value threshold of 10-5 (Table 2). We also found that 7,027 (15.45 %) unigenes showed similarity to genes found in rice, 11.33% were similar to those found in Brachypodium distachyon, and we also found a significant proportion of the unigenes that were similar to those found in Setaria italica, Oryza brachyantha, and Zea mays (Fig. 2a). We identified 24,847 (52.52%), 28,317 (59.86%) and 43,909 (92.82%) unigenes that showed significant matches to entries in the Swiss-Prot, Pfam, and eggnog databases, respectively (Table 2). Many unigenes expressed in the F. macclureana transcriptome were functionally annotated as regulators of plant responses to evolutionarily important phenotypes, including membrane stabilization, heat stress response and pathogen defense (Additional file 2: Table S2). Most BEUs were involved in genetic information processing, environmental adaptation and signal transduction As shown in the Venn diagram (Fig. 4a), we found nearly equal numbers of unigenes that were broadly and specifically expressed in I-spikelets, P-spikelets, F-branchlets, and F-leaves. COG analysis indicated that most BEUs were clustered in signal transduction mechanisms (T), replication, recombination and repair (L), and transcription (K), besides general function prediction only (R). GO enrichment analysis for these BEUs indicated that they were also mainly involved in reproduction, environmental adaptation and signal transduction, which was largely similar with that for all predicted unigenes (Additional file 4: Table S4-a). KEGG enrichment analysis also indicated that these BEUs were mainly enriched in pathways related to environmental adaptation (including circadian rhythm, endocytosis, and plant-pathogen interactions), signal transduction (including plant hormone signal transduction, phosphatidylinositol signaling system, and inositol phosphate metabolism) and genetic information processing (including spliceosome, mRNA surveillance, and RNA transport and degradation; Additional file 4: Table S4-b). Functional annotation indicated that many unigenes were involved in metabolism and genetic information processing We were able to annotate 13,128 unigenes (27.75% of the total) in 25 different categories of the COG (clusters of orthologous groups) classification database (Fig. 2b). Of these, the cluster for “General function prediction only” (3,277, representing 24.96% of the 13,128 unigenes annotated by this database) was the largest group, followed by “Replication, recombination and repair” (2,202, 16.77%), “Transcription” (1,571, 11.97%), and “Translation, ribosomal structure and biogenesis” (1,429, 10.88%). The “Signal transduction mechanisms”, “posttranslational modification, protein turnover, chaperones”, “carbohydrate and amino acid transport and metabolism” and “transport and metabolism” categories also contained a significant proportion of the annotated unigenes. GO enrichment analysis indicated that these predicted unigenes were categorized into three main categories— i.e. biological process (BP), cellular component (CC), and molecular function (MF). As shown in Fig. 2c, for unigenes that were enriched in the BP category, they were mainly involved in biological processes related to reproduction, posttranslational modification and signal transduction; as for those in the CC category, they were mainly involved in cellular components related to membrane, ubiquitin ligase complex, mitochondrion, chloroplast and etc.; while for those in the MF category, they were mainly involved in molecular functions related to signaling transduction (e.g. “ATP binding”, “zinc ion binding”, “protein kinase activity”, and etc.) (Additional file 3: Table S3). We also mapped 14,307 unigenes (representing 30.24% of the total) to six different KEGG subsystems, including metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. As shown in Fig. 3, the majority of these unigenes (7,922, representing 66.17% of the 14,307 unigenes classified using KEGG annotations) were assigned to metabolic pathways, including carbohydrate metabolism, energy metabolism, and others. In addition, 4,024 unigenes (28.13%) were assigned to genetic information processing, including transcription, translation, and folding, and 474 unigenes (3.31%) were found to be related to membrane transport and signal transduction. We also found 707 genes (4.94%) that were related to transport and catabolism and 377 genes (2.64%) related to environmental adaptation. Page 4/27 The SEUs were mostly involved in carbohydrate metabolism, energy metabolism, and environmental adaptation As shown in Fig. 4a, we identified 10,653 unigenes that were specifically expressed in spikelets, including 5,528 and 5,025 unigenes in I- and P-spikelets, respectively. We also found 9,067 and 7,437 unigenes that were specifically expressed in F-branchlets and F-leaves, respectively. COG annotation indicated that the distribution patterns of SEUs among the 26 terms were similar, with the number of SEUs within each term varying among the three tissues (Fig. 4b). The GO enrichment analysis indicated that these SEUs not only shared some common GO terms, but also had some particular ones. As shown in Fig. 4c and Additional file 4: Table S4-c, for those SEUs that were enriched in the BP category, they were broadly involved in several important biological processes, including “protein phosphorylation”, “regulation of flower development”, “protein ubiquitination”, “regulation of transcription, DNA- templated”, “reciprocal meiotic recombination” and “meiotic chromosome segregation”. In addition, SEUs in I- and P- spikelets were also involved in some processes related to reproduction; and those in F-branchlets were mainly involved in processes related to posttranslational modification; while those in F-leaves were mainly involved in processes related to plant-pathogen interaction. As for those in the CC category, they were broadly involved in several important cellular components, including “mitochondrion”, “plasma membrane” and “plastid”. In addition, SEUs in I- and P-spikelets were also involved in ribosome and mitochondria; and those in F-branchlets were mainly involved in endoplasmic reticulum and proteasome; and those in F-leaves were mostly involved in chloroplast. As for those in the MF category, they were broadly involved in several molecular functions, including “ATP binding”, “ubiquitin-protein transferase activity” and “protein tyrosine kinase activity”. In addition, SEUs in I- and P-spikelets were also involved in DNA and microtubule binding; those in F-branchlets were also enriched in oxidoreductases activities; and those in F-leaves were also enriched in enzymes involved in carbohydrate metabolism. As shown in Additional file 5: Fig. S1, KEGG pathway analysis indicated that SEUs in I- and F-spikelets mainly mapped to the ribosome pathway, with those in F-branchlets mainly mapped to the ribosome, amino acid biosynthesis, and carbon metabolism pathways, and those in F-leaves mainly mapped to KEGG pathways related to energy metabolism (including  oxidative phosphorylation, fatty acid metabolism, and Page 5/27 Page 5/27 photosynthesis), environmental adaptation (e.g. proteasomes), genetic information processing, and various unrelated metabolic pathways (e.g. tryptophan metabolism, beta-alanine metabolism, and N-glycan biosynthesis). DEUs were mostly involved in carbohydrate and energy metabolism, signal transition and environmental adaptation As shown in Table 3, many unigenes showed differential expressions across all 15 groups sampled. The number of DEUs in each sample pair ranged from 970 between I- vs P-spikelets to 13,577 in NF-leaves vs I- spikelets. For most pairwise comparisons, the number of up- and down-regulated DEUs was approximately the same, except for four groups, including I- vs P-spikelets, F-branchlets vs both I- and P- spikelets, and F-leaves vs P-spikelets. The Venn diagram of DEU sets shows that 5,494 unigenes were differentially expressed in F-branchlets/F- leaves vs I- and P-spikelets. For those DEUs that were up-regulated in spikelets, they are mainly mapped to KEGG pathways related to carbohydrate metabolism, plant-pathogen interactions and DNA repair (Fig. 5a). Notably, among the 970 DEUs identified between I- and P-spikelets, 916 up-regulated DEUs were mapped to KEGG pathways related to metabolic activity (Additional file 6: Table S5). A total of 5,494 unigenes were differentially expressed in the DEU sets of spikelets/F-leaves vs F- branchlets. Upregulated DEUs in F-branchlets were mapped to KEGG pathways including phenylalanine metabolism, phenylpropanoid biosynthesis, ABC transporters, and flavone and flavonol biosynthesis (Fig. 5b). Those that were upregulated in F- and NF-leaves vs F- branchlets were mainly mapped to plant hormone signal transduction, homologous recombination, base excision repair, and mismatch repair (Additional file 6: Table S5). Notably, 3,275 (50.20% of the total) DEUs found between NF- and F-branchlets were upregulated; these were mainly mapped to KEGG pathways related to replication and recombination (Additional file 6: Table S5). Those that were downregulated were mainly mapped to carbon fixation and photosynthesis (Additional file 6: Table S5). We also found that 6,966 (43.69% of the total) DEUs found in spikelets/F-branchlets vs F-leaves were up- regulated, and were mainly mapped to KEGG pathways related to carbohydrate metabolism (Fig. 5c). 2,492 (49.52%) DEUs in NF-vs F-leaves were up-regulated, and these were mainly mapped to starch and sucrose metabolism (Additional file 6: Table S5). In contrast, downregulated DEUs were mainly mapped to KEGG pathways related to photosynthesis (Additional file 6: Table S5). Two putative FT orthologs were regulated differently in the circadian rhythm–p Among the 5,032 DEUs identified between NF- and F-leaves, 70 were mapped to the circadian rhythm–plant KEGG pathway (Additional file 7: Fig. S2) and 10 of them showed differential expressions (Additional file 8: Table S6). Notably, c109220.graph_c0 and c110963.graph_c4 were both annotated as FT orthologs: the former was a putative bamboo ortholog of Heading date 3a (Swissprot: PE=1 SV=1), and the latter was another ortholog of rice FT; we designated them as FmHd3a and FmFT, respectively. As shown in Fig. 6a, protein sequence alignment indicated that both FmFT and FmHd3a had high amino acid sequence similarities (77.14%) with the known FT/TFL1 proteins and had the critical amino acids of FT/Hd3a Page 6/27 Page 6/27 proteins. For example, they both carry a conserved phosphatidylethanolamine-binding protein (PEBP) domain, D-P-D-x-P and G-x-H-R motifs, and the invariant histidine (asterisk), all of which are relevant to the ability of PEBP proteins to bind phosphoryl ligands, hence interfering with certain kinases and effectors [27-28]. Furthermore, all five proteins carry tyrosine-139 and tryptophan-143 (triangles) in the PEBP domain, two conserved sites in FT homologs acting as flowering inducers [29]. Whereas, our comparison also revealed that there were differences in amino acid sequences between FTs and Hd3as. proteins. For example, they both carry a conserved phosphatidylethanolamine-binding protein (PEBP) domain, D-P-D-x-P and G-x-H-R motifs, and the invariant histidine (asterisk), all of which are relevant to the ability of PEBP proteins to bind phosphoryl ligands, hence interfering with certain kinases and effectors [27-28]. Furthermore, all five proteins carry tyrosine-139 and tryptophan-143 (triangles) in the PEBP domain, two conserved sites in FT homologs acting as flowering inducers [29]. Whereas, our comparison also revealed that there were differences in amino acid sequences between FTs and Hd3as. Additionally, phylogenetic analysis indicated that these ten proteins were subdivided into two distinct subgroups (Fig. 6b). FT and Hd3a proteins from O. sativa (OsFT, OsRFT1 and OsHd3a), F. macclureana (FmFT and FmHd3a) and A. thaliana (AtFT) were clustered in a branch. Furthermore, OsFT and FmFT were clustered together, just like OsHd3a and FmHd3a; they were both closer to each other than they were to AtFT. Interestingly, two FT orthologs were regulated differently in NF- vs F- leaves. FmHd3a was significantly upregulated in F-leaves (FDR = 4.23, log2FC = 5.55), while FmFT was significantly downregulated in F-leaves (FDR = 4.25E-07, log2FC = -4.81). WGCNA results identified gene modules related to specific tissues WGCNA results showed that unigenes expressed in the six different tissues of flowering and nonflowering plants tested here clustered into 18 branches representing 18 different genetic modules (Additional file 9: Fig. S3a). Unigenes within each module were highly co-expressed, while those in different modules were co- expressed to a lower degree (Additional file 9: Fig. S3b). In six of the samples collected, we identified nine significant gene modules including 1,344 unigenes. Here, correlation coefficient of a module with a related trait > 0.7 was used as a threshold of significance (Additional file 9: Fig. S3c). Notably, these six tissues were more strongly divided into clades according to whether they were flowered or not rather than by the differences among tissues (Additional file 9: Fig. S3d). In addition, the unigenes in gene modules relating to I - and P- spikelets were most strongly enriched in KEGG pathways related to carbohydrate metabolism, genetic information processing, and environmental information processing. In contrast, those related to F- and NF- branchlets were mostly enriched in KEGG pathways related to metabolism, plant hormone signal transduction, and genetic information processing. The gene modules related to F-leaves were enriched in pathways related to plant hormone signal transduction and protein processing, while the gene modules related to NF-leaves were enriched in KEGG pathways related to oxidative phosphorylation (Additional file 10: Table S7). Two putative FT orthologs were regulated differently in the circadian rhythm–p RT-qPCR analysis also showed that FmHd3a was significantly more highly expressed in I- /P- spikelets and F-leaves than in NF-leaves or NF- branchlets (Fig. 6c). Identification of SSRs We detected a total of 9,296 SSRs in 7,668 unigenes longer than 1,000 bp (Additional file 11: Table S8). 1,628 (21.23%) unigenes contained more than one SSR. Mono-nucleotide repeats were the most common (46.28% of all SSRs) at a density of 71 SSRs per Mb, followed by tri- (26.32%) and di- (22.06%) nucleotide repeats, with densities of 40 and 32 SSRs per Mb, respectively (Fig. 7). Activated Hd3a expression probably accelerates flowering in F. macclureana FT is a key floral regulator that controls the timing of flowering and seasonal growth cessation in response to light and the circadian clock in many plant species [8, 10, 30]. In this study, FmHd3a, a bamboo ortholog of rice FT, was significantly expressed only in tissue samples collected from flowering plants. In rice, Hd3a functions as a major photoperiodic flowering regulator and participates in the OsGI–Hd1–Hd3a module, which is similar to the GI-CO-FT module in Arabidopsis [31]. Hd1 activates and suppresses Hd3a expression by promoting heading under the short day (SD) and long day (LD) conditions, respectively [32-33]. As F. macclureana rarely blossoms on the QTP, which experiences a long photoperiod with a low ratio of red to far-red light, it may have evolved specific reproductive strategies involving flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. It is probably that the weak light intensity with a low proportion of blue light might activate Hd3a expression even in the LD conditions, thereby accelerating flowering. Notably, reproduction pathways play an important role in the mechanisms of plant adaptation to extreme environments. Previous studies showed that the phytochrome and flowering time regulatory protein 1 (PFT1) from Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the QTP, showed signs of positive selection for adaptive divergence [34]. We presume that the function of Hd3a in promoting flowering is likely to be conserved between bamboo and rice, because both of them belong to the Poaceae. Interestingly, FmFT and FmHd3a were regulated differently in NF- vs F- leaves, although they were both annotated as FT regulators in the circadian rhythm-plant pathway. Basically, FT sub-family protein acts as florigen; the diverse functions of the FT gene family in flowering regulation had been demonstrated in many different plant species [29]. For example, sugar beet BvFT1 repressed flowering and the divergence within three amino acids of an external loop in PEBP domain was demonstrated to be the major cause [35]. Similarly, a single amino acid exchange in PEBP domain was sufficient to convert FT to TFL1, an activator and a repressor [36-37]. In our study, five proteins carry invariant amino acids in positions where variation occurred; however, there were many differences in predicted amino acid sequences between them in PEBP domain. Especially for some positions, amino acids of AtFT and FmHd3a are the same, but different from that of FmFT (Fig. Discussion Page 7/27 Page 7/27 Carbohydrate metabolism and signal transport may be major factors in floral transition, organogenesis, and death after flowering Bamboo exhibits excellent flexibility and fracture toughness, and so far, the presence of fibers within the bamboo culm was thought responsible for these remarkable mechanical properties [38]. And the development of plant fibers is accompanied by the of carbohydrate metabolism [39]. Perhaps this is the reason why many DEUs were involved in carbohydrate metabolism pathway. Interestingly, our results indicated that starch and sucrose metabolism was a major enriched KEGG pathway for the DEUs from several combination pairs, including NF- vs F-leaves, branchlets/leaves vs I- & P- spikelets. Transcripts and metabolic signatures of maize leaves have shown that the balance between transitory starch and sucrose is associated with the autonomous floral transition [40]. And in Lilium, carbohydrates have been found to be transported from the vascular bundles to floral organs during reproduction [41]. Yang et al., (2017) also reported that the deficiency in the resources in male flowers reduced pollen viability in Tapiscia sinensis due to biased carbohydrate transport toward the female flowers [42]. Therefore, we suspect there may be a correlation between DEUs related to starch and sucrose metabolism and arrow bamboo floral organ development. In rice, excessive uridine 5´-diphosphoglucose-glucose (UDPG) can result in programmed cell death, accumulation of reactive oxygen species and an increase in the caspase-like activity [43] and inactivate starch synthase disrupted normal male reproduction by delaying programmed cell death in cotton [44], suggestive of a correlation between starch and sucrose metabolism and death. Bamboo flowering, especially in masting species, often causes plants to wilt and die after setting seed. It is possible that increased starch and sucrose metabolism might trigger the excessive accumulation of reactive oxygen species and result in the altered activity of key enzymes in important biological pathways. In the present study, unigenes related to the signal transduction pathways were significantly upregulated in the tissues of flowering arrow bamboo plants. The transcriptomic profiles of Posidonia oceanica also showed a strong metabolic activation of hormones in the heat stress-induced flowering plants [45]. Signal transduction- related genes were also found to have undergone both significant positive selection and expansion events on the adaptive evolution of Crucihimalaya himalaica [34] and cyanobacterium Trichormus sp. NMC-1 [46] on the QTP. In the present study, unigenes related to the signal transduction pathways were significantly upregulated in the tissues of flowering arrow bamboo plants. Activated Hd3a expression probably accelerates flowering in F. macclureana 6a). Possibly, FmFT acts as a repressor FT due to the conversion of certain amino acids in PEBP domain, while FmHd3a acts as an inducer one. Thus, we suspect that the floral transition of F. macclureana is regulated by a complex regulatory network in which at least two unique FT orthologs interact with the circadian clock pathways. However, how these circadian clock pathways mediate the activation of FmHd3a and FmFT in response to light signalling remains to be elucidated by future research. Notably, we detected FmHd3a expressions in all four tissues collected from the flowering plants, but not in NF- leaves or NF-branchlets. We also noticed that all plants were sampled from the same provenance and grown in chambers under the same conditions after transplantation, but all non-flowering plants each had a longer section of rhizome than the flowering ones. Given that rhizome can provide nutrients for plants to maintain normal growth, thus, we hypothesize that the broken balance between vegetative and reproductive growth after transplantation result in the floral transition. Additionally, the DEUs upregulated in F-leaves relative to NF-leaves were mainly enriched in KEGG pathways related to starch, sucrose, and galactose metabolism, and corresponding down-regulated DEUs were mapped to the light and carbon fixation, plant circadian rhythm, and photosynthesis pathways. Therefore, we speculate that bamboo FT orthologs might be regulated by upstream regulators involved in carbohydrate metabolism. corresponding down-regulated DEUs were mapped to the light and carbon fixation, plant circadian rhythm, and photosynthesis pathways. Therefore, we speculate that bamboo FT orthologs might be regulated by upstream regulators involved in carbohydrate metabolism. Page 8/27 Carbohydrate metabolism and signal transport may be major factors in floral transition, organogenesis, and death after flowering We suspect that this may be due to the long distance transport of the FT proteins, which ensures floral promotion at the shoot apex [47], or the phytohormone signaling and calcium signaling, which play diverse roles in the specification of flower organs during arrow bamboo reproductive development [48-49]. Conclusions In the present study, we constructed a novel de novo transcriptome analysis for F. macclureana. Based on two major KEGG pathways of carbohydrate metabolism and signal transduction that DEUs were enriched in, as well as the different regulation of FmFT and FmHd3a in NF- vs F- leaves, we speculate that both environmental signals and physiological status have effect on floral transition in F. macclureana. Significant expressions of unigenes enriched in DNA repair and plant-pathogen interaction pathways may reflect the adaptation of F. macclureana to its high radiation and pathogen-specific environment on the QTP. We identified both similarities and differences in adaptive mechanisms (e.g., disease-resistance and DNA repair pathways) and stress- induced flowering mechanisms (e.g., carbohydrate metabolism and signal transduction pathways) among plants that grow at high altitudes. Although further experimental verification is needed, our results provide insight into the regulation of flowering time in highland bamboo as well as how this species adapts to harsh and extreme environments. F. macclureana has presumably evolved an integrated mechanism to adapt to the harsh environment of the QTP We detected the broad expressions of unigenes encoding putative Hsp proteins, such as heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). Both Hsp70 and Hsp90 are important for maintaining cellular protein homeostasis under stress conditions and they function by activating other targets [50-52]. It is likely that the sudden exposure to the higher temperature of the lab (i.e. outside the QTP) triggered the expressions of these unigenes. Warmer temperatures can greatly reduce flowering synchrony among individuals from 72 woody and herbaceous species [53]. It was also reported that P. oceanica, a highly clonal and long-lived Page 9/27 Page 9/27 species, massively bloomed after a simulated heatwave [45]. Given the cold temperatures present at the high- altitude regions of the QTP [26], it is reasonable to presume that F. macclureana has developed into a heat- sensitive but not heat-tolerant bamboo species and flowering is probably a stress-induced response to the higher temperature in the lab. DNA repair and disease-resistance pathways have been found to play a crucial role in the highland adaptation of Tibetan highland plants [46, 54]. In the present study, we detected significant expressions of many unigenes related to pathogen response that contained either a nucleotide binding (NB)-ARC domain or a leucine-rich repeat (LRR) domain, which were present in most resistance (R) proteins [55-57]. We also identified many differentially expressed unigenes that were significantly enriched in the DNA repair pathways. Since relatively fewer species of pathogenic microorganisms and intense UV radiation exist on the QTP [58], it is reasonable to presume that F. macclureana has evolved a relatively narrow range of pathogen specificity and specific DNA- repair mechanisms. Sudden exposure to the lab environment, which contains a heavier load of pathogens and weak light intensity, may have induced an innate defensive response of F. macclureana, and those that were enriched in the plant-pathogen interaction and DNA repair response pathways may be important for F. macclureana to cope with the new environment present in the lab. Although further studies are needed to investigate the molecular mechanisms responsible for the putative adaptive evolutionary changes, this study provides insights into how plants adapt to harsh and extreme environments. Tissues collection The studied plant species is highland arrow bamboo (Fargesia macclureana), and it grows mainly as an underbrush of coniferous forest or coniferous and broad-leaved mixed forest, and sometimes forms a pure population in the QTP at an altitude of approximately 2,000 ~ 3800 m above sea level (Fig. 1). F. macclureana was formally identified by Stapleton in 1993 [25] and detailed explanations are provided in the volume 22 of Flora of China (http://foc.iplant.cn/) [59]. A voucher specimen of this material has been deposited in the Bamboo Research Institute of Nanjing Forestry University. A total of six seedlings of F. macclureana were Page 10/27 Page 10/27 sampled from the wild with the permission of the local forestry department and collected from the Bayi District, Linzhi City, Tibet, China (29˚46′ 0.95″ N, 94̊ 44′46.36″ E, altitude: ~2,200 m). They were dig out of the ground, each with a section of rhizome, which can provide nutrients for plants. Then they were all transferred to individual pots at the State Forestry Administration Key Open Laboratory at the International Center for Bamboo and Rattan in Beijing (N: 39˚59′ 17.52″, E: 116̊ 28′46.06″, altitude ~34 m). During growth, the plants were maintained at 28 ± 1 °C and 50-55% relative humidity under a 16/8 h (light/dark) photoperiod regimen with a light intensity of 200 μmol · m-2 · s-1. All the seedlings were watered with a 1/3 B5 macronutrient nutrient solution three times a week. After twenty days, four seedlings which had shorter rhizomes flowered, while the other two didn't blossom until the time of sampling. One month later, we collected samples of six tissues for further de novo sequencing, including inflorescences in the initial flower stage (I-spikelets), inflorescences at the peak flower stage (P-spikelets), branchlets of the flowering plants (F-branchlets), leaves of the flowering plants (F-leaves), branchlets of the non-flowering plants (NF-branchlets) and leaves of the non-flowering plants (NF- leaves). We collected three independent replicates of each tissue type. RNA extraction, quantification, and qualification Total RNA was extracted from each of the six unique tissues mentioned above using a RNeasy plant RNA extraction kit (Qiagen, Dusseldorf, Germany), and the extraction procedure was performed according to the manufacturer’s instructions. RNA degradation and contamination were monitored using 1% agarose gels. RNA purity was checked using a NanoPhotometer® spectrophotometer (Implen GmbH, Munich, Germany). RNA concentration was measured using a Qubit® RNA Assay Kit and a Qubit®2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using an RNA Nano 6000 Assay Kit run on an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Library preparation for transcriptome sequencing Library construction and RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). A total of 3 μg RNA per sample was used for RNA preparation. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, followed by fragmentation carried out using divalent cations at elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via the exonuclease and polymerase activities. Next, the 3´ ends of the DNA fragments were adenylated and ligated to the NEBNext adaptors with hairpin loop structures to prepare samples for hybridization, this was to select cDNA fragments that are 150-200 bp in length. Library fragments were then purified using an Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA), and 3 μl USER enzyme (New England Biolabs, Ipswich, MA, USA) was added to the size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by five min at 95°C before PCR. PCR was performed using Phusion High-Fidelity DNA polymerase (Thermo Fisher, Waltham, MA, USA), universal PCR primers, and the Index (X) Primer. Finally, the PCR products were purified using the AMPure XP system and library quality was assessed on the Agilent Bioanalyzer 2100. Clustering and sequencing Clustering and sequencing Expression analysis of broadly and specifically expressed unigenes For all unigenes, those that were expressed in all six tissues were defined as broadly expressed unigenes (BEUs). Similarly, unigenes that were specifically expressed in only one tissue were defined as specifically expressed unigenes (SEUs). The identification of BEUs and SEUs was conducted by using tools on the BMKCloud platform (http://www.biocloud.net). Clustering and sequencing Page 11/27 The clustering of index-coded samples was performed using a cBot Cluster Generation System and a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), and all experimental procedures were performed according to the manufacturer’s instructions. After that, library preparations were sequenced on an Illumina Hiseq 2000 platform and paired-end reads were generated. The clustering of index-coded samples was performed using a cBot Cluster Generation System and a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), and all experimental procedures were performed according to the manufacturer’s instructions. After that, library preparations were sequenced on an Illumina Hiseq 2000 platform and paired-end reads were generated. De novo transcriptome assembly Raw data in fastq format were first processed using in-house perl scripts. Clean data were obtained by removing low-quality reads and reads that contain the adapters or poly-N sequences. Meanwhile, we checked the quality of our unassembled read dataset by examining various measures including Q20, Q30, GC-content, and sequence duplication. All the downstream analyses were performed using high-quality clean data. The transcriptome was assembled using clean reads from all libraries and samples. The assembly was produced using Trinity [60] with min_kmer_cov set to 2 and all other parameters set to their respective default values. Functional annotation of the transcriptome Gene function was annotated using the following databases: Nr (NCBI non-redundant protein sequences), Pfam (Protein family), KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins), Swiss-Prot (a database of manually annotated and reviewed protein sequences), KEGG (the Kyoto Encyclopedia of Genes and Genomes), and the GO (Gene Ontology) database. Quantification of gene expression levels Gene expression levels were estimated using RSEM [61] for each sample: clean data were mapped back onto the assembled transcriptome, followed by a read count for each gene. The expression levels of unigenes were calculated and normalized using FPKM (fragments per kb per million fragments) [62]. Weighted gene co-expression network analysis (WGCNA) WGCNA was performed on all unigenes identified using the WCGNA R package. We calculated the adjacency matrices and performed the topological overlap measures (TOMs), which show the degree of overlap in shared neighbors between pairs of genes in the network to define gene clusters in our transcriptome dataset. 1 − TOM was used as a dissimilarity measure for hierarchical clustering and module detection. Modules of the clustered genes were then selected using the Dynamic Tree Cut algorithm as implemented by WGCNA. To identify modules that are significantly related to particular tissues, expression profiles of each module were summarized by a module eigengene (ME) and the correlations between the modules and corresponding tissues were calculated. Page 12/27 Page 12/27 Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) p To understand the higher-level functions of the observed unigenes, we performed GO term annotation and KEGG pathway enrichment analysis using BMKCloud (http://www.biocloud.net/; [64]). We used KOBAS 2.0 [65] to test the statistical enrichment of differentially expressed genes in KEGG pathways. Pathways with P values < 0.05 were considered significantly enriched. Protein-protein interactions (PPIs) The DEU and SEU sequences were queried using BLASTX against the related species to predict PPIs that the DEUs and SEUs may be involved in. This search procedure was capable of identifying PPIs that may be similar to any others found in the STRING database (http://string-db.org/). These PPIs were then visualized using Cytoscape [66]. Detection of SSRs Picard-tools version 1.41 and samtools version 0.1.18 were used to sort data, remove duplicated reads, and merge the bam alignment results of each sample. SSRs were identified using MISA (https://webblast.ipk- gatersleben.de/misa/). Expression analysis of differently expressed unigenes (DEUs) Before analysis, we conducted a principal component analysis (PCA) and removed one replicate that showed an inconsistent expression pattern in the NF-branchlets and NF-leaves to ensure consistency in the expression patterns of unigenes between replicates (Additional file 12: Figure S4). Expression analysis of the DEUs between pairs of tissues/groups was performed using the DESeq package (1.10.1) in R. DESeq provides statistical routines for identifying differential expression in the digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini-Hochberg method for controlling the false discovery rate [63]. Here, uni-transcripts with an absolute value of log2 ratio ≥ 2, an FDR significance score < 0.01, and an adjusted P-value < 0.05 were deemed to be differentially expressed. Funding This work was supported by the Special Funds for Fundamental Scientific Research on Professional Work Supported by International Center for Bamboo and Rattan (No. 1632019008) and the Sub-Project of National Science and Technology Support Plan of the Twelfth Five-Year in China (No. 2015BAD04B01). The funder is the corresponding author of this manuscript and he played an important role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Consent for publication Not applicable. Abbreviations BEUs: Broadly expressed unigenes; BP: Biological process; CC: Cellular component; COG: Clusters of orthologous groups; DEUs: Differentially expressed unigenes; F-branchlets: Branchlets of the flowering plants; F- leaves: Leaves of the flowering plants; FPKM: Fragments per kb per million fragments; GO: Gene ontology; I- spikelets: Inflorescences in the initial flower stage; KEGG: Kyoto Encyclopedia of Genes and Genomes; ME: Module eigengene; MF: Molecular function; NF-branchlets: Branchlets of the non-flowering plants; NF-leaves: Leaves of the non-flowering plants; Nr: NCBI non-redundant protein sequences; PCA: Principal component analysis; Pfam: Protein family; PPIs: Protein-protein interactions. P-spikelets: Inflorescences at the peak flower stage; QTP: Qinghai–Tibet Plateau; SEUs: Specifically expressed unigenes; SSRs: Simple sequence repeats; TOMs: topological overlap measures. WGCNA: Weighted gene co-expression network analysis. Authors’ contributions Conceived and designed the experiments: ZM G. Performed the experiments: KB Y and JJ S. Analyzed the data: CX Z, YL D and Y L. Interpreted the results and wrote the paper: Y L. All authors have read and approved the final manuscript. Availability of data and materials The RNA sequencing dataset generated during the current study have been submitted to NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra) with the accession number PRJNA544133. Validation of FmHd3a transcript levels by RT-qPCR To verify the expression pattern of the FmHd3a, we used RT-qPCR to assess the expressions of FmHd3a in six distinct tissues. First-strand cDNA was synthesized from total RNA extracted by using a reverse transcription system (Promega, Madison, WI, USA) following the manufacturer’s instructions. Each RT-qPCR amplification was performed at least three times, and NTB and TIP41 were used as internal controls [67]. Primers for these genes are listed in Additional file 13: Table S9. The relative expression levels of FmHd3a in different tissues were calculated using the 2-ΔΔCT method [68]. The statistical significance of differences in the mean levels of expression was tested using a one-way ANOVA. Significant differences in transcript abundance between different tissues were then compared using Duncan’s multiple range tests as implemented by SPSS version 17.0 (IBM SPSS, Chicago, USA). We considered mean differences at P < 0.05 and P < 0.01 to be statistically significant and highly statistically significant, respectively. Page 13/27 Page 13/27 Page 13/27 Page 13/27 Ethics approval and consent to participate Not applicable. Competing interests The authors declare that they have no competing interests. Declarations Ethics approval and consent to participate Acknowledgements Page 14/27 Page 14/27 Page 14/27 Not applicable. References 1. Putterill J, Varkonyi-Gasic E. 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Revisiting the Origin of Plant NBS-LRR Genes. Trends Plant Sci. 2019;24:9- 12. 57. Takken FL, Albrecht M, Tameling WI. Resistance proteins: molecular switches of plant defence. Table 1 Length range of transcripts and unigenes identified in the transcriptome of F. macclureana. Table 1 Length range of transcripts and unigenes identified in the transcriptome of F. macclureana. Page 18/27 Page 18/27 Length Range Transcripts  Unigenes  200-300 36,390 (12.59%) 25,291 (25.53%) 300-500 47,515 (16.43%) 21,257 (21.46%) 500-1,000 78,453 (27.13%) 23,806 (24.03%) 1,000-2,000 77,456 (26.79%) 16,752 (16.91%) 2,000+ 49,308 (17.05%) 11,950 (12.06%) Total number 289,122 99,056 Total length 341,956,623 91,685,618 N50 length 1,765 1,587 Mean length 1,182.74 925.59 Total number 289,122 99,056 Total length 341,956,623 91,685,618 N50 length 1,765 1,587 Mean length 1,182.74 925.59 Table 2 Statistics of annotation analysis of unigenes Anno_Database Annotated_Number percentage 300<=length<1,000 length>=1,00 COG_Annotation 13,128 27.75 3,261 7,515 GO_Annotation 34,055 71.99 10,659 17,855 KEGG_Annotation 14,307 30.24 4,550 7,397 KOG_Annotation 23,492 49.66 6,863 12,779 Pfam_Annotation 28,317 59.86 7,823 16,896 Swissprot_Annotation 24,847 52.52 7,450 14,500 eggNOG_Annotation 43,909 92.82 14,040 21,568 Nr_Annotation 45,516 96.22 15,031 22,271 All_Annotated 47,306 100.00 15,602 22,437 Table 3. Differentially expressed unigenes (DEUs; Fold change > 2; FDR < 0.01) among tissues of F. macclureana. DEUs_total: the total number of DEUs; DEUs_up (%): the number (and percentage) of up-regulated DEUs; DEUs_down (%): the number (and percentage) of down- regulated DEUs). Table 3. Differentially expressed unigenes (DEUs; Fold change > 2; FDR < 0.01) among tissues of F. macclureana. DEUs_total: the total number of DEUs; DEUs_up (%): the number (and percentage) of up-regulated DEUs; DEUs_down (%): the number (and percentage) of down- regulated DEUs). Table 3. Differentially expressed unigenes (DEUs; Fold change > 2; FDR < 0.01) among tissues of F. macclureana. DEUs_total: the total number of DEUs; DEUs_up (%): the number (and percentage) of up-regulated DEUs; DEUs_down (%): the number (and percentage) of down- regulated DEUs). Page 19/27 Number Group DEUs_total DEUs_up (%) DEUs_down (%) 1 I-spikelets vs P-spikelets 970 916 (94.43) 54 (5.57) 2 F-branchlets vs I-spikelets  4,970 3,046 (61.29) 1,924 (38.71) 3 F-branchlets vs P-spikelets 5,124 3,338 (65.14) 1,786 (34.86) 4 F-branchlets vs F-leaves 8,467 3,967 (46.85) 4,500 (53.15) 5 F-leaves vs I-spikelets  12,829 6,791 (52.93) 6,038 (47.07) 6 F-leaves vs P-spikelets 10,791 6,625 (61.39) 4,166 (38.61) 7 NF-branchlets vs I-spikelets  11,628 6,135 (52.76) 5,493 (47.24) 8 NF-branchlets vs P-spikelets 10,809 5,893 (54.52) 4,916 (45.48) 9 NF-branchlets vs F-branchlets 6,524 3,275 (50.20) 3,249 (49.80) 10 NF-branchlets vs F-leaves 11,670 5,902 (50.57) 5,768 (49.43) 11 NF-branchlets vs NF-leaves 3,853 1,946 (50.51) 1,907 (49.49) 12 NF-leaves vs I-spikelets  13,577 6,921 (50.98) 6,656 (49.02) 13 NF-leaves vs P-spikelets 11,718 6,130 (52.31) 5,588 (47.69) 14 NF-leaves vs F-branchlets 11,659 5,606 (48.08) 6,053 (51.92) 15 NF-leaves vs F-leaves 5,032 2,492 (49.52) 2,540 (50.48) Supplementary File Legends Additional file 1: Table S1. Statistic of sequencing and assembly data. Additional file 1: Table S1. Statistic of sequencing and assembly data. Additional file 2: Table S2. 47,306 unigenes were annotated and their predicted functions. Additional file 4: Table S4. The most enriched GO terms and KEGG pathways for the specially expressed unigenes (SEUs) and broadly expressed unigenes (BEUs) in all tissues collected from flowering plants. Additional file 4: Table S4. The most enriched GO terms and KEGG pathways for the specially expressed unigenes (SEUs) and broadly expressed unigenes (BEUs) in all tissues collected from flowering plants. Additional file 5: Figure S1. KEGG annotation of unigenes that were specifically expressed in P-spikelets (a), F- branchlets (b) and F-leaves (c) of arrow bamboo flowering plants. The size of dots is proportional to the number of unigenes. Additional file 6: Table S5. KEGG enrichment of differentially expressed unigenes (DEUs) between different tissues. Additional file 7: Figure S2. Hub unigenes in regulatory networks of flowering identified based on analysis of DEUs among tissues. Unigenes c109220.graph_c0 and c110963.graph_c4, showing differential expressions between NF-leaves and F-leaves, are both bamboo orthologs of FLOWERING LOCUS T (FT), which was marked with a red square; while unigenes down-regulated were marked with green squares. Page 20/27 Additional file 8: Table S6. Expressions of 10 unigenes that were differentially expressed between F-leaves vs NF-leaves and mapping into the circadian phythm-plant KEGG pathway. Additional file 9: Figure S3. Weighted gene co-expression network analysis (WGCNA) of all unigenes identified in the transcriptome of F. macclureana. (a) The phylogenetic tree diagram and the heat map related to the traits. This diagram is divided into three parts: the cluster tree of gene system, the module color of corresponding genes, and the correlation between genes related to each trait in tested samples and its module. The redder the color, the more positive the correlation; conversely, blue is negatively correlated. (b) Gene co- expression network heatmaps drawn by randomly selected 1,500 genes, in which the left and the upper sides are the symmetrical system clustering tree of gene network/module, and the lower right area indicates the dissimilarity between genes, and the smaller the value is, the darker the color is. (c) Module and trait correlation heat map showing the relationship between a module and a given trait. Additional file 11: Table S8. 9,296 SSRs identified from 7,668 unigenes. Additional file 11: Table S8. 9,296 SSRs identified from 7,668 unigenes Additional file 12: Figure S4. Principal component analysis (PCA) of unigenes expressions for 18 samples collected from inflorescences in the initial and peak flower stage (I- and P- spikelets), branchlets and leaves of flowering and non-flowering bamboo plants (F/NF-branchlets and F/NF-leaves). Additional file 13: Table S9. Primer pairs used for RT-qPCR. Seedlings of Fargesia macclureana flowered shortly after being transferred from the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3,800 m above sea level to a low altitude lab. (a-b) Floret and spikelet of a flowering seedling maintained at the low altitude lab outside the QTP. (c-d) The seedling and shoot of plants growing on the QTP. (e) The original growing environment of F. macclureana. Supplementary File Legends The closer the correlation between a shape and a module is to the absolute value of 1, it is likely that this trait is related to the module gene work. (d) Systematic clustering tree of samples based on unigenes expressions. Additional file 10: Table S7. KEGG enrichment of unigenes in nine significant gene module relating to P- spikelets. Figure 1 Seedlings of Fargesia macclureana flowered shortly after being transferred from the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3,800 m above sea level to a low altitude lab. (a-b) Floret and spikelet of a flowering seedling maintained at the low altitude lab outside the QTP. (c-d) The seedling and shoot of plants growing on the QTP. (e) The original growing environment of F. macclureana. Page 21/27 Figure 2 Fi 2 Figure 2 Figure 2 Function annotation and classification of unigenes identified from the transcriptome of F. macclureana. (a) Nr annotation. (b) Clusters of orthologous groups (COG) annotation. Out of 45,516 Nr hits, 13,128 unigenes had a COG classification. A: RNA processing and modification B: Chromatin structure and dynamics C: Energy production and conversion D: Cell cycle control, cell division, chromosome partitioning E: Amino acid transport and metabolism F: Nucleotide transport and metabolism G: Carbohydrate transport and metabolism H: Coenzyme transport and metabolism I: Lipid transport and metabolism J: Translation, ribosomal structure and biogenesis K: Transcription L: Replication, recombination and repair M: Cell wall/membrane/envelope biogenesis N: Cell mobility O: Posttranslational modification, protein turnover, chaperones P: Inorganic ion transport and metabolism Q: Secondary metabolites biosynthesis, transport and metabolism R: General function prediction only S: Function unknown T: Signal transduction mechanism U: Intracellular trafficking, secretion, and vesicular transport V: Defense mechanisms W: Extracellular structures Y: Nuclear structure Z: Cytoskeleton. (c) GO annotation. Results were summarized in three main categories: biological process, cellular Function annotation and classification of unigenes identified from the transcriptome of F. macclureana. (a) Nr annotation. (b) Clusters of orthologous groups (COG) annotation. Out of 45,516 Nr hits, 13,128 unigenes had a COG classification. A: RNA processing and modification B: Chromatin structure and dynamics C: Energy production and conversion D: Cell cycle control, cell division, chromosome partitioning E: Amino acid transport and metabolism F: Nucleotide transport and metabolism G: Carbohydrate transport and metabolism H: Coenzyme transport and metabolism I: Lipid transport and metabolism J: Translation, ribosomal structure and biogenesis K: Transcription L: Replication, recombination and repair M: Cell wall/membrane/envelope biogenesis N: Cell mobility O: Posttranslational modification, protein turnover, chaperones P: Inorganic ion transport and metabolism Q: Secondary metabolites biosynthesis, transport and metabolism R: General function prediction only S: Function unknown T: Signal transduction mechanism U: Intracellular trafficking, secretion, and vesicular transport V: Defense mechanisms W: Extracellular structures Y: Nuclear structure Z: Cytoskeleton. (c) GO annotation. Results were summarized in three main categories: biological process, cellular Page 22/27 component and molecular function. The right and left y-axes indicated the number and percentage of unigenes in a certain category, respectively. Figure 3 KEGG annotation of unigenes in the transcriptome of F. macclureana. The x-axis indicated the number of unigenes in a certain category. The right y-axis showed the main clusters of KEGG pathways. Figure 3 KEGG annotation of unigenes in the transcriptome of F. macclureana. The x-axis indicated the number of unigenes in a certain category. The right y-axis showed the main clusters of KEGG pathways. Page 23/27 Page 23/27 Figure 4 Unigenes that were specifically expressed in different tissues collected from flowering plants of F. macclureana. (a) Venn diagram of unigenes expressed in spikelets in the initial flower stage (I-spikelets, A) and the peak flower stage (P-spikelets, B), branchlets (F-branchlets, C) and leaves (F-leaves, D) of flowering plants. (b) COG annotation of unigenes that were specifically expressed in I-spikelets, P-spikelets, F-branchlets and F-leaves. (c) GO enrichment of unigenes that were specifically expressed in I- & P- spikelets, F-branchlets and F-leaves. BP: biological process; CC: cellular component; MF: molecular function. Page 24/27 Page 24/27 Figure 5 KEGG annotation of unigenes that were specifically expressed in P-spikelets (a), F-branchlets (b) and F-leaves (c) of arrow bamboo flowering plants. The size of dots is proportional to the number of unigenes. Figure 5 KEGG annotation of unigenes that were specifically expressed in P-spikelets (a), F-branchlets (b) and F-leaves (c) of arrow bamboo flowering plants. The size of dots is proportional to the number of unigenes. Page 25/27 Figure 6 Figure 6 Alignment (a) and phylogenetic analyses (b) of FT/TFL1 proteins from F. macclureana and other plants, as well as the expression pattern of FmHd3a across six different tissues collected by RT-qPCR (c). The involving sequences and their accession numbers are: three FT/TERMINAL FLOWER 1 (TFL1) proteins from A. thaliana: AtFT (AT1G65480.1), AtTFL1 (NP_196004.1) and BROTHER OF FT AND TFL1 (AtBFT, AT5G62040.1); and four FT/TFL1 proteins from Oryza sativa: OsFT (XP_015619436.1), OsRFT1 (rice RICE FLOWERING LOCUS T 1 (RFT1), BAO03220.1), OsHd3a (XP_015611892.1) and two RCNs (putative TFL1/CENTRORADIALIS (CEN) orthologs): OsRCN1 (AAD42895.1) and OsRCN3 (AAD42896.1). Amino acid sequences with double underline and single underline indicate the critical motifs in PEBP proteins [27]; asterisk and triangles indicate the invariant histidine in the PEBP domain [28] and two conserved sites in inducer FTs [29], respectively. Hollow circles indicate the position where the critical amino acid variation occurred between Arabidopsis TFL1 and FT, an activator and a repressor of flowering [36]. Relative expression levels were calculated using the 2−ΔΔCT method to reflect expressions relatively more veritably. The statistical significance was tested by one-way ANOVA, considering P < 0.05 and P < 0.01 as statistically significant and extremely significant, respectively. Significant differences in transcript abundance between different tissues were then compared by Duncan’s multiple range test using SPSS 17.0 (SPSS Inc., Chicago, USA). Figure 7 Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats, respectively. Figure 7 Figure 7 Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats, respectively. Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats, respectively. Page 26/27 Page 26/27 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableS5.xlsx TableS6.xlsx FigureS6.docx FigureS1.tif FigureS2.tif FigureS4.tif TableS4.xlsx TableS3.xlsx TableS7.xlsx FigureS3.tif TableS2.xlsx TableS1.docx TableS8.xlsx FigureS5.docx TableS9.xlsx TableS5.xlsx TableS6.xlsx FigureS6.docx FigureS5.docx TableS9.xlsx Page 27/27
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From Connectivity to Advanced Internet Services: A Comprehensive Review of Small Satellites Communications and Networks
Wireless communications and mobile computing
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Hindawi Wireless Communications and Mobile Computing Volume 2019, Article ID 6243505, 17 pages https://doi.org/10.1155/2019/6243505 Hindawi Wireless Communications and Mobile Computing Volume 2019, Article ID 6243505, 17 pages https://doi.org/10.1155/2019/6243505 Scott C. Burleigh,1 Tomaso De Cola ,2 Simone Morosi ,3 Sara Jayousi,3 Ernestina Cianca ,4 and Christian Fuchs 2 Ernestina Cianca , and Christian Fuchs 1Jet Propulsion Laboratory, California Institute of Technology, USA 2German Aerospace Center (DLR), Institute of Communications and Navigation, 82234 Oberpfaffenhofen, Germany 3Information Engineering Department, University of Florence, 50139 Florence, Italy 4Department of Electronic Engineering, University of Rome Tor Vergata, Italy 1Jet Propulsion Laboratory, California Institute of Technology, USA 2German Aerospace Center (DLR), Institute of Communications and Navigation, 82234 Oberpfaffenhofen, Germa 3Information Engineering Department, University of Florence, 50139 Florence, Italy 4Department of Electronic Engineering, University of Rome Tor Vergata, Italy Correspondence should be addressed to Simone Morosi; simone.morosi@unifi.it Received 27 December 2018; Accepted 18 March 2019; Published 2 May 2019 Academic Editor: Pham Tien Dat Academic Editor: Pham Tien Dat Copyright © 2019 Scott C. Burleigh et al. Thisis an openaccessarticledistributed under the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recently, the availability of innovative and affordable COTS (Commercial Off-The-Shelf) technological solutions and the ever- improving results of microelectronics and microsystems technologies have enabled the design of ever smaller yet ever more powerful satellites. The emergence of very capable small satellites heralds an era of new opportunities in the commercial space market. Initially applied only to scientific missions, Earth observation and remote sensing, small satellites are now being deployed to support telecommunications services. This review paper examines the operational features of small satellites that contribute to their success. An overview of recent advances and development trends in the field of small satellites is provided, with a special focus on telecommunication aspects such as the use of higher frequency bands, optical communications, new protocols, and advanced architectures. Review Article From Connectivity to Advanced Internet Services: A Comprehensive Review of Small Satellites Communications and Networks Scott C. Burleigh,1 Tomaso De Cola ,2 Simone Morosi ,3 Sara Jayousi,3 Ernestina Cianca ,4 and Christian Fuchs 2 1Jet Propulsion Laboratory, California Institute of Technology, USA 2German Aerospace Center (DLR), Institute of Communications and Navigation, 82234 Oberpfaffenhofen, Germany 3Information Engineering Department, University of Florence, 50139 Florence, Italy 4Department of Electronic Engineering, University of Rome Tor Vergata, Italy 1. Introduction Figure 1: INTELSAT, EUTELSAT, and INMARSAT annual revenues (left). Annual sales of VSAT terminals by LSAT, EUTELSAT, and INMARSAT annual revenues (left). Annual sales of VSAT terminals by type (right) [7]. a matter of fact, in the last of few decades, amazing and unexpected progresses and changes have been obtained in the diverse fields of broadcasting, mobile communications, Earth observation and remote sensing, interplanetary explo- ration, transport, and remote monitoring, so encompassing commercial, civil, and military applications [7]. However, it is worth stressing that starting from the postwar times to the today scenarios, satellite systems have undergone themselves a radical and systemic evolution which proves the fact that their abilities perfectly adapt to the ever-changing needs of both the society and the market; particularly, while in the first decades, governments and national agencies were the main players in the start of space race, in the design of satellite missions, and in the development of satellite-based systems, more recently private companies have largely increased their role in this strategic industry [8].h the telecommunication aspects such as the use of higher- frequency bands and optical communications, protocols, and architectures. It is worth outlining that some surveys about small satellites have been published recently [3–6]: whereas [5] focuses on the evolution of the antennas for small sats, [6] concentrates on intersatellite link and related communica- tion protocols for small sat constellations. On the other hand, [4] reviews the history of small satellite development and summarizes its capabilities and applications. A rather comprehensive review is provided in [3], which deals with many aspects, from hardware components and structures, to network topology and communication protocols; moreover, [3] focuses on Cubesat class of small sats. With respect to pre- vious surveys, this paper provides a more extensive overview on telecommunication aspects and aims at describing this rapid evolving field, giving more insights into new protocols, architectures, and technology developments.h This trend has also been enforced by the privatization of the main international satellite organizations which has taken place at the end of the last century and produced high revenues as shown by Figure 1 [7]. As far as the VSAT and broadband satellite systems are concerned, the same trends of deregulation and stimulation of the market forces have been experienced from their launch to the final successful spreading as reported by the graphs in Figure 1 [7]. 1. Introduction gy p The paper is organized as follows: in Section 2, a brief history of the evolution of the small satellites is provided, trying to unveil the commercial reasons of their success. An overview of the services and applications which are enabled by the small satellite is given in Section 3. Sec- tion 4 is devoted to a description of the evolution of the payloads, focusing on the used frequencies and the Soft- ware Defined Radio (SDR) concept. New telecommunication architectures and the suitable protocols for small satellites based systems are described in Sections 5 and 6, respec- tively. Finally, the perspectives and the open challenges are discussed in Section 7 and the conclusions are drawn in Section 8. On the other hand, the end of the twentieth century has also seen the birth and the first steps of a new paradigm that is based on the exploitation of the so-called small satellites, whose size and weight are much smaller than the huge geostationary orbit (GEO) or the big medium Earth orbit (MEO) and low Earth orbit (LEO) ones. These new systems are identified as micro-, nano-, and picosatellites according to their dimensions [3]. The early missions of small satellites were mainly organized and performed by research groups of Universities and Research Organizations with the goals of enabling a technology demonstration or an application validation [9]. 1. Introduction technological achievements with hundreds of small satellites being launched in the last few years and even more envisaged to be commissioned in the near future. In the short span of the first two decades of the new millennium, a revolution has taken place in the field of satellite systems: the availability of innovative and affordable COTS technological solutions and the ever-improving results that are produced by microelectronics and microsystems technologies have paved the way to a process of size reduction for the satellite components and to the design of smaller and smaller satellites that have been defined as small satellites (whose weight is less or equal to 1000 kg), microsatellites (from 10 to 100 kg), nanosatellites (1 −10 kg), and picosatel- lites (0.1 −0.99 kg) [1].h So far, the main drivers of small satellites develop- ments have been Earth observation and remote sensing, as they may greatly contribute to filling the gap of data poverty in many industry verticals (e.g., agriculture, disas- ter management, forestry, and wildlife). Nevertheless, new investments in developing mega-constellations (hundreds) of pico/nanosatellites [1] for providing global communica- tions, the increased role of satellites in Machine-to-Machine (M2M) communications [2], and the interest in taking advantage of one of the main possibilities enabled by small satellites, which is the development of distributed systems with interconnected satellites, are moving the attention also towards the telecommunication aspects. Therefore, this paper provides an overview of recent advances and development trends in the field of small satellites, with a special focus on These technological trends have allowed new oppor- tunities in the space market and the implementation of long-awaited projects that have been postponed or sup- pressed for years due to high inherent costs. More impor- tantly, a new space rush has been originated by these Wireless Communications and Mobile Computing 2 2 4500 4000 3500 3000 2500 2000 1500 1000 500 0 Revenue ($million) 1964 1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 Year INMARSAT EUTELSAT INTELSAT 900,000 800,000 700,000 600,000 500,000 400,000 300,000 200,000 100,000 0 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Consumer Internet SME Broadband enterprise Figure 1: INTELSAT, EUTELSAT, and INMARSAT annual revenues (left). Annual sales of VSAT terminals by type (right) [7]. Wireless Communications and Mobile Computing Wireless Communications and Mobile Computing Specific prices into SSO for two launcher categories Source : Euroconsult’s 2016 Prospects for the Small Satellite Market k$/kg 80 70 60 50 40 30 20 10 0 Falcon 9 Soyuz Dnepr Rockot PSLV Vega Firefly Rocket Lab Virgin Dedicated smallsat launches Shared smallsat launches Figure 2: Specific prices for two launcher categories [10]. Specific prices into SSO for two launcher categories Source : Euroconsult’s 2016 Prospects for the Small Satellite Market k$/kg 80 70 60 50 40 30 20 10 0 Falcon 9 Soyuz Dnepr Rockot PSLV Vega Firefly Rocket Lab Virgin Dedicated smallsat launches Shared smallsat launches Figure 2: Specific prices for two launcher categories [10]. Moreover, three constellations for satcom and Earth observation accounted for 38% of the total and this share should grow up to 68% in the next 5 years driven by several large projects. As a result, this analysis unveils the main booster of the massive development of the small satellites: the ability to relatively easily build a constellation. This peculiar aspect will be considered in the following sections with a specific focus on the intersatellite communications. pi Finally, it is worth introducing the main player of the massive increase of the number of small satellites: the Cube- Sat. The CubeSat has been designed as the goal of a Stanford University program which was started in 1999 to obtain a very low-cost/weight satellite which could be quickly developed and used for educational purposes [3]. Together with the California Polytechnic State University, Stanford University developed CubeSat specifications with the goal to obtain a customizable satellite, but with standard shape and weight, in order to simplify launch and deployment operations. As it is known, a CubeSat is made by one (1U) or more (nU) 10 cm X 10 cm X 10 cm units, with a mass of up to 1.33 kg per unit [11]. The nature of CubeSats has enabled the standardised production of subsystems that can even be purchased as a COTS product from online shops, so keeping the mission cost very low [9].h Figure 2: Specific prices for two launcher categories [10]. Wireless Communications and Mobile Computing Smallsat <500kg demand between 2010 2020 OneWeb Cubesat constellations Single cubesat Single satellite mission >10 kg Large EO constellation >20# Other constellation Source : Euroconsult’s 2016 Prospects for the Small Satellite Market 400 350 300 250 200 150 100 50 0 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 Figure 3: Number of small satellites in the last 15 years. [10]. Smallsat <500kg demand between 2010 2020 These peculiar features of the CubeSat solution have been very important in the fast increase of the small satellite missions and in the huge development of companies whose main core is in the new space market, such as Terra Bella (formerly Skybox Imaging), Spire, Planet Labs, and OneWeb, who are developing mega-constellations of small spacecrafts in LEO orbit [9]. 3. Overview of Services and Applications Around the year 2000, the SmallSats were able to properly exploit innovative COTS technological solutions (hardware and software), achieving the ability to compete effectively and to make profit. The successful growth of the modern Small- Sats services, encompassing a large variety of application contexts, shall be analysed also based on a new management approach the small satellite organizations started to adopt: the agile methodology. This paradigm comes from the IT indus- try and it is based on a highly iterative design technique: well- defined objectives, missions and requirements, incremental changes to the design for a continuous improvement of the system performance, short timescale, and reduced cost. Agile approaches and the exploitation of the latest off-the-shelf technologies represent the two main drivers of the New Space Age [4]. More interestingly, these early attempts have paved the way to a new philosophy which has been aimed at implement- ing and exploiting very-low-cost satellites [10]. As reported in [3], the estimated production and launch cost for a single small satellite can be approximately assumed to span from 100, 000 to 200, 000 USD: particularly, in the case of shared small satellite, the launch unitary costs per 𝑘𝑔can lower down to few kEuros, as sketched in Figure 2. As a result, these unprecedented features of small satellites have been favourably considered by market forces which have been largely stimulated in the last thirty years and pushed to start a new gold rush in space, with original objectives and well-targeted applications such as Earth observation and communications, in the civil, military, and commercial dominions. Since the possible applications will be reviewed in the following section, it is now important to provide some rough numbers that can give the idea of how strong the new race to the space exploitation is. In the following, a brief overview of the main applications and services of the SmallSats is provided. Since some of the following acronyms may be unknown to the reader, a comprehensive list is provided in Table 1. Overall, 551 satellites (< 400𝑘𝑔) have been launched between 2010 and 2015, while the expectations for the succes- sive five years are targeted on other 1380 launches [10]: these trends are shown in Figure 3. These numbers are confirmed by the 300 launches of nano/microsatellites which have been recorded in 2017 [1]. (1) Earth Observation and Remote Sensing. 2. A Brief History of Small Sats Evolution From the dawn of the space era to their latest developments, satellite communications have been one of the most reli- able indicators of the technical and societal evolution: as 3 3. Overview of Services and Applications AcronymDefinition Acronym Definition ACM Adaptive Coding and modulation ADC Analog to Digital Converter BP Bundle Protocol CCSDS Consultative Committee for Space Data Systems COTS Commercial Off the Shelf DLR German Aerospace Center DSA Dynamic Spectrum Access DSP Digital Signal Processor DTN Delay Tolerant Network DVB-S2 Digital Video Broadcasting - Satellite 2nd generation EDRS European Data Relay System ELaNa Educational Launch of Nanosatellites EO Earth observation ESA European Space Agency FEC Forward Error Correction FPGA Field Programmable Gate Array FSK Frequency Shift Keying GEO Geostationary Orbit GPP General Purpose Processor GRACE Gravity Recovery and Climate Experiment GSTP General Support Technology Programme HTS High Throughput Satellite HTTP Hypertext Transfer Protocol HW Hardware ICN Information-centric networking IOT Internet of things IP Internet Protocol ISS International Space Station IT Information technology JPL Jet Propulsion Lab LDPC Low Density Parity Check LEO low Earth Orbit LTP Licklider Transmission Protocol LUCE LUnar Cubesats for Exploration LUMIO Lunar Meteoroid Impacts Observer MAC Mean Access Control MarCO Mars Cube One MEC Multi-Access Edge Computing MEO medium Earth Orbit MMIC Monolithic Microwave Integrated Circuit M2M Machine-to-Machine NASA National Aeronautics and Space Administration NC Network Coding NEA Scout Near-Earth Asteroid Scout NFV Network function Virtualization OPALS Optical Payload for Lasercomm Science OSIRIS Optical Space Infrared Downlink PICASSO Pico-Satellite for Atmospheric and Space Science Observations PRETTYPassive REflecTomeTrY QARMAN QubeSat for Aerothermodynamic Research and Measurements on Ablation QKD Quantum Key Distribution (QKD) QoS Quality of Service QoE Quality of Experience SCAN Space Communications and Navigation SDN Software Defined Networking SDLS Space Data Link Security SDR Software Defined Radio SOTA Small Optical Transponder SW Software TBIRD Terabyte Infrared Delivery TCP Transmission Control Protocol TTL Time-To-Live UHF Ultra High Frequency USD US Dollar USLP Unified Space Link Protocol VHF Very High Frequency VLC Visible Light Communications VMMO Volatile and Mineralogy Mapping Orbiter ESA CubeSat missions have been funded under the In- Orbit Demonstration part of the General Support Tech- nology Programme (GSTP): GOMX-3 and GOMX-4B for demonstrating new capabilities of nanosatellites, QARMAN (QubeSat for Aerothermodynamic Research and Measure- ments on Ablation) for demonstrating re-entry technologies, PICASSO (Pico-Satellite for Atmospheric and Space Science Observations) for the analysis of the ozone distribution in the stratosphere, the temperature profile up to the mesosphere and the electronic plasma characterization in the ionosphere, RadCube for real-time monitoring of the cosmic radiation and space weather environment, and PRETTY (Passive REflecTomeTrY), a nanosatellite to measure and register ice on the glaciers or on the poles and wave movements of the oceans. 3. Overview of Services and Applications So far, the primary use for nano/microsatellites has been Earth observation (EO) and remote sensing. The implementation of large satellite constellations allows performing many simultaneous and distributed measurements or observations (Earth resources Wireless Communications and Mobile Computing 4 Table 1: List of the acronyms. 3. Overview of Services and Applications monitoring, weather monitoring, and disaster monitoring) with an increased temporal resolution of collected data (i.e., shorter revisit times) [12]. A more extensive use of small satellites for EO and remote sensing calls for higher and higher data rate links to download the acquired information in a short time. (2) Science and Technology Demonstration Missions. Micro- and nanosatellites enable a wider access to space and rep- resent an affordable test for young engineers and scientists to prove prototype systems and experience the idea of a future satellite. To this aim, NASA created the NASA Educational Launch of Nanosatellites (ELaNa), an initia- tive oriented to students of several disciplines (science, technology, engineering, and mathematics). A number of Wireless Communications and Mobile Computing 5 58% 26% 12% 4% 50% 16% 10% 22% 3% Historical (2013 – 2017) Communications EO/RS Scientific Future (2018 – 2022) Technology Novel Applications EO/RS Scientific Technology Communications Figure 4: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Figure 4: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Fleet Space, and Myriota are some of the main commu- nications operators offering IOT/M2M and data relaying services. (3) Interplanetary Exploration Missions. Small Satellite Plat- forms have led to a new era of space exploration espe- cially thanks to new enabling technologies and new highly capable launch vehicles, which open many opportunities for future lunar and planetary exploration. National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) have adopted the Interplanetary CubeSat Model, supporting missions and studies, ranging from Mars and Lunar observation to the study of meteoroids and asteroids. Some of them are MarCO (Mars Cube One) [13], NEA Scout (Near-Earth Asteroid Scout) [14], LUCE (LUnar Cubesats for Exploration), LUMIO (Lunar Mete- oroid Impacts Observer), VMMO (Volatile and Mineralogy Mapping Orbiter), Lunar Flashlight [15], and Arkyd series [16]. An overview of the nano/microsatellite trends by appli- cation in the near term is provided in Figure 4 [1]. Although the analysis highlights that the primary use for nano/microsatellites will remain Earth observation and remote sensing, an increase of communications constella- tions is expected. SpaceWorks estimates that about 700 com- munications nano/microsatellites will require launch over the next 5 years. (5) Commercial, Civil, and Military Applications. 3. Overview of Services and Applications Transport, smart environments (including remote monitoring), quality of life, safety, and security represent the main application con- texts of the adoption of small, micro-, and nanosatellites [17]. As examples of commercial constellation of nanosatellites, Aerial & Maritime and Sky & Space Global are two GomSpace commercial missions: the former is oriented to aircraft and vessel tracking for situational awareness, while the latter will provide a global communication infrastructure in space. Moreover, Astrocast has a project for offering global M2M services as remote monitoring, geolocalization, intelligent data collection, and predictive maintenance [18]. (4) Communications Services. Small micro- and nanosatellites organized in constellations can be used for providing data distribution (broadcasting applications) and data exchange (Internet of things and Machine-to-Machine paradigm) and also for extending the Internet access to the entire Earth [11]. According to Space Works Market outlook, in the next years, communication constellations of micro- and nanosatellites, which are now in the technology demonstration phase, will be used to serve and support the rapidly growing Internet of things (IOT) and Machine-to-Machine (M2M) market. Sky & Space Global, Kepler Communications, Hiber (Magnitude Space), Helios Wire, Astrocast, Blink Astro, Figure 5 shows the SpaceWorks analysis on the Nano/ Microsatellite Operator Trends: Military Operators (aiming to support national defense activities), Commercial Opera- tors (whose purpose is profit revenue generating activities), 6 Wireless Communications and Mobile Computing 0% 25% 50% 75% 100% Historical (2013 - 2017) Future (2018 - 2022) Percentage of Satellites Civil Military Commercial Commercial operators are expected to encompass over 70% of nano/microsatellites launched in the next 5 years Despite rapid commercial market share growth, civil and military operator demand is expected to remain consistent over the next 5 years Figure 5: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Figure 5: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Figure 5: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWo and Civil Operators (nonmilitary or non-profit activities) are considered than S-band, the efficiency of solid-state high-power ampli- fiers drops from 80% (at UHF) to 30%. At such frequencies, most common antennas are still wire antennas or planar antennas, such as patch and slot antennas. Patch antennas have gained special attention for CubeSats, as they are relatively easy to fabricate. A variety of patch antenna designs have been investigated at S bands. 3. Overview of Services and Applications Downlinks on S-Band would be expected to be able to implement data rates from 100 kbps to 1 Mbps. Larger data rates require the use of higher- frequency bands such as Ku-, K-, and Ka-band, which are the state of the art for large spacecrafts, but they are still young technologies in the small satellite world. A Ka-band trans- mitter on a CubeSat began orbital operations in 2015 [21]. At such higher frequencies, it is possible to implement also high- gain reflector antennas which can meet the strict size and weight requirements of a small satellite. Reflectarray antennas are also very suitable, as they provide high gain and can be easily integrated with the CubeSat structure. Their structure consists of flat panels, which can be folded and stowed on the CubeSat [5]. In [22], the development of a reflectarray for the Mars Cube One (MarCO) is described. MarCO is the first CubeSat mission designed for Mars operation. The frequency of operation in this case was 8.425 GHz, with a measured gain of 29.2dB. 4. Payload Evolution In the early implementation of small satellites, mainly used as a platform for university and technology development projects [19], the payload was supposed to perform very simple operations such as transmission of a beacon, storing data or transmitting data collected by simple sensors at very low data rate (1 to 9.6kbps). Amateur frequencies at UHF were mainly used and operated via the standard AX.25 [20]. At such low frequencies, wire antennas (dipoles, monopoles, and helical) are especially common as the wavelength is long and achieving good radiation efficiency within a small volume is challenging. A considerable number of the CubeSats that are currently in space use wire antennas for their simplicity of implementation. Moreover, the omnidirectionality of dipoles makes them viable candidates for intersatellite communi- cations. Emerging applications and the associate need for transmitting at higher data rate or performing more complex tasks, keeping low mass and weight, have raised the need for larger bandwidths and higher-frequency bands, an increasing request of digital implementation and SW control. As a matter of fact, small satellites also represent a viable and cost-effective way to test new frequency bands for satellite communications (both in terms of HW components and propagation channel), such as W-band [23]. The investigation of such high-frequency bands is mainly motivated by the need of bandwidths in High Throughput Satellites [24]. On the other hand, those frequencies could be an interesting option for intersatellite links of small sats [25]. At Q/V and W-band, horn antennas can be a viable option for small sats as they provide good gain and could be fabricated also for uni- versity experiments [26]. A potential horn design that could 4.1. Evolution in the Frequency Bands and Antennas. In recent years, the use of higher frequencies than the common VHF/UHF bands such as S-Band (mainly for telemetry) and X-Band (for data transmission) has become more widely available thanks to the advent of commercially available Monolithic Microwave Integrated Circuits (MMICs). The shift towards higher-frequency bands implies other require- ments on the spacecraft design, mainly on the power system and the antennas. For instance, already at frequencies higher 7 Wireless Communications and Mobile Computing Figure 6: Left: CAD-model of OSIRIS4Cubesat terminal. Right: Artist’s impression of terminal integration in 1U Cubesat [35]. CAD-model of OSIRIS4Cubesat terminal. Right: Artist’s impression of terminal integration in 1U Cubesat [35]. 4. Payload Evolution be considered for future Ka-band CubeSat communication is discussed in [27, 28].h the order of 300 g, consumes 8 W of electrical power, and requires only 0.3U of space within the CubeSat. It reaches a data rate of 100 Mbps. OSIRIS4CubeSat will be offered on the market by DLR’s commercialization partner Tesat Spacecom under the market name CubeL. The need of higher data rates, low cost, and small size has also moved the attention towards FSO communications, especially for intersatellite links as presented in the next section. An important challenge in optical satellite-to-ground communications is the limited availability due to clouds. This can be overcome by employing a world-wide network of optical ground stations. By using a sufficient buffer memory onboard the satellites, this enables overcoming the issues due to limited availability of the space-to-ground link [36]. Although most optical ground stations available to date have been developed mainly for research purposes, both new and established ground segment operators have expressed strong interest in building up the required infrastructure. Thus, it is only a matter of time until optical links can be used in an operational manner, even in small-satellite applications. 4.2. Laser Communication Terminals for Small Satellites and CubeSats. In recent years, free-space optical communica- tions have become a mature alternative to traditional RF com- munication systems. With the use of laser communication terminals in systems like the European Data Relay System (EDRS) [29] for intersatellite links, the technology has passed the barrier from research to the operational application. Concerning downlinks from satellite to Earth, a number of demonstrations have been performed in recent years, like the Small Optical Transponder (SOTA) experiment [30] of the National Institute of Information and Communications Technology, which used a dedicated satellite, or the Optical Payload for Lasercomm Science (OPALS) experiment [31] of the NASA’s Jet Propulsion Lab (NASA-JPL), which demon- strated optical downlinks from the International Space Sta- tion (ISS). The Aerospace Corporation demonstrated an optical downlink from a 1.5U-CubeSat [32]. Even optical links from the Moon to Earth have been demonstrated [33]. 4.3. Towards SDR Payloads. Since the early development of small satellites, one trend in the payload design can be identified: privileging the use of low-cost COTS and in general HW components and moving towards a digi- tal implementation. Wireless Communications and Mobile Computing 8 to Digital Converters (ADCs), General Purpose Processors (GPPs), Digital Signal Processors (DSPs), and FPGAs. SDR payloads are considered as a needed technological step in traditional satellite systems for assuring a longer lifetime and a more efficient resource utilization [37], even if so far, few SDR payloads have flown on big satellites. For small satellites, which are designed with few years of lifetime in mind, the reason for moving towards SDR payloads is mainly related to the offered flexibility to adapt to new science opportunities and potentially reducing development cost and risk through reuse of common space platforms to meet specific mission requirements. SDR can be used to support multiple signals, increase data rates over reliable intersatellite and ground links to Earth, and also help in facing the shortage of available frequencies for communications in the more crowded bands. As a matter of fact, the use of an SDR approach also allows implementing Dynamic Spectrum Access (DSA) techniques and hence a more efficient spectrum utilization. To date, no satellite application of DSA is in use, although companies such as Tethers Unlimited (U.S.), with funding from NASA, are looking at upgrading SDR platforms with advanced cognitive radio.h (i) They are increasingly used to form application- focused segments of the infrastructure supporting existing communication architectures, notably the Internet. (ii) They also form and/or utilize altogether new, distinct communication architectures. (ii) They also form and/or utilize altogether new, distinct communication architectures. 5.1. As Supporting Infrastructure. The use of Earth-orbiting satellites to conduct Internet traffic is of course not new. From TELSTAR in 1962 through Iridium, Globalstar, ViaSat, and EchoStar, the market for relaying data via radio links to satellites has grown rapidly. But historically, those satellites have been large and expensive, whether operating in LEO or GEO orbits. What is new is the use of large numbers of small satellites for this purpose. The field has grown rapidly in recent years as new concepts are proposed; many of them highly ambitious: (i) The OneWeb constellation is initially expected to comprise 882 small Internet service delivery satellites in LEO orbit, potentially growing to 2620 satellites [42]. The challenge of this more digital approach is related to one of the strong limitations of a small satellite: power consumption. Wireless Communications and Mobile Computing For this reason, FPGA has been preferred so far, especially for higher data rates in the X- and Ka-band, as they allow performing compute intensive tasks in parallel and use more efficiently every clock cycle [38]. Additionally, modern FPGAs have embedded processing systems, such as ARM cores, integrated inside the FPGA. Few SDRs have already flown in small satellites and other are under development, e.g., AstroSDR, NanoDock SDR, GAMALINK, and STI-PRX-01. It is definitely a hot topic of research and development, and there is growing interest in developing and testing new solutions. Pinto et al. [39] exploited SDR in small satellite systems to design an intersatellite communication model which could be easily reconfigured to support any encoding/decoding, modulation, and other signal processing schemes. In [40], a novel SDR architecture on an embedded system is proposed, whose potential applications are the ground station for multisatellite communications, deployable mobile ground station network, and can be further extended to distributed satellite system. (ii) Samsung has proposed a 4600-satellite constellation, projected to be able to carry one billion terabytes of Internet data per month [43]. (iii) The SpaceX corporation’s Starlink constellation is envisioned to comprise up to 12,000 small satellites in LEO orbit, with the capacity to carry up to 10% of local Internet traffic in densely populated areas [44]. 5.2. As Participants in New Architectures. In addition to supporting the propagation of traffic within the Internet, however, small satellites require new, increasingly capable telecommunication architectures to sustain their own oper- ations. Coordination among satellites in LEO orbit relies on cross-links between satellites, relay services provided by ground stations (typically via the terrestrial Internet), or a combination of both. This capability is critical for constellations such as the GRACE (Gravity Recovery and Climate Experiment) mission and the QB-50 project.t 5.2. As Participants in New Architectures. In addition to supporting the propagation of traffic within the Internet, however, small satellites require new, increasingly capable telecommunication architectures to sustain their own oper- ations. Coordination among satellites in LEO orbit relies on cross-links between satellites, relay services provided by ground stations (typically via the terrestrial Internet), or a combination of both. Wireless Communications and Mobile Computing This capability is critical for constellations such as the GRACE (Gravity Recovery and Climate Experiment) mission and the QB-50 project.t Moving farther, the twin MARCO spacecrafts (each a 6U CubeSat) accompanying the InSight spacecraft on its mission to Mars will primarily serve to relay information from the InSight lander to its mission operations center on Earth, while the lander is engaged in entering the atmosphere of Mars, descending to the surface, and landing. As shown in Figure 7, the link from InSight to each MARCO orbiter will be in the UHF band, while the MARCO vehicles communication with Earth will be by X-band radio transmission. Each MARCO can use only one of these links at a time, so the communication architecture will be very different from the continuous end-to-end connectivity that characterizes Internet traffic.fi A new generation of SDR technologies have been inte- grated in the SCAN Testbed (Space Communications and Navigation Testbed), which is an advanced integrated com- munications system and laboratory facility to be installed on the International Space Station (ISS), to develop, test, and demonstrate new communications, networking, and naviga- tion capabilities in the space environment [41]. The SCAN Testbed consists of reconfigurable and reprogrammable SDR transceivers/transponders operating at S-band, Ka-band, and L-band, along with the required RF/antenna systems neces- sary for communications. fi Projecting that deviation from the Internet traffic model back to high-volume terrestrial communications, a satellite communications architecture that is designed to tolerate the associated delays in end-to-end communication on a large scale has been proposed. The “Ring Road” architecture [45] 4. Payload Evolution Thanks to the availability of modern high-speed and low-power digital signal processors and high speed memories, the trade-off between the HW/SW implementation is moving more and more towards the SW implementation and the concept of SDR. SDR is an evolution of flexible and reconfigurable payloads. An early adopter of reconfigurable technology for space applications was the Australian FedSat microsatellite communications payload launched in 2002. The FedSat communications payload uti- lized Field Programmable Gate Array (FPGA) components for baseband digital signal processing and included a code upload mode allowing it to be reprogrammed while in orbit [9]. The evolution from reconfigurable and reprogrammable devices to SDR has been driven by the demand for flexible and reconfigurable radio communications in support of military and public safety operations and it has been pushed by advances in the enabling technologies such as Analog A number of further demonstration missions are cur- rently planned, such as NASA’s Terabyte Infrared Delivery (TBIRD) mission, aiming at demonstrating a 100 Gbps link from a CubeSat to ground [34], or within the Optical Space Infrared Downlink (OSIRIS) programme of the German Aerospace Center (DLR), which aims at demonstrating opti- cal downlinks from small satellites and CubeSats to Earth [35]. Practical implementations of current optical commu- nication systems for small satellite applications may reach data rates of about 10 Gbps with a terminal weight in the order of 5 kg and a power consumption of about 50 W. For applications on CubeSats, Figure 6 shows OSIRIS4 CubeSat implementation as an example. The terminal weighs in Wireless Communications and Mobile Computing The constellation operates as follows: The constellation operates as follows: The constellation operates as follows: (i) A user at a cold spot node issues data in a bundle (such as an email message or an HTTP proxy query). The node queues the bundle up for transmission to the next courier that flies overhead [48]. (ii) Eventually, a courier flies over the cold spot. The courier’s orbit is well known, so the contact between the courier and the cold spot can be scheduled far in advance. The courier and cold spot begin communi- cations using BP over LTP (Licklider Transmission Protocol [49]; see more details in the next section) over whatever radio frequencies are available. Bundles from elsewhere that are destined for this cold spot called “forward traffic” are transmitted from the courier to the cold spot node for forwarding within the local network, if any. Bundles issued from the cold spot called “return traffic” are transmitted to the courier and queued on-board for future transmission. Figure 7: The MARCO communication architecture. Image credit: NASA/JPL-CalTech. D B E A 2 3 4 1 “cold spot” (no Internet connectivity) “hot spot” (connected to Internet) “courier” (nanosatellite, a “data mule”) C non-RingRoad node on Internet Y non-RingRoad node on local area net X Figure 8: The Ring Road network architecture [45]. D B E A 2 3 4 1 C Y X (iii) The courier computes a route for each bundle it receives from the cold spot. It knows about its own future scheduled contacts, so any bundle that is destined for some other cold spot that the courier will reach before the bundle’s TTL (Time-To-Live) expires is queued for future transmission to that cold spot. All other bundles are queued for transmission to the next hot spot the courier will fly over. (iv) When the courier flies over a hot spot, the queued bundles are transmitted to the hot spot and the courier receives bundles that the hot spot node has queued for transmission to that courier. (v) When a hot spot node receives bundles from a courier, it computes a route for each bundle. If the bundle’s destination endpoint is directly reachable via the Internet (e.g., a database server in Montreal), then the hot spot uses BP over TCP/IP to send the bundle immediately to that endpoint. 5. New Telecommunication Architectures Small satellites are playing an increasingly important role in telecommunication architectures in two main ways: 9 Wireless Communications and Mobile Computing Figure 7: The MARCO communication architecture. Image credit: NASA/JPL-CalTech. (i) Router satellites, called “courier” nodes, in polar orbit (ii) Nodes residing in computers that are attached to the Internet, called “hot spots” (iii) Nodes residing in computers that are highly isolated, with no electronic connectivity, called “cold spots.” Wireless Communications and Mobile Computing As more satellites are added, the frequency of coverage of any given cold spot increases and N drops, which increases the carrying capacity of the network as a whole (the aggregate storage capacity of all the couriers), so that the number of cold spots supported can increase. Adding more hot spots on the ground would also incrementally increase the carrying capacity of the network, by enabling earlier drainage of the return-traffic bundles in couriers’ on- board storage and thereby making room for more bundles, which would further increase the number of supportable cold spots you could support. (i) Unlike a crosslink-based routing-fabric constellation, there is no need to orbit the whole constellation all at once in order to get data moving. The network could begin with one hot spot, one cold spot, and one courier. In that case, the round-trip time for the cold spot would be very long as there would be only one contact per N orbits of the satellite, where N is however many orbits would be needed to bring the cold spot back into the satellites ground track. Nonetheless, bidirectional data flow between the cold spot and any point on the Internet would be reliably supported, albeit at very low effective data rates. As more satellites are added, the frequency of coverage of any given cold spot increases and N drops, which increases the carrying capacity of the network as a whole (the aggregate storage capacity of all the couriers), so that the number of cold spots supported can increase. Adding more hot spots on the ground would also incrementally increase the carrying capacity of the network, by enabling earlier drainage of the return-traffic bundles in couriers’ on- board storage and thereby making room for more bundles, which would further increase the number of supportable cold spots you could support. Still related to the objective of distributing small satellite data across the Internet is providing the network architecture with content-oriented functions in order to differentiate QoS management and routing functions applied to the data objects obtained from the small satellite systems. The constellation operates as follows: Otherwise, the hot spot consults the contact schedule to determine which courier has the earliest scheduled contact with the destination cold spot and then reconsults the contact schedule to determine which hot spot has the earliest scheduled contact with that courier. If the first hot spot that will see that courier is the local hot spot itself, then the hot spot simply queues the bundle locally for future transmission to that courier; otherwise, it uses BP over TCP/IP to send the bundle immediately to that computed best-way-forward hot spot. (v) When a hot spot node receives bundles from a courier, it computes a route for each bundle. If the bundle’s destination endpoint is directly reachable via the Internet (e.g., a database server in Montreal), then the hot spot uses BP over TCP/IP to send the bundle immediately to that endpoint. Otherwise, the hot spot consults the contact schedule to determine which courier has the earliest scheduled contact with the destination cold spot and then reconsults the contact schedule to determine which hot spot has the earliest scheduled contact with that courier. If the first hot spot that will see that courier is the local hot spot itself, then the hot spot simply queues the bundle locally for future transmission to that courier; otherwise, it uses BP over TCP/IP to send the bundle immediately to that computed best-way-forward hot spot. “cold spot” (no Internet connectivity) “hot spot” (connected to Internet) “courier” (nanosatellite, a “data mule”) non-RingRoad node on Internet non-RingRoad node on local area net Figure 8: The Ring Road network architecture [45]. is based on the use of delay-tolerant networking (DTN) protocols [46], discussed later. The basic idea is to deploy, gradually, one satellite at a time, a constellation of DTN Bundle Protocol (BP) [47] routers in LEO orbit. As shown in Figure 8, the network encompasses three classes of DTN nodes: (vi) When a hot spot node receives bundles from some node in the Internet (possibly another hot spot), it computes a route for each bundle as above. When a courier flies overhead, it exchanges bundles with the Wireless Communications and Mobile Computing Wireless Communications and Mobile Computing 10 authorities, and single users for different applications (e.g., space data exploitation, education purposes, surveillance and monitoring, etc.). In this context, data retrieval will likely happen over Internet terrestrial infrastructure, hence calling for proper integration strategies to be deployed between the ground segment of the small satellite system and the core terrestrial network. This integration task can be easily considered in the broader plan of converging satellite and 5G networks [50] (and papers included in that special issue), which has recently become a hot topic for the satellite industry. Without entering the details of the architecture proposals [51] elaborated to meet this goal, it is of pivotal importance to provide a flexible integrated architecture. Network flexibility is indeed recommended in order to ensure proper coexistence of existing Internet flows and small satellite data retrieval, which may be regarded in terms of different network slices, each characterised by diverse QoS/QoE characteristics. To this end, the implementation of proper SDN (Software Defined Networking) and NFV (Network Function Virtualization) solutions is desirable, so as to achieve also the “softwarisation” of the satellite network, whose understanding is however still not complete and will deserve additional studies for the case of small satellite constellations. courier. When the courier subsequently flies over a cold spot, it exchanges bundles with it in the same way, and so on. courier. When the courier subsequently flies over a cold spot, it exchanges bundles with it in the same way, and so on. The concept offers a number of advantages: (i) Unlike a crosslink-based routing-fabric constellation, there is no need to orbit the whole constellation all at once in order to get data moving. The network could begin with one hot spot, one cold spot, and one courier. In that case, the round-trip time for the cold spot would be very long as there would be only one contact per N orbits of the satellite, where N is however many orbits would be needed to bring the cold spot back into the satellites ground track. Nonetheless, bidirectional data flow between the cold spot and any point on the Internet would be reliably supported, albeit at very low effective data rates. Wireless Communications and Mobile Computing This may suggest the application of the existing Information Cen- tric Networking architectures [52], whose baseline concept should be however adapted in order to meet the content characteristics of the data objects retrieved from the satellite systems and to interface with the network architecture (e.g., DTN-based) proposed for the satellite network (as illustrated in the previous subsection). (ii) While the routing is somewhat complex, it takes place in potentially powerful ground-based computers at hot spots, not in the courier satellites. This means that small, mass-produced satellites can be suitable as couriers. (iii) All elements of the architecture are, therefore, rela- tively inexpensive. In more detail, ICN-based architectures build on publish- subscribe (pub-sub) paradigms, so that users subscribe to content distributions services and accordingly contents are distributed upon request reception. One of the main peculiar- ities of ICN networks is in that contents are explicitly mapped to object names, which enable more advanced content-aware routing and security schemes. Moreover, this approach helps implement a content-centric networking approach, hence superseding the typically employed host-centric approach (i.e., as implemented in IP-based systems), where locations and content descriptions are mapped into a unique iden- tifier (e.g., IP address), hence posing some limitations on implementing content-based networking functions. Another intrinsic key advantage of ICN networks is to implement distributed caching functionalities throughout the entire network, hence possibly simplifying the integration of MEC (Multi-Access Edge Computing) and Cloud Computing func- tionalities, which are pivotal building blocks in the modern communication networks. As a conclusion, this SmallSat-based architecture could enable very widely available network data service at low cost, starting with a very modest initial investment. 5.3. Integration with Terrestrial Architectures. The potentials offered by Small- and CubeSats constellations from a service point of view have to be analysed from a wider angle in order to consider the data availability from different stakeholders. In the case of processing centres placed nearby control centres or in any case directly connected to them via dedicated terrestrial infrastructure, the architecture design may essentially consist in the extension of the exemplary one illustrated in the previous subsections. This can be achieved by terminating the proposed DTN architecture directly at the processing centres or by making use of specialised gateways capable of interfacing native DTN architectures with non- DTN aware counterpart (i.e., in the case of legacy networks building on pure TCP/IP protocol architectures). Wireless Communications and Mobile Computing ICN functionalities are typically supported by spe- cialised networking elements, i.e., ICN routers, which can be deployed not only in terrestrial networks, but also in the space counterpart, provided that satellites offer the necessary On the other hand, the increasing interest towards the service provided by small satellite constellations may result in distributing data to enterprises, universities, schools, public Wireless Communications and Mobile Computing 11 11 End-Users MEC/Cloud Computing Satellite constellation Message Formats Message Formats Message Formats Message Formats Message Formats Content request Content retrieval 5G Network ICN/DTN terrestrial router ICN/DTN space router Figure 9: Integrated satellite-5G network based on ICN/DTN concepts for content delivery [52]. Satellite constellation ICN/DTN space router 5G Network Content request Figure 9: Integrated satellite-5G network based on ICN/DTN concepts for content delivery [52]. capabilities are provided at multiple layers of the protocol stack. storage and computing capabilities. As a matter of fact, the coexistence of DTN- and ICN-based protocol architectures in the same network deployment is possible in order to exploit the main advantage offered by the two with respect to disrup- tion resilience and caching, although specific adaptations of the protocol interfaces are necessary (not treated in this paper as beyond the scope). 6.1. Physical Layer. Originally, the only communication links supported for CubeSat satellites were UHF links operated via AX.25. Given the low requirements in terms of data rate of most of the original, mainly scientific, missions, simple modulation schemes have been used, such as binary-FSK [53, 54]. It is also worth outlining that the AX.25 protocol allows detecting errors but not correcting them. The emerging need for transmitting at higher data rate and keeping low mass and weight is pushing to use larger bandwidths and higher-frequency bands, as reported in Section 4, but also to use more efficiently the available bandwidths through more advanced modulations schemes. Moreover, the shift towards SDR payload and ground stations, made possible by the rapid evolution of digital electronics, opens the opportu- nity to implement more advanced communication protocols and modulation schemes, including error correction capa- bilities and dynamic adaptation of modulation parameters In general, the overall network architecture encompass- ing 5G and satellite segments, building on ICN/DTN archi- tectures, and interacting with MEC and cloud computing elements is exemplified in Figure 9, where the case of a satellite constellation complementing a 5G access network to boost the content delivery is sketched. Wireless Communications and Mobile Computing 12 depending on the current link conditions [55]. This has motivated some theoretical studies on the choice of the most appropriate modulations [56, 57]. However, there are already some innovative transceiver designed for CubeSat and Small Satellites using higher-frequency bands such as X-band up to Ka-band, implementing Variable and Adaptive Coding and Modulation (VCM, ACM) capabilities [58, 59]. For instance, RADIOSAT is an innovative transceiver developed by ESA, working at a Ka-band and integrated with a DVB-S2 modem, overall characterized by low power consumption. On the design of intersatellite link, it is worth mentioning the recent studies on the use of Visible Light Communications (VLCs), which can provide higher data rates with smaller, light- weight nodes, while avoiding the usual interference problems associated with RF, as well as the apparent radio spectrum scarcity below the 6 GHz band. Furthermore, the electronics required for achieving precision pointing accuracy for laser communication systems will be avoided. With approximately 300 THz of free bandwidth available for VLC, high capacity data transmission rates could be provided over short dis- tances using arrays of LEDs [60]. central fact in both circumstances is the potential inability of each network node to request timely assistance from any other, for any purpose, and at any given moment. The unifying principle in the design of the features of DTN, then, is recognition of this fact. Nodes must be able to make their own operational decisions locally, on their own, with global information that may well be stale or incomplete, and the network must be able to continue to operate at some useful level even when these decisions are flawed. The core protocol of DTN is BP [47], a network-layer protocol that functions as the DTN analog to the Internet Protocol. BP is similar to IP in that a BP node receives data issued by an application entity, stores the data in some medium, and forwards the data through the network toward the node serving the application entity that is the destination of the data. It principally differs from IP in that a forwarding node does not immediately discard data items (called “bundles”) for which no onward communication link is currently available; instead, it may store bundles for a lengthy period of time, waiting for a link to become available. The DTN analog to the Internet’s TCP is the LTP [49]. Wireless Communications and Mobile Computing An LTP “engine” divides an outbound bundle into small “segments” and transmits the segments to the LTP engine serving the BP node that BP has determined to be the best next proximate destination for the bundle the next step on the bundle’s end-to-end path. Both LTP and TCP account for transmitted data, detecting data loss and automatically recovering from that loss by retransmitting segments as necessary. The principal difference between LTP and TCP is this: 6.2. Link Layer. While operators of Earth-orbiting CubeSats initially had few options beyond AX.25, new and more capable protocols that are suited for space flight operations at Earth and beyond Earth orbit are becoming available. The new CCSDS Unified Space Link Protocol (USLP) [61] is designed to be adaptable to a very wide range of space data transmission conditions. It includes a “virtual channels” concept that enables a single physical link to be transparently shared among multiple data streams at higher layer, together with further multiplexing accommodation multiplexer access points that enable multiple data services to share a single virtual channel. It also provides mechanisms for aggregating small service data units and segmenting those aggregations, for extensive control over the sizes of protocol data units. CCSDS has also defined a security service at the link layer, called Space Data Link Security (SDLS). Security is rapidly becoming an urgent concern of space flight mission designers, as security breaches at ground stations and mission operations centers served by the Internet grow ever more troublesome. SDLS provides a security standard for simple space flight missions, where a single spacecraft is in contact with its control center through a ground station. It includes data origin authentication, connection and connectionless confidentiality, connection integrity with and without recov- ery, and connectionless integrity. (i) In TCP, the entity that discovers and reports data loss is the TCP instance serving the application entity that is the destination of the data, and the data loss is reported to the TCP instance that serves the application entity that was the source of the data. That is, retransmission is “end-to-end” and TCP is situated above IP in the Internet protocol stack. (ii) In a space flight mission scenario, end-to-end retrans- missions could result in extremely lengthy delays in data delivery because the source and destination of data might be on different planets separated by many light minutes of propagation latency. 6. Advances in Communications and Network Protocols New protocols for communication with and among small satellites have emerged rapidly in the past decade. The new Wireless Communications and Mobile Computing Wireless Communications and Mobile Computing In LTP, data loss is instead reported to the LTP instance at the prox- imate source of the data (the immediately prior BP node on the end-to-end path), which retransmits the lost segments as early as possible. LTP retransmission is “point-to-point” within the network, and LTP is situated below BP in the DTN protocol stack. 6.3. Network Layer. DTN concepts date back to the early days of the Interplanetary Networking Research Group of the Internet Research Task Force. DTN is a network architecture that is aimed at eking as much data communication as possible out of inhospitable networks—in particular, those where link interruptions (whether anticipated or not) are frequent and significant and/or where signal propagation latency is high. The effects of high delay and of disconnection are in fact similar in many ways, and the network architecture features developed for DTN serve to mitigate both. The 6.3. Network Layer. DTN concepts date back to the early days of the Interplanetary Networking Research Group of the Internet Research Task Force. DTN is a network architecture that is aimed at eking as much data communication as possible out of inhospitable networks—in particular, those where link interruptions (whether anticipated or not) are frequent and significant and/or where signal propagation latency is high. The effects of high delay and of disconnection are in fact similar in many ways, and the network architecture features developed for DTN serve to mitigate both. The Complementary to the use of DTN protocol solution is the exploitation of network coding (NC) [62] for improving the robustness of data transmission as well as optimised use of the available network resources (i.e., bandwidth). Taking as reference the case of Ring Road network model for small satellite constellations, network coding can be applied on all the network nodes (i.e., on the space and ground segments) [63]. In this case, network coding functionalities would actually consist in online (on fly) encoding and decoding functions. In more detail, each NC-enabled node will be in 13 Wireless Communications and Mobile Computing entire network, but only limited to the network legs exhibiting more challenges from a communication reliability point of view. As such, no re-encoding functionalities are necessary (as those made possible by random linear network coding) and on the contrary classical packet layer FEC solutions can be considered, i.e., based on LDPC or Reed-Solomon codes. Wireless Communications and Mobile Computing In this respect, some proposals have been already worked out by CCSDS with reference to the case of erasure codes applied space downlinks [67], where the potential of LDPC-based erasure codes was exploited especially for the case of free-space optical link communications. Although in this case, network coding is implemented only on specific links, the node capability to implement encoding/decoding functionalities as well as to store data prior to processing functions is certainly an important requirement to be taken into account in the system design phase in the light of the typically resources-constrained implementations of nodes in space. Other activities looking into implementation of net- work coding for intersatellite links have been also considered, although the aforementioned constraints coming from the space segments were not completely taken into account, hence requiring additional study for a deeper understanding of all underlying implications and requirements. charge of collecting a given number of information packets and to encode them so as to generate a certain number of redundancy packets, where the overall network coding configuration plays an important role in what concerns both the specific number of input information and output redundancy packets as well as the adopted coding strategy [62]. In this respect, the use of random linear network coding has gained quite some popularity in the last two decades, so that it is nowadays considered on the most appealing approach to implement NC in real network deployments. In particular, the application on random linear network coding of data chunks to be dumped to ground stations would help increase the reliability of data exchange against sporadic fluctuations of the transmission channel quality. Moreover, the network coding can be also exploited to transmit a reduced number of data packets, hence improving the actual bandwidth utilisation. This advantage can be particularly relevant if multicast data communications are exploited [64, 65], so that the performance advantages recognised for network coding can be fully exploited. g y p On the other hand, in spite of the aforementioned advantages, it is also worth considering the complexity implications arising from the implementation of network coding on the space segment [66]. As a matter of fact, network coding implementation requires some dedicated computation capability for online coding functions as well as specific on-board storage to keep temporary copies of the data chunks being subject to encoding or decoding procedures. Wireless Communications and Mobile Computing Moreover, some attention has to be also paid to the protocol layer wherein network coding is being applied, so that often either (i) layered or (ii) integrated approaches are considered [67]. In the former, NC is implemented as a dedicated shim layer placed in between existing protocol layers in order to have a limited increase in the overall system implementa- tion. In the latter, instead, NC functionalities have to be incorporated in an existing protocol, hence increasing the overall implementation complexity. Another point relates to the actual position of NC functionalities in a protocol stack, for which no specific consensus has been reached yet. On the one hand, it would be desirable to keep NC implementation as much closer as possible to the lower layers of the protocol stack (i.e., datalink) in order to have a more efficient recovery of possible packet losses. On the other hand, implementing NC in the upper layers of the protocol stack would help matching more precisely the characteristics of data services and eventually also meet the corresponding QoS requirements. In this respect, a good compromise could be to implement NC functionalities directly within the bundle protocol or immediately beneath it as part of any of the convergence layers (i.e., UDP or LTP) considered for that specific mission design. As such, it is immediate to see that all these requirements have to be properly taken into account in the full system design, with respect to the capabilities offered by existing satellite payloads and the actual service requirements to be targeted by the considered system. 7. Perspectives and Open Challenges The paper has reviewed the state of the art of small satel- lite systems, highlighting the distinctive features enabling novel applications and focusing on telecommunication services.h The provision of advanced Internet services through mega-constellations of pico/nanosatellites is going to become reality in the near future. However, several challenges must be faced yet, which are summarized in the following. (i) Physical Layer. (a) The use of frequency bands higher than Ka- band and the use of free space optical (FSO) communications for Earth-satellite links (i.e., not only for intersatellite links), as reported in Section 4, raise one important challenge: the propagation channel can be strongly attenuated. Both for high frequency RF transmission and for FSO, this issue could be overcome by pro- viding a ground network with a high number of ground stations at highly diverse sites. The concept of site diversity has been extensively studied in the field of High Throughput Satel- lite (HTS), and recent works have highlighted the fact that SDN paradigm could provide the gateways implementing the concept of Smart Diversity, a high level of reconfigurability that could allow efficient resources allocation during traffic switching events [68]. (b) Besides the few theoretical studies mentioned in Section 6.1, and some transceiver implementing ACM techniques, much more work is needed to design optimized modulation and coding (b) Besides the few theoretical studies mentioned in Section 6.1, and some transceiver implementing ACM techniques, much more work is needed to design optimized modulation and coding Another interesting point related to the use of network coding in the proposed network architecture is about their use in the form of [67] for mitigating packet losses. In this case, network coding is not implemented throughout the 14 Wireless Communications and Mobile Computing 14 schemes able to satisfy strict requirements in terms of mass, weight, size, and power con- sumption. which show the feasibility of ground-to-space quan- tum key distribution (QKD) [75–78]. QKD uses individual light quanta in quantum superposition states to guarantee unconditional communication security between distant parties. Satellite-based QKD promises to establish a global-scale quantum network by exploiting the negligible photon loss and decoher- ence in the empty outer space. No eavesdropping can take place as the distribution of entangled photons between the ground and the satellite is used to certify the quantum nature of the link. By placing the entangled photon source on the ground, the space segments contain “only” the less complex detection system, enabling its implementation in a compact enclosure, compatible with the 12U CubeSat standard [75]. (vi) Adoption of the SDN/NFV. It is clear that SDN/NFV paradigms will play a key role in the integration of satellite systems with 5G. However, the use of SDN/NFV in a network of small satellites has yet to be investigated; as discussed in Section 6, it could be important. Indeed, small satellite network deployments could accelerate the infusion of SDN concepts into satellite systems. For instance, on- board SDN-compatible routers could be developed and operated on small satellites as router functions migrate into software. It is worth mentioning that 3GPP Service and system Aspects (SA) activities have identified satellite systems both as a possible solution for stand-alone infrastructure and as complements to terrestrial networks [79]. In this framework, HTS systems could play a key role in some of the 5G application scenarios once they will be able to provide extremely high data rate. However, in many other 5G applications scenarios that focus on M2M communications or require extremely low latency, only small satellite constellations can really provide an effective complement to terrestrial systems. It is crucial to effectively face the challenges discussed above in order not to miss the opportunities offered by the 5G ecosystem. The challenge is still open. Interesting works are ongoing on the use of physical layer approaches to security in satellite communications [73, 74]. No specific work on application of physical layer security to CubeSat can be found; even this could open novel solutions to overcome the challenges of security in the small satellite framework. (i) Physical Layer. In [76], a LEO satellite has been developed and launched to implement decoy-state QKD with over kHz key rate from the satellite to ground over a distance up to 1200 km, which is up to orders of magnitudes more efficient than that expected using an optical fiber (with 0.2 dB/km loss) of the same length. In [78], it was demonstrated that a 4 kg CubeSat can generate a quantum-secure key, which has so far only been shown by a much larger 600 kg satellite mission. (ii) MAC Layer. In view of emerging system constraints, the imple- mentation in small satellites of the scheduled and random-access MAC protocols adopted in existing satellite networks needs further investigation. (iii) Upper Layers. Definition is needed for interoperable application- layer protocols to be employed on top of the lower layer satellite protocols, addressing a wide range of application scenarios and traffic data configurations. (iv) Routing over Time. Due to the frequent topology changes in a CubeSat network, successful data delivery will require ample long-term storage at intermediate nodes to deal with satellite link disruptions. (v) Security Issues in LEO Satellite Networks. v) Security Issues in LEO Satellite Networks. Telemetry, command and control messages, and mis- sion specific data are sent through radio links. There- fore, security concerns arise. CubeSats are susceptible to Denial of Service (DoS) attacks as well as eaves- dropping and data can be accessed by unauthorized user. The attacker could send spurious commands causing excessive resources consumption, data loss, or mission failure. Security challenges are exasperated by the use of SDR payload which opens the possibility of placing new software on the SDR unit through unau- thorized and potentially malicious software installed on the platform [69]. Another security concern that has been raised recently is related to the use of small satellites that have propulsion systems and they could be hacked and endangering other satellites [70]. As also reported in Section 6, communication protocols currently implemented for CubeSat have almost no security features. Security mechanisms developed for conventional terrestrial networks, characterized by lengthy handshake exchanges and substantial com- putational effort, can hardly be directly applied to networks of small satellites. Power, space, and weight constraints related to CubeSat pose challenges in implementing complicated encryption schemes and computational expensive mechanisms [3]. Scientists are already working on them [71, 72]. (vi) Adoption of the SDN/NFV. References [19] T. Delovski, K. Werner, T. Rawlik, J. Behrens, J. Bredemeyer, and R. 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The effect of <i>Cassia alata</i> L. leaf extract on egg hatching and survival of <i>Pangasianodon hypophthalmus</i> larvae
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The effect of Cassia alata L. leaf extract on egg hatching and survival of Pangasianodon hypophthalmus larvae Iko Imelda Arisa1,3,4*, Sri Agustina2,3,5, and Lisa Handayani1 1 Departement of Aquaculture, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 2 Departement of Marine Science, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 3 Research Center for Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 1 Departement of Aquaculture, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 2 Departement of Marine Science, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 3 Research Center for Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 4Animal Histology and Fish Nutrition Laboratory, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111 I d i y y 3 Research Center for Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 4Animal Histology and Fish Nutrition Laboratory, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111 Indonesia , y , , , 4Animal Histology and Fish Nutrition Laboratory, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111 Indonesia 5Laboratory of Marine Chemistry and Biotechnology of Fisheries, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111 Indonesia Abstract. This study aims to determine the effect of using Cassia alata L. leaf extract on the prevention of fungus on fish eggs Pangasianodon hypophthalmus This research was conducted at the Blangpidie Fish Seed Center. Aceh Barat Daya from July to August 2020. Statistical analysis used a completely randomized design (CRD) method with 7 treatment levels and 3 replications. The treatments were conducted by soaking the fish eggs in the leaf extract for 20 minutes. Those treatments were A (control), B (2.5 ppm), C (5 ppm), D (7.5 ppm) E (10 ppm), F (12.5 ppm), and G (15 ppm). ANOVA test results showed that the addition of leaf extract C. alata had a significant (P <0.05) effect on hatchability and survival of P. hypophthalmus larvae. A concentration of 15 ppm is the best with a hatchability value of 85.33% and a concentration of 10 ppm is the most optimum for the survival value of P. hypophthalmus larvae, namely 92.99%. 2 Material and methods Pangasianodon hypophthalmus is one of the potential aquaculture commodities to be developed. However, according to Muhlis et al. [1], P. hypophthalmus hatchery activities encountered several obstacles, including disease attacks in the egg phase caused by fungi. Fungal infection at the time of hatching eggs affects the damage to the eggs and causes a decrease in hatchability. Furthermore Larasati et al. [2] also mentions that the problem that is often faced in catfish farming is the low degree of egg hatching. Low egg hatchability will have an impact on the supply of catfish seeds which are low, so that aquaculture production activities are also low. The research was carried out in July to August 2020 at the Blang Pidie Fish Seeds Center, Aceh Barat Daya District, Aceh Indonesia. The research design used was a completely randomized design (CRD) consisting 7 treatments and 3 replications. As for the types of treatments tested were as follows: A=immersion without C. alata leaves extract (0 ppm); B= immersion with C.alata leaves extract at a dose of 2.5 ppm; C= immersion with C.alata leaves extract at a dose of 5 ppm; D= immersion with C.alata leaves extract dose of 7.5 ppm; E= immersion with C.alata leaves extract at a dose of 10 ppm; F= immersion with C.alata leaves extract at a dose of 12.5 ppm; G= immersion with C. alata leaves extract at a dose of 15 ppm. Efforts to control disease in cultured fish eggs due to pathogenic infections that use chemicals or antibiotics in the long term can have negative impacts, among others, can cause bacterial and fungal resistance and can pollute the environment [3,4]. So the disease requires alternative treatments such as the use of natural ingredients. One of the natural ingredients that has the potential to inhibit the growth of pathogens in fish is the use of Cassia alata leaves. This plant is known by different names in different countries, namely as Gelenggang (Malaysia), Ketepeng Badak or Ketepeng Cina (Indonesia) and bush ringworm (Australia) and has been used as as traditional medicine to treat various diseases [5,6]. E3S Web of Conferences 339, 01003 (2022) 10thICMR-2ndINSAEF 2021 E3S Web of Conferences 339, 01003 (2022) 10thICMR-2ndINSAEF 2021 https://doi.org/10.1051/e3sconf/202233901003 *Corresponding author: ikoimeldaarisa@unsyiah.ac.id © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) 2.3.2 Hatching rate (HR) Calculation of egg hatching rate parameter in research based on Arfiah et al. [9]:  =          100%  =          100% The data from this study showed that the highest hatchability of fish eggs was found in the 15 ppm treatment, which was 85.33%. This indicates that the antifungal content of Cassia alata leaf extract with a concentration of 15 ppm is optimal in inhibiting the fungus Saprolegnia sp. to infect P. Hypophthalmus fish eggs. C. alata is a traditional medicine that has excellent antibacterial and antifungal properties [20-21]. Previous research Bowo et al. [22] stated that a concentration of 60 ppm C. alata leaf extract showed the best results in inhibiting the growth of the fungus Saprolegnia sp, while for egg hatchability and survival of gouramy larvae the highest was obtained at a concentration of 10 ppm. Where: HR = Hatching Rate (%) 2.3.1 Eggs condition Observation of eggs started after soaking using C. alata leaf extract until the eggs hatched. Eggs infected with fungi will show clinical symptoms in the form of threads like cotton covering the eggs. Observations of eggs that recovered due to fungal infection after soaking were observed every 1 hour interval using a microscope. 2.1 Cassia alata L. leaves extract Cassia alata leaves were washed with clean water and dried under the sun light until the leaves were easily crushed. The dried leaves are ground into flour. A total of ±500 g of C. alata leaves powder was soaked in 3 L 96% PA absolute ethanol for 24 hours. The results of the immersion are filtered (Whattman No.41), the results are extracted using a rotary evaporator, then the extract is stored at -5 °C until it is used for testing. E3S Web of Conferences 339, 01003 (2022) 10thICMR-2ndINSAEF 2021 https://doi.org/10.1051/e3sconf/202233901003 2.3.3 Survival rate Calculation of survival rate parameter in research based on Effendie [10]: ]  =    100 (2)  =    100 (2) Where: SR = Survival Rate (%); Nt = number of test dead fish that died during rearing (ind); No =the total number of test fish observed (ind). 2.3.4 Water quality parameters Several water quality parameters measured during the study were pH, temperature, and dissolved oxygen. pH parameter measurement using a pH meter (PCSTestrTM 35), temperature parameter measurement was carried out using a thermometer (PCSTestrTM 35) and dissolved oxygen parameter measurement using a DO meter (Thermo Scientific Eutech DO 6+). Water quality parameters measurements are carried out every day. Tabel 1. Hatching rate and survival rate of P. hypophthalmus Tabel 1. Hatching rate and survival rate of P. hypophthalmus Treatments Parameters Hatching Rate (%) Survival Rate (%) A (0 ppm) 22.00 ± 4.0a 55.32±16.4a B (2.5 ppm) 37.33 ± 6.1b 74.45±12.3ab C (5 ppm) 45.33 ± 5.0b 81.75±10.7ab D (7.5 ppm) 61.33 ± 7.6c 90.56±9.4b E (10 ppm) 67.33 ± 8.1cd 92.99±8.7b F (12.5 ppm) 75.33 ± 3.0de 90.29±3.9b G (15 ppm) 85.33 ± 5.0e 92.73±1.3b Note:writing different superscripts in the column indicates a significant difference between treatments (P<0,05). hypophthalmus Treatments Parameters Hatching Rate (%) Survival Rate (%) A (0 ppm) 22.00 ± 4.0a 55.32±16.4a B (2.5 ppm) 37.33 ± 6.1b 74.45±12.3ab C (5 ppm) 45.33 ± 5.0b 81.75±10.7ab D (7.5 ppm) 61.33 ± 7.6c 90.56±9.4b E (10 ppm) 67.33 ± 8.1cd 92.99±8.7b F (12.5 ppm) 75.33 ± 3.0de 90.29±3.9b G (15 ppm) 85.33 ± 5.0e 92.73±1.3b 3. Results and Discussion Fish eggs Pangasianodon hypophthalmus 50 healthy eggs that have been fertilized are put into each treatment container. Futhermore, as many as 25 fish eggs that have been infected with fungi and put into each of these containers and then left for 6 hours. After the healthy eggs were infected with the fungus, they were transferred to each container which was given a solution of C. alata leaf extract in accordance with the provisions of the treatment concentration. The eggs were soaked in a solution of C. alata leaf extract for 20 minutes. Cassia alata extract has enormous pharmacological value in supporting the use of traditional medicine in curing several important diseases, including antioxidant, anti-fungal and anti-bacterial properties [11]. Based on the results of the study, it was found that the use of C. alata leaf extract could increase egg hatchability and survival of P. hypophthalmus (Table 1), where the higher the dose of extract used in the treatment will also increase the hatchability of fish eggs. Fatmawati et al. [6] stated that C. alata leaf extract contains chemical compounds that can be used as antimicrobials. Furthermore, Makinde et al [12] said that the antibacterial and antifungal activity of the aqueous and methanolic extracts of C. alata leaves found that they had more antifungal properties than antibacterial. Phytochemical analysis of C. alata extract including flavonoids, tannins, steroids, phenols and alkaloids [13, 14, 15]. Then Roshida et al. [16] stated that tannins are antibacterial by presipitating protein. The antimicrobial effect of tannins through reactions with cell membrane inctivation of enzymes, destruction or inactivation of functions. Meanwhile, flavonoids have antimicrobial properties can form complex compounds with dissolved extracellular proteins and microbial cell walls. Falvonoids also have antibacterial and antioxidant properties that work by increasing the immune system, where leukocytes as antigen-eaters are produced faster and activate the lymphoid system more quickly [17-19]. Note:writing different superscripts in the column indicates a 2.4 Data analysis Research parameter data such as egg hatchability and survival rate of fish were analyzed using Analysis of Variance (ANOVA), if the results were influential then Duncan's test was performed with a 95% confidence level. While the water quality parameter measurement data is presented descriptively. 2 E3S Web of Conferences 339, 01003 (2022) 10thICMR-2ndINSAEF 2021 https://doi.org/10.1051/e3sconf/202233901003 Tabel 2 Water quality parameters measurement data Treatments Temperature (oC) pH DO (mgL-1) A (0 ppm) 26.4 – 26.8 6,7 – 6.5 5.3 – 5.5 B (2.5 ppm) 26.5 – 26.9 6.6 5.4 – 5.6 C (5 ppm) 26.5 – 26.9 6.5 – 6.7 5.4 – 5.5 D (7.5 ppm) 26.6 – 26.8 6.5 – 6.7 5.4 – 5.5 E (10 ppm) 26.6 – 26.9 6.6 – 6.7 5.4 – 5.5 F (12.5 ppm) 26.6 – 26.9 6.5 – 6.7 5.4 – 5.6 G (15 ppm) 26.5 – 26.9 6.5 – 6.7 5.4 – 5.5 5. Y.Y. Ling, P.S. Fun, A. Yeop, M.M. Yusoff, J. Gimbun, Material Todays: Proceedings 19 (2019) 6. S. Fatmawati, Yuliana, A.S. Purnomo, M.F.A. Bakar, Helion 6 (2020) 7. M. Bahi., R. Mutia., E. Mustanir, Lukitaningsih, J Natural 14, 1 (2014) 8. S. Saena, S. yanto, A. Amirah, J Pendidikan Teknologi Pertanian 6, 1 (2020) 9. H. Arfiah, Muftucha, O. Caman, J Akuakultuur Indonesia 5 (2006) The results of the analysis of variance in use of C. alata leaf extract had an effect on survival of catfish larvae (P<0.05). During the study, the observation of the survival rate of P. hypophthalmus larvae was categorized as good. It is suspected that the chemical content in leaf extractof C. alata was able to inhibit the growth of fungi in P. hypophthalmus eggs. 10. M.I. Effendie, Metode Biologi Perikanan (Yayasan Dewi Sri, Bogor, 1979) 11. O.T. Tcheghebe, V.R.S. Tala, F.N. Tatong, G.K. Tchuente, Medicine and Medical Sciences (LRJMSS) 4, 2 (2017) 12. A.A. Makinde, J.O. Igoli, L. TA’Ama, S.J. Shaibu, A. Garba, African Journal of Biotechnology 6, 13 (2007) g g yp p gg Good water quality management can inhibit the spread of fungi in hatchery activities both at time of hatching eggs and rearing post larvae in aquaculture activities. Good management of aquaculture media water quality will suppress the spread of fungal diseases through water management is carried out for all water quality parameters, both chemical, physical and biological. 4. Conclusion 19. R.A. Nugroho, R. Aryani, W.D. Anggraini, E.H. Hardi, R. Rudianto, E. Kusumawati, S. Sudrajat, F.M. Nur, H. Manurung. IOP Conference Series: Earth and Environmental Science 348, 1(2019) The use of Cassia alata L. leaf extract make an impact to hatchability of fish eggs and the survival of Pangasianodon hypophthalmus fish larvae. The highest fish egg hatchability value of 85.33% was obtained by soaking C. alata leaf extract at a dose of 15 ppm and the survival value the highest fish larvae of 92.99% was obtained at a dose of 10 ppm. 20. A. Happy, M.Soumya, S.V. Kumar, S. Rajeshkumar, R.D. Sheba, T. Lakshmi, V.D. Nallaswamy, Biochemistry and Biophysics Reports 17 (2019) 21. S.A. Gaddam, V.S. Kotakadi, D.V.R.S. Gopal, Y.S. Rao, A.V. Reddy, J of Nanostructure Chemistry 4 (2014) 2.4 Data analysis During the research, it was found that the water quality parameters measurement value data still within the tolerance range of P. hypophthalmus (Table 2). Based on the Indonesian National Standard [23], the optimal pH value for catfish rearing ranges from 6.5 - 8.5. The value of dissolved oxygen in the media for hatching eggs and rearing P. hypophthalmus larvae is > 5 mgL-1. Furthermore, the research of Putra et al. [24] states that the water quality for hatching and rearing catfish larvae is a temperature of 26.2 – 27.90 and dissolved oxygen 4.7 – 5.5 mgL-1. 13. R.P.S. Kumar, V. Malayaman, S. Shinduja, J of Chemical and Pharmaceutical Research 5, 11 (2013) 14. R. Dewi, Y. Firza, M. Nashiry, F.S. Al-Suede, A.S.A. Majid, Australian Herbal Insight 2 (2019) 15. J.M.N. Abille, A Review on The Medicinal Plant Cassia alata (L.) ROXB. (FABACEAE) (Biological Sciences Department, College of Science & Computer Studies De La Salle University – Dasmarinas, Cavite, Philippines pp 1- 12, 2019) 16. Rosidah, W.M. Afizia J Akuatika 3, 1 (2012) 17. R.S. Hardhini, Amir, D.N. Setyowati J Perikanan 8, 1 (2018) 18. F.M. Nur, R.A. Nugroho, S. Fachmy. AIP Conference Proceedings 1813, (2017) Reference 22. A.T. Bowo, Sunarto, Rachimi, J Ruaya 1, 1 (2014) 1. Muhlis, M. Ghofur, M. Sugihartono, J Akuakultur Sungai dan Danau 4, 1 (2019) 23. Standar Nasional Indonesia,Produksi Benih Ikan Patin Siam (Pangasius hyphthalamus) Kelas Benih Sebar (SNI : 01- 6483.4 – 2000) 2. S. Larasati, F. Basuki, T. Yuniarti, J of Aquaculture Management and Technology 6, 4 (2017) 24. P.L. Putra, D. Jubaedah, M. Syaifudin, J Akuakultur Rawa Indonesia 8, 1 (2020) 3. Noga, Pestisida Nabati untuk Penyakit Ikan (Penebar Swadaya, Jakarta, 2010) 3. Noga, Pestisida Nabati untuk Penyakit Ikan (Penebar Swadaya, Jakarta, 2010) 4. D.Q. Pham, H.T. Pham, J.W. Han, T.H. Nguyen, H.T. Nguyen, T.D. Nguyen, T.T.T. Nguyen, C.T. Ho, H.M. Pham, H.D. Vu, G.J. Choi, Q.L. Dang, Industrial Crops & Products 166 (2021) 4. D.Q. Pham, H.T. Pham, J.W. Han, T.H. Nguyen, H.T. Nguyen, T.D. Nguyen, T.T.T. Nguyen, C.T. Ho, H.M. Pham, H.D. Vu, G.J. Choi, Q.L. Dang, Industrial Crops & Products 166 (2021) 3
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*Charles H King, Alison P Galvani chk@cwru.edu Cognitive deficits and educational loss in children with schistosome infection—a systematic review and meta-analysis. PLoS Negl Trop Dis 2018; published online Jan 12. DOI:10.1371/journal.pntd.0005524. Correspondence Correspondence Correspondence this in three parts. First, we thank the commentator for the suggestion to consider adding genital lesions as an outcome of schistosomiasis. We plan to review the associated literature on this outcome in a subsequent iteration of the GBD Study. We also will review the new evidence on the cognitive deficits associated with schistosomiasis, as presented in their referenced article. Until now, our assessment has been that the evidence for a causal link between schistosomiasis infection and loss of cognition was not strong enough to warrant inclusion in the GBD studies. Second, anaemia is a sequela of schistosomiasis that has been included since GBD 2010. Finally, the “minimal disability weights” assigned to schistosomiasis infection are derived from large-scale population surveys. Until new evidence emerges that challenges the values of these disability weights, we will continue to use the current values. used a boosted regression tree method to generate environmental suitability maps for schistosomiasis that used georeferenced data and geospatial covariates (including vegetation, precipitation, and population). Geotagged epidemiological infor­ mation was paired with several hypothesised environmental drivers of schistosomiasis presence to define the environment that best characterises the reported occurrences of the parasite. We produced species-specific maps and determined the optimal thresholds for transmission using the geopositioned dataset. Species-specific maps were combined into one all- species schistosomiasis map to show areas that are environmentally suitable for transmission of schistosomiasis. We overlaid this map with population data at a scale of 25 km² to generate a national estimate of the total population living within areas that are suitable for schistosomiasis transmission. We used DisMod-MR 2.1, the Bayesian meta-regression tool developed for GBD 2016 estimations, to estimate prevalence within this PAR, such that environmental factors and localised data were both used to inform our prevalence estimates. CHK reports a grant from the Schistosomiasis Consortium for Operational Research and Evaluation. APG declares no competing interests. Institute for Health Metrics and Evaluation, Seattle, WA 98121, USA Charles King and Alison Galvani cite three specific concerns in their commentary regarding the Global Burden of Disease (GBD) Study1 2016 and we respond briefly to them. 1 GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390: 1211–59. pl y King and Galvani state that environ­ mental factors were not considered within the revised Bayesian meta- regression of the GBD Study 2016. However, we did use environmental factors in our model. The schistoso­ miasis prevalence model in the GBD Study 2016 underwent a substantial revision that included a systematic review and literature data extraction and required dev­eloping models of both environ­mental suitability for schistosomiasis transmission (used as a proxy for identifying populations at risk [PAR]) and prevalence within those at-risk populations. To estimate PAR, we *Charles H King, Alison P Galvani chk@cwru.edu Center for Global Health and Diseases, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA (CHK); and Center for Infectious Disease Modeling and Analysis (CIDMA), Yale School of Public Health, New Haven, CT, USA (APG) 1 GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390: 1211–59. 1 GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390: 1211–59. 2 GBD 2015 Mortality and Causes of Death Collaborators. Global, regional, and national life expectancy, all-cause mortality, and cause- specific mortality for 249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet 2016; 388: 1459–544. 2 GBD 2015 Mortality and Causes of Death Collaborators. Global, regional, and national life expectancy, all-cause mortality, and cause- specific mortality for 249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet 2016; 388: 1459–544. 3 Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis 2006; 6: 411–25. 3 Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis 2006; 6: 411–25. 4 Kjetland EF, Ndhlovu PD, Kurewa EN, et al. Prevention of gynecologic contact bleeding and genital sandy patches by childhood anti- schistosomal treatment. Am J Trop Med Hyg 2008; 79: 79–83. 4 Kjetland EF, Ndhlovu PD, Kurewa EN, et al. Prevention of gynecologic contact bleeding and genital sandy patches by childhood anti- schistosomal treatment. Am J Trop Med Hyg 2008; 79: 79–83. JS reports grants from Merck outside the submitted work. All other authors declare no competing interests. 5 Ezeamama AE, Bustinduy AL, Nkwata AK, et al. Cognitive deficits and educational loss in children with schistosome infection—a systematic review and meta-analysis. PLoS Negl Trop Dis 2018; published online Jan 12. DOI:10.1371/journal.pntd.0005524. 5 Ezeamama AE, Bustinduy AL, Nkwata AK, et al. Authors’ reply King and Galvani state that “many of the case count data remain inferred”. Although the prevalence model infers prevalence information from countries in similar regions, this prevalence model is representative of the PAR and only uses data in those at risk. These prevalence estimates were then scaled to represent prevalence at the GBD location level through the PAR estimates extracted from the environmental suitability maps, which used the association between environmental factors and more than 12 000 geolocated records. Institute for Health Metrics and Evaluation, Seattle, WA 98121, USA Department of Error Sinharay R, Gong J, Barratt B, et al. Respiratory and cardiovascular responses to walking down a traffic- polluted road compared with walking in a traffic-free area in participants older than 60 years with chronic lung or heart disease and age-matched healthy controls: a randomised, crossover study. Lancet 2017; 391: 339–49—In this Article (published online first on Dec 5, 2017), the corresponding author has been corrected, the middle initial for Frank Kelly has been added, the role of the funding source has been updated, and author initials have been updated throughout. These corrections have been made to the online version as of Jan 25, 2018, and the printed Article is correct. King and Galvani also state that “severe and yet common clinical outcomes of schistosomiasis were not integrated into the disability weights” and “serious clinical manifestations of intestinal schistosomiasis were not incorporated”. We will address 308 www.thelancet.com Vol 391 January 27, 2018
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Aldosterone-to-renin ratio is related to arterial stiffness when the screening criteria of primary aldosteronism are not met
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Aldosterone‑to‑renin ratio is related to arterial stiffness when the screening criteria of primary aldosteronism are not met OPEN Eeva Kokko1, Pasi I. Nevalainen1,2, Manoj Kumar Choudhary1, Jenni Koskela1,2, Antti Tikkakoski1,3, Heini Huhtala4, Onni Niemelä1,5, Marianna Viukari6, Jukka Mustonen1,2, Niina Matikainen6 & Ilkka Pörsti1,2* Aldosterone-to-renin ratio (ARR) is a screening tool for primary aldosteronism (PA), but the significance of ARR when the PA criteria are not met remains largely unknown. In this cross-sectional study we investigated the association of ARR with haemodynamic variables in 545 normotensive and never-medicated hypertensive subjects (267 men, 278 women, age range 19–72 years) without suspicion of PA. Supine haemodynamic data was recorded using whole-body impedance cardiography and radial tonometric pulse wave analysis. In sex-adjusted quartiles of ARR, determined as serum aldosterone to plasma renin activity ratio, the mean values were 282, 504, 744 and 1467 pmol/ µg of angiotensin I/h, respectively. The only difference in haemodynamic variables between the ARR quartiles was higher pulse wave velocity (PWV) in the highest quartile versus other quartiles (p = 0.004), while no differences in blood pressure (BP), heart rate, wave reflections, cardiac output or systemic vascular resistance were observed between the quartiles. In linear regression analysis with stepwise elimination, ARR was an independent explanatory factor for PWV (β = 0.146, p < 0.001, ­R2 of the model 0.634). In conclusion, ARR was directly and independently associated with large arterial stiffness in individuals without clinical suspicion of PA. Therefore, ARR could serve as a clinical marker of cardiovascular risk. Trial registration: ClinicalTrails.gov: NCT01742702. Primary aldosteronism (PA) is the most common form of secondary ­hypertension1, yet most cases remain ­undiagnosed2,3. The estimated prevalence of PA has conventionally ranged from 5 to 15% among hypertensive ­patients4–6, but according to a recent study, the prevalence might be as high as 16% to 22%7. It is known that PA is associated with significantly higher risk of cardiovascular events and target organ damage than essential hypertension at comparable levels of blood ­pressure5,8,9. Aldosterone excess increases the accumulation of growth factors and collagen fibres in arterial ­wall10,11 and it has been shown that PA patients have an increased arterial wall stiffness in comparison with patients who have essential ­hypertension12,13.h f p p yp Screening of PA is based on the aldosterone-to-renin ratio (ARR). www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2020) 10:19804 Methods St d b Study subjects. The recruitment of the study subjects has previously been described in ­detail21–23. All subjects were examined by a physician and routine laboratory analyses for elevated blood pressure (BP) were ­taken24. The medical history, lifestyle behaviour and family history were documented. Alcohol use was evalu- ated as standard drinks (~ 12 g of absolute alcohol) per week, and smoking amount was estimated in pack-years. The exclusion criteria were history of coronary artery disease, stroke, cardiac insufficiency, valvular heart dis- ease, chronic kidney ­disease25, secondary hypertension, alcohol or substance ­abuse26, psychiatric illnesses other than mild to moderate depression or anxiety, heart rhythm other than sinus rhythm, use of antihypertensive or uric-acid-lowering medications, and ongoing pregnancy. Altogether 606 subjects, aged 19–72 years, were eligible for the study. Data of ARR was missing in 48 subjects while 13 subjects met the screening criteria of PA (serum aldosterone > 550 pmol/l and ARR > 750 pmol/µg of angiotensin I/h, plasma potassium concentration < 3.3 mmol/l, BP > 150/100 mmHg) and were therefore excluded. The Endocrine Society screening for PA is based on BP > 150/100 mmHg on three different ­days1,5. In the present study the BP criteria were applied on the 5-min laboratory BP measurements (see below), and the results were confirmed by applying the criteria for the office BP measurements on a single occasion. The final study population consisted of 545 subjects.h fi ghi y p p j Signed informed consent was obtained from all participants. The study complies with the declaration of Helsinki and was approved by the Ethics Committee of the Pirkanmaa Hospital District (study code R06086M). Laboratory analyses and indexes glucose tolerance. Blood and urine samples were taken after 12 h of fasting. Plasma renin activity (GammaCoat Plasma Renin Activity 125-I RIA Kit, DiaSorin, Saluggia, Italy) and aldosterone concentration (Active Aldosterone RIA, Beckman Coulter, Fullerton, CA, USA) were deter- mined using commercial kits. Plasma C-reactive protein (CRP), sodium, potassium, glucose, creatinine, cystatin C, uric acid, triglyceride, and total, HDL and LDL (high- and low-density lipoprotein, respectively) cholesterol concentrations were determined using Cobas Integra 800 (F. Hoffmann-LaRoche Ltd, Basel, Switzerland). Insu- lin and parathyroid hormone (PTH) were determined using electrochemiluminescence immunoassay (Cobas e411, Roche Diagnostics). Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease – Epidemiology collaboration (CKD-EPI) cystatin C ­equation27. www.nature.com/scientificreports/ events at a young age (< 40 years), hypertension together with sleep apnoea, and all first-degree relatives of patients with ­PA1,5.hi p The role of ARR for screening of PA is robust, but there is a scarcity of data evaluating the significance of ARR in subjects who remain below the screening threshold for PA. Previously, ARR has been associated with the development and severity of hypertension, even in patients without excessive aldosterone ­levels14–18. A Japanese long-term observational analysis demonstrated that elevated ARR is associated with an increased incidence of cardiovascular disease in patients with essential ­hypertension19. In 60 healthy adults with a mean age 43 years, Shapiro et al. reported a direct correlation ­(rP = 0.298) between ARR and pulse wave velocity (PWV)20. In 2000 participants of the Framingham study, Lieb et al. found that ARR was a significant independent correlate of PWV (β = 0.20). However, the participants were not screened for the presence of PA, 33% were ingesting antihyper- tensive medications, and 12% had diabetes ­mellitus18. , In this cross-sectional study we analysed the association of ARR with several cardiovascular variables in nor- motensive subjects and previously undiagnosed never-medicated hypertensive patients. We tested the hypothesis whether ARR correlates with haemodynamic parameters in subjects who are not fulfilling the clinical criteria for the screening of PA. Aldosterone‑to‑renin ratio is related to arterial stiffness when the screening criteria of primary aldosteronism are not met OPEN The Endocrine Society recommends the screening in the following cases with clinical suspicion pf PA: Blood pressure > 150/100 mmHg on three different days, drug resistant hypertension that is uncontrolled with ≥ three antihypertensive drugs, controlled hyperten- sion with ≥ four antihypertensive drugs, hypertension with spontaneous or diuretic-induced hypokalaemia, hypertension with adrenal incidentaloma, hypertension with a family history of hypertension or cerebrovascular 1Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland. 2Department of Internal Medicine, Tampere University Hospital, Tampere, Finland. 3Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland. 4Faculty of Social Sciences, Tampere University, Tampere, Finland. 5Clinical Laboratory and Medical Research Unit, Seinäjoki Central Hospital, Seinäjoki, Finland. 6Endocrinology, Helsinki University Hospital and Research Programs Unit, Clinical and Molecular Medicine, University of Helsinki, Helsinki, Finland. *email: ilkka.porsti@tuni.fi | https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 www.nature.com/scientificreports/ Methods St d b Insulin sensitivity was evaluated by the quantitative insulin sensitivity check index (QUICKI)28, and homeostatic model assessment of insulin resistance (HOMA-IR)29. In addition, the subjects were invited to participate in a standard 75-g oral glucose tolerance test (OGTT) for the determination of the Matsuda ­index30. Experimental protocol. Haemodynamic recordings were performed in a noiseless, temperature-controlled laboratory by research ­nurses21,22,31. Products containing caffeine, smoking or heavy meal were not allowed for ≥ 4 h, and alcohol consumption was not allowed for ≥ 24 h before the investigation. The subjects rested supine on the examination table with impedance cardiography electrodes placed on body surface, tonometric sensor for pulse wave analysis on left radial pulsation, and oscillometric brachial cuff for BP calibration to the right upper arm. The left arm with the tonometric sensor was stabilized to 90° in a support, which held the measurement probe at the heart level.h p The measurement consisted of a 5-min period, during which haemodynamic data was captured continuously. For the statistical analyses, the mean values of each 1-min period of recording were calculated. The repeatability and reproducibility of the protocol have been demonstrated to be ­good21,22,31. Pulse wave analysis. Radial BP and pulse wave were recorded by a tonometric sensor (Colin BP-508 T, Colin Medical Instruments Corp., USA) fixed on the radial ­pulse21,22. The radial BP signal was calibrated approx- imately every 2.5 min by brachial BP measurements. Aortic BP was derived using the SphygmoCor pulse wave monitoring system (SphygmoCor PWMx, AtCor medical, Australia)32. Aortic pulse pressure, augmentation index (AIx, augmented pressure/pulse pressure * 100), and AIx adjusted to heart rate 75/min (AIx@75) were also determined. The central forward wave amplitude was defined as the difference between waveform foot and first systolic inflection point pressure in the aortic ­waveform33,34. Whole‑body impedance cardiography. We used the CircMon device (JR Medical Ltd., Tallinn, Estonia) to assess changes in body electrical impedance during cardiac cycles to record heart rate, stroke volume, cardiac Scientific Reports | (2020) 10:19804 | https://doi.org/10.1038/s41598-020-76718-7 www.nature.com/scientificreports/ Table 1. Results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio: demographic data and clinical characteristics. BP, blood pressure; mean (standard deviation) or median [25th–75th percentile]. *P < 0.05 versus Q1; †P < 0.05 versus Q2. a Office BP was available from 123–129 participants per group. Methods St d b b Haemodynamic measurement systolic/diastolic BP values were 12.7–15.6/16.0–17.3 mmHg, and heart rate 2.8–4.5 beats/min, lower than in the office with no significant differences between the quartiles. Quartile 1 (n = 135) Quartile 2 (n = 137) Quartile 3 (n = 137) Quartile 4 (n = 136) Male/female (n) 66/69 67/70 67/70 67/69 Number of female hormone users 16 12 21 21 Number of oestrogen users 11 10 18 16 Age (years) 41.9 (11.4) 44.8 (11.5) 46.0 (11.5)* 49.5 (10.9)*† Weight (kg) 78.7 (15.8) 79.6 (15.6) 80.0 (14.8) 81.8 (15.0) Height (cm) 173.2 (9.5) 172.2 (9.6) 173.8 (8.8) 172.7 (9.2) Body mass index (kg/m2) 26.1 (4.2) 26.8 (4.6) 26.4 (4.1) 27.3 (3.9) Alcohol (standard drinks/week) 3 [0–5] 3 [1–6] 3 [1–6] 2 [0–4] Current smokers (n) 22 18 11 13 Office measurementsa Systolic BP (mmHg) 135.3 (20.0) 137.5 (20.2) 139.7 (18.6) 144.1 (21.0)* Diastolic BP (mmHg) 86.2 (13.1) 87.6 (12.2) 88.2 (10.7) 91.8 (11.6)*† Heart rate (bpm) 67.5 (9.6) 67.0 (9.3) 65.2 (9.6) 67.8 (9.1) Normotensive/hypertensive (n) 75 / 60 71 / 66 67 / 70 49 / 87* Haemodynamic measurementsb Systolic BP (mmHg) 127.4 (18.1) 129.0 (18.1) 129.1 (17.8) 132.0 (18.3) Diastolic BP (mmHg) 72.5 (11.9) 73.4 (12.0) 73.4 (12.0) 76.1 (12.8) Heart rate (bpm) 63.9 (10.2) 63.8 (10.3) 62.1 (9.3) 63.6 (8.1) Normotensive/hypertensive (n) 107 / 28 103 / 34 100 / 37 95 / 41 Table 1. Results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio: demographic data and clinical characteristics. BP, blood pressure; mean (standard deviation) or median [25th–75th percentile]. *P < 0.05 versus Q1; †P < 0.05 versus Q2. a Office BP was available from 123–129 participants per group. b Haemodynamic measurement systolic/diastolic BP values were 12.7–15.6/16.0–17.3 mmHg, and heart rate 2.8–4.5 beats/min, lower than in the office with no significant differences between the quartiles. output, and ­PWV35–37. Systemic vascular resistance was calculated from radial BP and cardiac output measured by CircMon. Stroke volume, cardiac output and systemic vascular resistance were presented as indexes related to body surface area calculated using the DuBois ­equation38. The method and electrode configuration has been previously ­reported36,37. p y p With CircMon the recorded stroke volume and cardiac output are in good agreement with values obtained utilizing 3-dimensional ­echocardiography31 and the thermodilution and direct oxygen Fick ­methods35,36, and the PWV values show very good correlation with values measured using ultrasound or the tonometric ­method37,39. Statistics. Methods St d b To illustrate the influence of ARR on the haemodynamic variables, the study participants were divided into quartiles of ARR. As serum aldosterone concentration was slightly higher in women than in men (see ‘Results’), the ARR quartiles 1–4 were constructed separately for women and men. q p y Analyses of normally distributed data were performed using analysis of variance (ANOVA) and ANOVA for repeated measurements, and non-normally distributed data using Kruskal–Wallis test with Mann–Whitney U-test in the post-hoc analyses. The Bonferroni correction was applied in all post-hoc analyses. The ANOVA for repeated measures analyses were adjusted for sex, age, BMI, and eGFR, as appropriate, by using these variables as covariates in the analyses. Normally distributed variables in the tables were presented as means and stand- ard deviations and non-normally distributed variables as medians and 25th–75th percentiles. The figures were depicted as means and standard errors of the mean (SEM). Pearson’s correlations were calculated for normally distributed and Spearman’s correlations for non-normally distributed variables. The values of the haemodynamic variables for the statistics in Table 1 and regression analysis were calculated as averages from the minutes 3–5 of the recordings when the signal was most stable. P < 0.05 was considered significant. g g gi To investigate factors independently associated with PWV in the whole study population, linear regression analysis with stepwise elimination was applied. The covariates in the analysis were age, sex, BMI, categorised smoking status (never, present, previous), categorised alcohol consumption (low, moderate, high); plasma aldos- terone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid, calci- triol, PTH, QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. Coefficients (B) and standard coefficients (Beta) of regression were calculated, and assumptions of linearity were confirmed by the analysis of residuals. IBM SPSS Statistics Version 26 (IBM Corporation, Armonk, NY, USA) was used for statistics. Results S d Study population and laboratory values. The proportion of male subjects was 49%. Plasma renin activ- ity and ARR did not significantly differ between women versus men [0.99 (0.90) vs. 0.97 (0.95) ng of angiotensin I/ml/h; 779 (527) vs. 719 (531) pmol/µg of angiotensin I/h, respectively; mean (SD)]. However, serum aldoster- one concentration was higher in women than in men (561 (481) vs. 449 (208) pmol/l, respectively, p < 0.001). Therefore, the ARR results were examined in sex-adjusted quartiles (Q) (Table 1). https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ Table 2. Laboratory results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio. Results shown as mean (standard deviation) or median [25th–75th percentile]; GFR, estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI ­formula27. HDL, high-density lipoprotein; LDL, low-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; QUICKI, quantitative insulin sensitivity check index. *P < 0.05 versus Q1; †P < 0.05 versus Q2; ‡P < 0.05 versus Q3. a n = 101–118 for 24-h urine excretion results. b n = 88–98 for Matsuda index results in each quartile. Results S d Quartile 1 (n = 135) Quartile 2 (n = 137) Quartile 3 (n = 137) Quartile 4 (n = 136) Aldosterone-to-renin ratio (pmol/µg of Ang I/h) 287 [227–342] 497 [451–560]* 740 [674–799]*† 1302 [1085–1614]*†‡ Serum aldosterone (pmol/l) 419 [297–552] 448 [324–599] 431 [336–636] 427 [323–575] Plasma renin activity (ng of Ang I/ml/h) 1.47 [1.12–2.17] 0.89 [0.67–1.21]* 0.58 [0.46–0.88]*† 0.29 [0.20–0.44]*†‡ Sodium (mmol/l) 140.4 (1.8) 139.9 (1.8) 140.1 (2.1) 140.7 (1.9)† 24-h urine sodium excretion (mmol)a 153 (62) 155 (61) 154 (65) 148 (57) Potassium (mmol/l) 3.8 (0.2) 3.8 (0.3) 3.8 (0.3) 3.8 (0.3) 24-h urine potassium excretion (mmol)a 85 (30) 87 (30) 86 (27) 79 (22) Calcium (mmol/l) 2.30 (0.09) 2.29 (0.10) 2.30 (0.10) 2.29 (0.10) Parathyroid hormone (pmol/l) 4.37 (1.59) 4.37 (1.41) 4.43 (1.49) 4.71 (1.59) 25OH-D3 (nmol/l) 74.2 (33.5) 66.1 (28.3) 74.9 (45.4) 70.7 (30.0) 1,25(OH)2-D3 (pmol/l) 110.2 (31.4) 109.3 (36.3) 107.6 (33.8) 106.7 (34.9) C-reactive protein (mg/l) 0.7 [0.5–1.5] 1.0 [0.5–2.2] 0.5 [0.4–1.3]† 1.0 [0.5–2.1]‡ Creatinine (µmol/l) 74 (13) 72 (13) 75 (14) 73 (14) Cystatin C 0.81 (0.14) 0.83 (0.14) 0.85 (0.15) 0.86 (0.15) Estimated GFR (ml/min/1.73 ­m2) 104.7 (18.1) 100.9 (17.8) 98.3 (18.0)* 95.3 (17.1)* Total cholesterol (mmol/l) 5.0 (1.0) 5.1 (1.0) 5.0 (1.0) 5.3 (1.0)* Triglycerides (mmol/l) 1.11 [0.67–1.38] 1.22 [0.74–1.43] 1.21 [0.67–1.46] 1.27 [0.87–1.54] HDL cholesterol (mmol/l) 1.6 (0.4) 1.6 (0.5) 1.6 (0.4) 1.5 (0.4) LDL cholesterol (mmol/l) 2.9 (0.9) 3.0 (1.0) 2.9 (0.9) 3.2 (0.9) Uric acid (µmol/l) 302 (71) 297 (65) 292 (71) 303 (86) Glucose (mmol/l) 5.3 (0.5) 5.5 (0.5) 5.4 (0.5) 5.6 (0.8)* Insulin (mU/l) 6.8 [5.0–10.4] 6.8 [5.3–9.7] 5.9 [4.3–8.1] 7.0 [4.7–9.4] Matsuda ­indexb 5.74 [3.74–8.18] 5.94 [3.50–8.78] 7.62 [4.44–9.69] 5.80 [3.89–7.72] HOMA-IR 1.56 [1.14–2.61] 1.70 [1.23–2.37] 1.43 [1.02–2.11] 1.69 [1.13–2.40] QUICKI 0.357 [0.331–0.375] 0.352 [0.335–0.371] 0.362 [0.341–0.382] 0.353 [0.335–0.376] Table 2. Laboratory results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio. Results shown as mean (standard deviation) or median [25th–75th percentile]; GFR, estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI ­formula27. HDL, high-density lipoprotein; LDL, low-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; QUICKI, quantitative insulin sensitivity check index. *P < 0.05 versus Q1; †P < 0.05 versus Q2; ‡P < 0.05 versus Q3. a n = 101–118 for 24-h urine excretion results. b n = 88–98 for Matsuda index results in each quartile. None of the subjects used anti-hypertensive medicines. Results S d The proportions of female hormone users did not dif- fer between the ARR quartiles. Mean participant age was 46 years, with an age range of 19–72 years in male and 21–72 years in female subjects. Participant age was higher in Q3 versus Q1, and in Q4 versus Q1 and Q2. Mean BMI was 26.6 kg/m2 with no differences between the quartiles. Average alcohol use was moderate 4.3 standard drinks/week, and the prevalence of present and previous smokers was 12% and 31%, respectively. Alcohol intake and the prevalence of smokers were corresponding in all quartiles (Table 1).fif p p g q ( ) When applying the office cut-off values of hypertension (systolic BP ≥ 140 or diastolic BP ≥ 90)24, the number of never-medicated hypertensive subjects was 283 (51.9%). Office systolic BP was higher in Q4 than in Q1, and office diastolic BP in Q4 than in Q1 and Q2. The proportion of hypertensive subjects in the office measurements was higher in Q4 than in Q1. g During the haemodynamic measurements, systolic and diastolic BP values were 13–16 and 16–17 mmHg lower, respectively, and heart rate 3–5 beats/min lower, than in the office measurements, with no significant differences between the quartiles. With the office cut-off values of hypertension, no significant differences were found in the proportion of hypertensive subjects between the quartiles during the haemodynamics measure- ments (Table 1). In sex adjusted quartiles of ARR, the mean values were 282, 504, 744 and 1467 pmol/µg of angiotensin I/h, respectively (Table 2). The differences in ARR were explained by variations in plasma renin activity, as aldoster- one concentrations did not differ between the quartiles. Plasma renin activity was different in all quartiles with highest values in Q1 and lowest values in Q4. In our study, age was inversely correlated both with plasma renin activity and serum aldosterone concentration ­(rS =  − 0.369 and ­rS =  − 0.253, respectively, p < 0.01 for both), while age and ARR were directly correlated ­(rS = 0.238, p < 0.01). g y ( S p ) Average measures of plasma electrolytes, PTH, CRP, uric acid, glucose metabolism and renal function were within the normal range in all quartiles (Table 2). In seven participants fasting plasma glucose ranged 7.0–10.3 mmol/l, i.e. in the diabetic range, but none of them presented with glucosuria. Altogether 53 participants had impaired fasting plasma glucose concentration ranging 6.2–6.9 mmol/l. Scientific Reports | (2020) 10:19804 | Results S d Plasma sodium concentration was minimally higher in Q4 than in Q2. No differences were observed in 24-h excretion of sodium or potassium to the urine, plasma potassium, calcium, PTH, creatinine, uric acid, triglycerides, HDL cholesterol or LDL https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ Figure 1. Radial systolic (a) and diastolic (b) blood pressure calibrated from brachial blood pressure measurements, and aortic systolic (c) and diastolic (d) blood pressure in quartiles (n = 135–137) of aldosterone- to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements results are depicted as mean and standard error of the mean Figure 1. Radial systolic (a) and diastolic (b) blood pressure calibrated from brachial blood pressure measurements, and aortic systolic (c) and diastolic (d) blood pressure in quartiles (n = 135–137) of aldosterone- to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, results are depicted as mean and standard error of the mean. Figure 1. Radial systolic (a) and diastolic (b) blood pressure calibrated from brachial blood pressure measurements, and aortic systolic (c) and diastolic (d) blood pressure in quartiles (n = 135–137) of aldosterone- to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, results are depicted as mean and standard error of the mean. cholesterol. However, total cholesterol was somewhat higher in Q4 when compared to Q1. Estimated GFR was lower in Q3 and Q4 when compared to Q1. Although fasting plasma glucose concentration was higher in Q4 than in Q1, all the evaluated insulin sensitivity indices (QUICKI, HOMA-IR, Matsuda index) were invariable between the ARR quartiles (Table 2). Haemodynamic measurements. ARR and haemodynamics. The results representing the haemodynamic variables in the sex-, age-, and eGFR-adjusted quartiles of ARR are shown in Figs. 1, 2 and 3. No differences in radial or aortic BP were observed between the quartiles (Fig. 1). Heart rate, stroke index, cardiac index and sys- temic vascular resistance index were also similar in all quartiles (Fig. 2). No differences were found in forward wave amplitude, augmentation index or extracellular water balance between the quartiles, either. However, in the absence of differences in central BP, aortic to popliteal PWV was clearly higher in Q4 versus other quartiles (p = 0.004) (Fig. 3). Results S d Among the study subjects, the Spearman correlation between ARR and PWV was 0.219 (p < 0.01). p Aldosterone and haemodynamics. When examined in sex-adjusted quartiles of serum aldosterone con- centration, the quartiles presented with differences in age and BMI (not shown). In the sex-, age- and BMI- adjusted quartiles of serum aldosterone concentration, no differences in the haemodynamic variables were detected between the quartiles except for PWV, the value of which was lower in Q1 versus Q4 (8.12 ± 0.12 vs. 8.58 ± 0.12 m/s, p = 0.045). p Renin activity and haemodynamics. In sex-adjusted quartiles of plasma renin activity, no additional adjust- ments were needed (not shown). Corresponding to the results in quartiles of aldosterone concentration, all haemodynamic variables in quartiles of renin activity were similar except for PWV. However, the difference in PWV was observed between Q1 and Q2 (8.65 ± 0.13 vs. 8.08 ± 0.12 m/s, p = 0.006), while PWV in Q1, Q3 and Q4 was corresponding (ranging from 8.37 ± 0.12 to 8.65 ± 0.12 m/s, p = 0.559–1.000). Determinants of large arterial stiffness. The results of the linear regression analyses with stepwise elimination are presented in Table 3. The independent explanatory factors for PWV were age, ejection duration, Scientific Reports | (2020) 10:19804 | https://doi.org/10.1038/s41598-020-76718-7 www.nature.com/scientificreports/ Figure 2. Heart rate (a), stroke index (b), cardiac index (c) and systemic vascular resistance index (d) in quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, mean and standard error of the mean. Figure 2. Heart rate (a), stroke index (b), cardiac index (c) and systemic vascular resistance index (d) in quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, mean and standard error of the mean. uric acid concentration, mean aortic pressure, ARR, BMI, low alcohol consumption, and heart rate ­(R2 = 0.634). The variables in the model included both aldosterone concentration and renin activity, yet neither of them turned out as independent predictors for PWV. QUICKI was used as an insulin sensitivity variable in the model, as the Matsuda index was not available from all participants (Table 2). Results S d In our study population the correla- tion between QUICKI and Matsuda index was 0.884 (p < 0.001) and between QUICKI and HOMA-IR-0.720 (p < 0.001). p When the office cut-off for hypertension (BP ≥ 140/90 mmHg) was applied for the laboratory ­measurements24, ARR was an independent explanatory factor for PWV in both the normotensive (p < 0.001, ­R2 = 0.580) and hypertensive (p = 0.012, ­R2 = 0.523) subgroups. The result did not change when applying the home BP measure- ment cut-off (BP ≥ 135/85 mmHg).fi f If the office BP measurements were applied for the exclusion of participants with potential PA, 499 subjects were eligible for the statistical analyses. Also, in this analysis ARR was an independent explanatory factor for PWV (p < 0.001), while the other explanatory factors were age, mean aortic pressure, heart rate, and plasma concentrations of HDL cholesterol, uric acid, and triglycerides ­(R2 = 0.572 of the model). Discussion In this study we investigated the association of ARR with several cardiovascular variables in normotensive sub- jects and never-medicated hypertensive patients without clinical suspicion of PA and without cardiovascular or renal comorbidities, and cardiovascular medications. In analyses adjusted for confounding factors, ARR was significantly associated with PWV but not with any other haemodynamic variable. The linear regression analyses confirmed that ARR was an independent explanatory factor for PWV, an acknowledged marker of large arterial ­stiffness40,41. As increased arterial stiffness is a strong independent predictor of cardiovascular ­events41, while higher ARR is associated with an increased incidence of cardiovascular ­disease19 and predicts future stroke in hypertensive ­patients42, a higher ARR may predispose to the future development of cardiovascular diseases.if yp p g y p p p Age is the most significant explanatory factor for large arterial ­stiffness43,44. In the present study, subjects in the highest ARR quartile were older than in the other quartiles. Consequently, the Figs. 1, 2 and 3 were adjusted https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ Figure 3. Forward wave amplitude (a), augmentation index (b), extracellular water balance (c) and aortic-to- popliteal pulse wave velocity (d) in quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, mean and standard error of the mean. Figure 3. Forward wave amplitude (a), augmentation index (b), extracellular water balance (c) and aortic-to- popliteal pulse wave velocity (d) in quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, mean and standard error of the mean. Table 3. Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid, calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR, estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI ­formula27. Table 3.   Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid, calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR, estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI ­formula27. www.nature.com/scientificreports/ for differences in age in addition to sex and estimated GFR. Moreover, despite the presence of age in the regres- sion model, ARR was an independent explanatory factor for PWV. The inverse correlation between age and plasma renin is known from previous ­studies45. High sodium intake would also lower ­renin1,5, but this was not the cause for lower renin in the ARR quartiles 2–4 versus quartile 1, as 24-h sodium excretion to the urine was similar in all quartiles.h q The screening positive cases with putative PA were excluded from our study population that was predomi- nantly normotensive during the haemodynamic measurements. We found that ARR was directly and indepen- dently associated with large arterial stiffness, while the linear regression model displayed no direct association between serum aldosterone concentration or plasma renin activity and PWV. This finding may reflect higher renin-independent aldosterone release in the participants with high ARR. Indeed, a continuum of renin-inde- pendent aldosteronism in normotensive subjects has been previously ­identified46,47. High aldosterone levels related to prevailing plasma renin activity may predispose to the chronic adverse effects of aldosterone in the vascular system already prior to the clinical diagnosis of PA or ­hypertension46. The molecular basis for this continuum of aldosterone secretion may be explained by the aldosterone-producing cell clusters that have been discovered in the zona fasciculata of morphologically normal non-neoplastic adrenal glands: these clusters may be found in > 50% of normal adrenal glands, leading to mild autonomous aldosterone secretion and increased ARR with increasing age far more commonly than previously ­perceived48,49.f g g y p y p Some former studies have suggested a direct association between ARR and arterial stiffness. Shapiro et al. reported a significant association of ARR with PWV in 60 normotensive subjects, but in contrast to our study, their approach did not provide evidence of ARR as an independent explanatory factor of ­PWV20. In the Framing- ham study, Lieb et al. found that ARR was directly associated with PWV and four other measures of vascular ­pathophysiology18. As a major difference to our study, medicated hypertensive subjects and patients with known diabetes were not excluded from their analyses. Also, there was a 3-year interval between the laboratory exami- nations and haemodynamic measurements, whereas in our study these procedures were performed within a median period of 8 days from one another (25th to 75th percentile 2–17 days). www.nature.com/scientificreports/ A small study in 24 patients with essential hypertension by Mahmud and Feely found no correlation between ARR and PWV, but showed that aldosterone antagonist-induced decrease in systolic BP correlated with pre-treatment ARR​50. However, in 102 patients with confirmed PA, no correlation was found between ARR and baseline PWV, or between ARR and the reduction in PWV following ­adrenalectomy51. Yet, the subsequent increase in plasma renin activity after adrenalectomy was an explanatory factor for the reduction in PWV during 6 months of follow-up51. Altogether, the effects of aldosterone excess on large arterial stiffness may be manifested early in the course of the disease, and the beneficial changes in the vasculature following surgical treatment do directly correlate with the plasma concentrations of aldosterone or ARR. Noteworthy, in the present study the systolic and diastolic BP values were 13–16 and 16–17 mmHg lower, respectively, during the haemodynamic laboratory measurements than in the office measurements. This resem- bles the white-coat effect in hypertension, which has been related to large arterial stiffness in both ­untreated52 and treated hypertensive ­patients53. Out-of-office BP monitoring provides better assessment of overall BP and response to treatment in patients with white-coat ­hypertension54. The average difference between the office and laboratory BP in our study was rather large, whereby haemodynamic measurements under standard conditions can also result in clearly lower BP when compared with the office measurements. The correlations between office and laboratory measurements were 0.70 and 0.65 for systolic and diastolic BP, respectively. We used the algo- rithm recommended by The Endocrine Society for the screening of PA ­patients1,5, in order to exclude potential PA patients from the study population. Regardless of whether office BP > 150/100 mmHg measured on a single occasion, or average BP > 150/100 mmHg during haemodynamic measurements, was applied for the exclusion of participants, ARR was an independent explanatory factor for PWV. Of note, the results were corresponding when studied in normotensive and hypertensive subjects with two different cut-off values for hypertension (140/90 or 135/85 mmHg). g Seven of the present participants had fasting plasma glucose in the diabetic range, 53 participants had impaired fasting plasma glucose, while 89% of the participants presented with glucose values within the normal range. PA has been linked to abnormalities in glucose metabolism, especially to insulin resistance and impaired glucose ­tolerance55–57. Discussion Pulse wave velocity (m/s) B Beta P R2 = 0.634 (constant) 5.046 Age 0.071 0.488 < 0.001 Ejection duration − 0.016 − 0.185 < 0.001 Uric acid 0.005 0.199 < 0.001 Mean aortic pressure 0.018 0.148 < 0.001 Aldosterone-to-renin ratio 4.68 × 10–4 0.146 < 0.001 Body mass index 0.041 0.103 0.001 Heart rate 0.017 0.098 0.010 Low alcohol consumption category − 0.220 − 0.062 0.025 Pulse wave velocity (m/s) B Beta P R2 = 0.634 (constant) 5.046 Age 0.071 0.488 < 0.001 Ejection duration − 0.016 − 0.185 < 0.001 Uric acid 0.005 0.199 < 0.001 Mean aortic pressure 0.018 0.148 < 0.001 Aldosterone-to-renin ratio 4.68 × 10–4 0.146 < 0.001 Body mass index 0.041 0.103 0.001 Heart rate 0.017 0.098 0.010 Low alcohol consumption category − 0.220 − 0.062 0.025 Pulse wave velocity (m/s) B Beta P R2 = 0.634 (constant) 5.046 Age 0.071 0.488 < 0.001 Ejection duration − 0.016 − 0.185 < 0.001 Uric acid 0.005 0.199 < 0.001 Mean aortic pressure 0.018 0.148 < 0.001 Aldosterone-to-renin ratio 4.68 × 10–4 0.146 < 0.001 Body mass index 0.041 0.103 0.001 Heart rate 0.017 0.098 0.010 Low alcohol consumption category − 0.220 − 0.062 0.025 Table 3. Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid, calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR, estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI ­formula27. Table 3. Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid, calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR, estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI ­formula27. https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ However, the approach to examine central haemodynamics instead of plainly focusing on brachial artery pressure, may be better related with the level of cardiovascular ­risk64,65. y p y In conclusion, a direct association between ARR and PWV was observed in 545 normotensive and never- treated hypertensive subjects when the screening criteria of PA were not met. Therefore, our results indicate that ARR is related to arterial stiffness in individuals without clinical suspicion of PA according to the prevail- ing guidelines. Altogether, increased ARR in the absence of stage II and more severe hypertension should be recognized as an indicator of increased cardiovascular risk. Data availability Analyses and generated datasets during the current study are not available publicly as our clinical database contains several indirect identifiers and the informed consent obtained does not allow publication of individual patient data. The datasets are available from the corresponding author on reasonable request. Received: 3 August 2020; Accepted: 2 November 2020 www.nature.com/scientificreports/ Even in the absence of PA, plasma aldosterone concentration was related to insulin resist- ance in 251 male African American ­subjects58. Higher ARR was also associated with insulin resistance in 483 young adult African Americans without cardiovascular or renal ­disease59. In our study, no differences in insulin sensitivity between the ARR quartiles were observed as the indices QUICKI, HOMA-IR and Matsuda did not deviate. Therefore, the differences in PWV between the ARR quartiles could not be explained by variations in insulin sensitivity. Also the plasma concentrations of uric acid, the levels of which are usually elevated in insulin resistance and metabolic ­syndrome23, were similar in quartiles of ARR.l Changes in calcium metabolism may influence ­BP60, and induce alterations in the components of the renin- angiotensin system in the ­vasculature61. Moreover, vitamin D receptor activation downregulates the synthesis of renin in the juxtaglomerular cells and influences the expression of other components of the renin-angiotensin system in the ­kidney62,63. In the present study, the plasma concentrations of calcium and vitamin D metabolites were similar in the ARR quartiles. Therefore, putative changes in the metabolism of calcium and vitamin D were not the explanations for the differences in arterial stiffness between the ARR quartiles.h ff Our study has limitations. The cross-sectional design cannot substantiate causality. A potential selection bias caused by the recruitment of voluntary subjects and the exclusion protocol must be acknowledged. The haemo- dynamic recordings lasted for five minutes and the values of the last three minutes were used for the analyses, which gives a rather short window of observation. Still, the analyses were based on average on 190 cardiac cycles in each subject (mean HR in the study population was 63.3 beats/min). In addition, we applied indirect non- invasive methods requiring mathematical processing to derive PWV, stroke volume and cardiac output from Scientific Reports | (2020) 10:19804 | https://doi.org/10.1038/s41598-020-76718-7 www.nature.com/scientificreports/ the bioimpedance ­signal36, and central aortic BP waveform from the applanation tonometry ­signal32. Therefore, the results must be interpreted with caution, albeit the methods have been validated against direct or invasive ­measurements31,35,37. However, the approach to examine central haemodynamics instead of plainly focusing on brachial artery pressure, may be better related with the level of cardiovascular ­risk64,65. the bioimpedance ­signal36, and central aortic BP waveform from the applanation tonometry ­signal32. Therefore, the results must be interpreted with caution, albeit the methods have been validated against direct or invasive ­measurements31,35,37. References p , , , , , , , g y,h g y and arterial stiffness in young healthy subjects. Clin. Endocrinol. (Oxf.). 68, 510–512 (2008). f 21. Tahvanainen, A. et al. Analysis of cardiovascular responses to passive head-up tilt using continuous pulse wave analysis and impedance cardiography. Scand. J. Clin. Lab. Invest. 69, 128–137 (2009). 22. Tikkakoski, A. J. et al. Hemodynamic alterations in hypertensive patients at rest and during passive head-up tilt. J. Hypertens. 31, 906–915 (2013). 23. Kangas, P. et al. Changes in hemodynamics associated with metabolic syndrome are more pronounced in women than in Sci. Rep. 9, 18377 (2019). p 24. Williams, B. et al. ESC/ESH guidelines for the management of arterial hypertension. Eur. Heart J. 39, 3021–3104 (2018). . Williams, B. et al. ESC/ESH guidelines for the management of a l l d l f h l d 25. KDIGO 2012 Clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int. Suppl. 3, (2013). ( ) 6. Treatment of alcohol abuse. Current Care Guideline by the Finnish Medical Society Duodecim and the Finnish Society of Addiction M di i (2015) htt // k h it fi/ b/kh/ it k t/ it ?id h i50028#K1 A d 10 A t 2020 6. Treatment of alcohol abuse. Current Care Guideline by the Finnish Medical Society Duodecim and the Finnish Society of Addiction Medicine. (2015). https​://www.kaypa​hoito​.fi/web/kh/suosi​tukse​t/suosi​tus?id=hoi50​028#K1. Accessed 10 August 2020.i p ypi g 7. Inker, L. A. et al. 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Acknowledgementsh g The authors are deeply grateful to Paula Erkkilä, RN and Reeta Kulmala, RN for invaluable contribution to the haemodynamic measurements. The authors wish to acknowledge CSC – IT Center for Science, Finland, for computational resources. The study was supported by the Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital, Finnish Foundation for Cardiovascular Research, Päivikki and Sakari Sohlberg Foundation, Pirkanmaa Regional Fund of the Finnish Cultural Foundation, Emil Aaltonen Foundation, and Ida Montin Foundation. References Koskela, J. K. et al. Association of resting heart rate with cardiovascular function: a cross-sectional study in 522 Finnish subjects. BMC Cardiovasc. Disord. 13, 102 (2013). https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | Competing interests h Competing interests  The authors declare no competing interests. p g The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to I.P. Author contributions E.K. and I.P. reviewed the literature and wrote the original version of the manuscript. I.P., E.K. and H.H. per- formed the statistical analyses. P.N., J.K., A.T. and I.P. carried out the clinical examinations of patients. E.K., P.N., M.K.C., J.K., A.T., J.M., M.V., N.M. and I.P. participated in the design of the technical details and methodology of the study. O.N. was responsible for hormonal laboratory analyses. All authors contributed to the discussion https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ and editing the manuscript. I.P. was responsible for designing and conducting the study. All authors take the responsibility for the contents of the manuscript. and editing the manuscript. I.P. was responsible for designing and conducting the study. All authors take the responsibility for the contents of the manuscript. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. © The Author(s) 2020 https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 |
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The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice
Frontiers in immunology
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The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice Jin Bu 1†, Shiyuan Huang 2†, Jue Wang 3, Tong Xia 2, Hui Liu 2, Ya You 2, Zhaohui Wang 2* and Kun Liu 4* 1 Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 2 Department of Geriatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 3 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 4 Institution of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Keywords: GABAA receptor, amphiregulin, macrophage, monocyte, pressure-overload hypertrophy Edited by: Guochang Hu, University of Illinois at Chicago, United States Background: Myocardial macrophages have key roles in cardiac remodeling and dysfunction. The gamma-aminobutyric acid subtype A (GABAA) receptor was recently found to be distributed in macrophages, allowing regulation of inflammatory responses to various diseases. This study aimed to clarify the role of GABAA receptor-mediated macrophage responses in pressure overload-induced heart failure. Reviewed by: Adam R. Wende, University of Alabama at Birmingham, United States Robert Morris Blanton, Tufts Medical Center, United States *Correspondence: Zhaohui Wang zhaohuiwang@hust.edu.cn Kun Liu liukun@hust.edu.cn †These authors share first authorship Reviewed by: Adam R. Wende, University of Alabama at Birmingham, United States Robert Morris Blanton, Tufts Medical Center, United States Reviewed by: Adam R. Wende, University of Alabama at Birmingham, United States Robert Morris Blanton, Tufts Medical Center, United States Methods and Results: C57BL/6J mice underwent transverse aortic constriction for pressure-overload hypertrophy (POH) and were intraperitoneally treated with a specific GABAA receptor agonist (topiramate) or antagonist (bicuculline). Echocardiography, histology, and flow cytometry were performed to evaluate the causes and effects of myocardial hypertrophy and fibrosis. Activation of the GABAA receptor by topiramate reduced ejection fraction and fractional shortening, enlarged the end-diastolic and end- systolic left ventricular internal diameter, aggravated myocardial hypertrophy and fibrosis, and accelerated heart failure in response to pressure overload. Mechanistically, topiramate increased the number of Ly6Clow macrophages in the heart during POH and circulating Ly6Chigh classic monocyte infiltration in late-phase POH. Further, topiramate drove Ly6Clow macrophages toward MHCIIhigh macrophage polarization. As a result, Ly6Clow macrophages activated the amphiregulin-induced AKT/mTOR signaling pathway, and Ly6ClowMHCIIhigh macrophage polarization increased expression levels of osteopontin and TGF-b, which led to myocardial hypertrophy and fibrosis. Conversely, GABAA receptor blockage with bicuculline reversed these effects. *Correspondence: Zhaohui Wang zhaohuiwang@hust.edu.cn Kun Liu liukun@hust.edu.cn †These authors share first authorship *Correspondence: Zhaohui Wang zhaohuiwang@hust.edu.cn Kun Liu liukun@hust.edu.cn †These authors share first authorship Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology Received: 20 February 2021 Accepted: 20 April 2021 Published: 31 May 2021 Received: 20 February 2021 Accepted: 20 April 2021 Published: 31 May 2021 ORIGINAL RESEARCH published: 31 May 2021 doi: 10.3389/fimmu.2021.670153 Animals and Treatments Six- to 8-week-old male C57BL/6J mice, weighing 22 to 24 g, were housed at room temperature under a 12-h light/dark cycle. Food and water were provided freely. The surgical and experimental procedures were approved by Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. p p Mechanical pressure-overload hypertrophy (POH) results in HF progression from EF preservation to EF reduction with myocardial hypertrophy and fibrosis. Recently, It was shown that macrophages acted as key innate immune cells with important roles in the initiation and development of cardiac remodeling and dysfunction in mice with POH (5, 6). Macrophages can be constitutively present within the myocardium like kupffer cells in the liver and derived from blood monocytes as well (7, 8). Macrophages of different origins exert distinct effects on either early or late phases of POH. The proliferation of cardiac resident Ly6Clow macrophages as a mechanism of self-renewal occurs within early-phase POH, and infiltrating macrophages arise from circulating classic Ly6Chigh monocytes in late-phase POH (9). Macrophages are sensitive to their surroundings and alter their physiology in response to environmental factors (10). A previous study showed that infiltration of microRNA-155–expressing macrophages in response to pressure overload promoted cardiac inflammation, hypertrophy, and failure (11). By contrast, depletion of macrophages attenuated left ventricle (LV) remodeling and dysfunction in POH mice (12). After 1 week of adaptive feeding, mice were assigned into either a sham surgery group or a transverse aortic constriction (TAC) group. Subsequently, mice in the TAC group were randomly assigned to one of the following treatments: (1) Sodium chloride (NaCl)-treated group (vehicle), NaCl at 8 ml/kg; (2) topiramate- treated group, topiramate at 35 mg/kg; (3) bicuculline-treated group, bicuculline at 2 mg/kg (provided by Sigma-Aldrich, USA). The four groups were named as follows: sham, vehicle, topiramate- treated TAC group, and bicuculline-treated TAC group. Starting 6 h after TAC surgery, mice were intraperitoneally (i.p.) injected once a day consecutively for 3 weeks. At the end of the study, the hearts were harvested and weighed to compare the heart weight/body weight (HW/BW, mg/g). Then, LV tissues were collected for further experiments. Neutralizing Antibody Administration Mice were intraperitoneally administered five micrograms of affinity-purified anti-mouse-AREG polyclonal antibody (AF989, R&D systems) or IgG immediately before TAC and every 2 days after TAC. INTRODUCTION In a transverse aortic constriction (TAC) model used to assess POH, we treated mice with a specific GABAA receptor agonist (topiramate) or antagonist (bicuculline). We demonstrated that activation of the GABAA receptor by topiramate accelerated HF. Conversely, blockade of the GABAA receptor by bicuculline protected POH mice against LV enlargement and EF reduction. The data indicated that GABAA receptor modulation would become a new strategy in the treatment of patients with pressure overload-induced HF. Heart failure (HF) is a leading cause of morbidity and mortality and has a poor prognosis (1). Chronic pressure overload, as seen in persistent hypertension or aortic stenosis, is a major risk factor for HF (2). The pressure-overload heart experiences the transitions from compensated hypertrophic remodeling with preserved contractile function to eccentric hypertrophy with contractile dysfunction (3). Notwithstanding the fact that current drugs capable of inhibiting neuroendocrine signaling can promote hypertrophy regression, HF progression and its poor prognosis remain unavoidable. Recent clinical evidence indicates that the progression from HFpEF to HFrEF has a worse prognosis than HFrEF and president HFpEF that is not accompanied by ejection fraction (EF) declines during follow-up (4). This underscores the need to unravel the mechanism of compensation to decompensation HF transition. Surgical Protocol TAC model was performed as described previously (17). In brief, mice were anaesthetized by isoflurane and artificially ventilated at a rate of 120 strokes per minute using a rodent ventilator with a mixture of O2 and air (1:2 vol/vol). Then, the aorta was constricted between the innominate and left common carotid arteries by placing a 5-0 nylon suture against a blunted 26-gauge needle. Blood pressure gradient across ligation site was confirmed by echography. Mice in sham group were subjected to identical operation without reducing aorta diameter to 26-gauge. Animals and Treatments Gamma-aminobutyric acid subtype A (GABAA) receptors are the principal neurotransmitter receptors of the central nervous system(CNS), and they are also found to be distributed in immune cells, such as neutrophils, monocytes, and macrophages (13). Recently, we published a study showing that activation or inhibition of the GABAA receptor exerted a pronounced effect on post-infarction ventricular remodeling by modulating monocyte/macrophage subsets (14). The GABAA receptor has also been shown to affect macrophage recruitment and activity in tumor microenvironments and stress-induced colon inflammation (15, 16). Thus, the GABAA receptor can influence pathological processes in various diseases via the modulation of macrophages. However, it remains unclear whether macrophage modulation affects pressure overload- induced cardiac remodeling and dysfunction, which have increasingly been associated with macrophage-mediated inflammation in POH. Citation: Bu J, Huang S, Wang J, Xia T, Liu H, You Y, Wang Z and Liu K (2021) The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice. Front. Immunol. 12:670153. doi: 10.3389/fimmu.2021.670153 Conclusions: Control of the GABAA receptor activity in monocytes/macrophages plays an important role in myocardial hypertrophy and fibrosis after POH. Blockade of the GABAA receptor has the potential to improve pressure overload-induced heart failure. May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 1 The GABAA Receptor and Macrophage Bu et al. Frontiers in Immunology | www.frontiersin.org Echocardiography A Vevo1100 high-resolution in vivo imaging system (Fujifilm Visualsionics) was used for echocardiographic analysis. Mice were anesthetized with isoflurane. The LV B-mode and M-mode Here, we sought to address the role of the GABAA receptor in pressure overload-induced cardiac remodeling and dysfunction. May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 2 The GABAA Receptor and Macrophage Bu et al. neubauer chamber (BD Bioscience, USA) with Trypan blue (Sigma-Aldrich, USA). Cell suspension were incubated with antibodies against CD45 (eBioscience, USA), Ly-6G (eBioscience, USA), Ly-6C (eBioscience, USA), and CD11b (eBioscience, USA) for 30 min at 4°C, and washed twice for flow cytometry (FCM) analysis. images were captured from parasternal short-axis or long-axis views at the level of the papillary muscles (18). The LV end- diastolic diameter (LVIDd) and LV end-systolic diameter (LVIDs) were measured from M-mode tracings of parasternal short-axis views at the mid and apical levels. The ejection fraction (EF) was calculated as EF (%) = 100 × [(LV Vol;d − LV Vol;s)/LV Vol;d]. LV Mass was calculated as LV mass (mg) = 1.053 × [(LVID;d + LVPW;d + IVS;d)3 −LVID;d3]. The fractional shortening (FS) was calculated as FS(%) = 100 × [(LVID;d −LVID;s)/LVID;d]. y y y To analyze macrophages and their subsets, heart macrophages were collected as described previously (14). The right atria, left atria, and atrioventricular valves were removed after the whole heart tissue was excised. Then, LV was cut into small pieces in PBS and digested with collagenase B. The cell suspension was filtered and resuspended to get single cell suspension. After total viable cells were calculated, they were conjugated with the following antibodies for the identification defined as Ly-6Chigh and Ly-6Clow macrophages according to Ly-6C (eBioscience, USA), CD45 (eBioscience, USA), F4/80 (eBioscience, USA), and CD11b (eBioscience, USA). After incubated with these antibodies for 30 min at 4°C, the cells were washed twice. To analyze the subsets of Ly-6Clow macrophages, cardiac single cell suspension was got according to the above. Cell suspension was incubated with antibodies against CD45 (eBioscience, USA), Ly-6C (eBioscience, USA), F4/80 (eBioscience, USA), MHCII (eBioscience, USA), and CD11b (eBioscience, USA) for 30 min at 4°C, and washed twice for FCM analysis. Similarly, to analyze the expression of Ki-67 protein, CD45 (eBioscience, USA), Ly-6C (eBioscience, USA), F4/80 (eBioscience, USA), Ki-67 (eBioscience, USA), and CD11b (eBioscience, USA) were incubated for FCM analysis. EdU Incorporation Assay To assess DNA synthesis in cardiac macrophages, 200 µg of EdU (5-ethynyl-2′-deoxyuridine) was injected into mice (i.p). Two hours after the injection, the heart was excised, and cell suspension was dispersed from the heart tissue and analyzed for incorporated EdU using FCM. For co-immunostaining of F4/80 and amphiregulin (Areg), antigen retrieval was performed by using boiling citrate buffered saline for 2 min after de-waxing and washing the sections in PBS. The heart slides were blocked for 1 h with 10% normal donkey serum. The tissue sections were incubated overnight with anti- Areg (A12680, ABclonal) and anti-F4/80 (Ab6640, Abcam) antibodies. Next day, the sections were washed and incubated for 2 h with Alexa-Fluor-488–conjugated anti-goat or Alexa- Fluor-594–conjugated anti-rat secondary antibodies (Invitrogen). DAPI was used to stain the cell nuclei. The stained samples were viewed using a confocal laser-scanning microscope. Western Blot Analysis Western blot was performed as described previously (21). LV tissue was homogenized in radioimmunoprecipitation buffer containing 1% protease inhibitor cocktail, and the concentration of the supernatant was measured by means of BCA. After the target proteins were loaded onto an SDS polyacrylamide gel, proteins were transferred from the gel to a PVDF membrane. Subsequently, the membranes were incubated overnight with Areg, mTOR (A2445, ABclonal), AKT (A18120, ABclonal), P- AKT (AP0655, ABclonal), or P-mTOR (AP0094, ABclonal) antibody after being blocked for 1 h. After washed in TBST, the membranes were incubated with HRP-linked secondary antibody for 60 min at room temperature. The protein bands were detected using an enhanced chemiluminescence agent (ECL reagents) and analyzed. Flow Cytometry To determine monocytes and their subsets or neutrophils, peripheral blood was collected in 50 mM EDTA (Sigma- Aldrich, USA) as anticoagulant at the day of sacrifice. Spleens and femurs were excised after vascular perfusion with sterile PBS. Spleens were triturated in PBS with the end of a 1-ml syringe, and filtered through 40 mm nylon mesh. Flushed bone marrow was also strained through the nylon mesh. Then, the cell suspension was centrifuged and resuspended to get single cell suspension. The total viable cell numbers were determined using Histological Analysis and Immunofluorescence The cross-sectional area of cardiomyocytes (CMs) was assessed on the basis of wheat germ agglutinin (WGA) staining. For that, 5 µm thick paraffin-embedded sections from mice hearts were deparaffinized, rinsed in PBS and subsequently stained with Alexa Fluor 488-conjugated WGA according to the manufacturer-provided protocol. For the quantification of the cross-sectional area, slides were imaged by a fluorescence microscope and only CMs that were aligned transversely were considered. Cell area was calculated in more than 200 cells. To determine collagen deposition, paraffin sections (5 µm thick) of hearts were stained with Masson’s trichrome as described previously (19). Perivascular fibrosis was calculated as the perivascular collagen area relative to the vessel area, and interstitial fibrosis was the percentage of the Azan’s trichrome- stained area per total area of cardiac tissue. The blinded measurements were made by two independent observers using ImagePro Plus 6.0 (Media Cybernetics, USA). To further detect collagen content, sections were stained in Sirius Red solution as described previously (20). Type I collagen fibrosis was determined as the ratio of red/yellow-stained area to total collagen-stained area. Statistical Analysis All values were expressed as the mean ± SEM and multiple group comparisons were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni’s test. The survival rate was analyzed by Kaplan–Meier survival analysis and compared by the log-rank test. A value of P < 0.05 was regarded to be statistically significant. The statistical analysis was performed with SPSS 17.0 and GraphPad Prism 7.0. Activation or Blockade of the GABAA Receptor Promotes or Alleviates Pressure Overload–Induced Myocardial Remodeling Activation or Blockade of the GABAA Receptor Promotes or Alleviates Pressure Overload–Induced Myocardial Remodeling Consistent with findings in cardiac function, topiramate-treated TAC mice showed more severe myocardial hypertrophy, and interstitial or perivascular fibrosis, compared with vehicle. As shown in Figures 2A–C, F, cross-sectional area of individual cardiomyocyte measured respectively by WGA or Masson trichrome staining increased as well as visible heart enlargement did in topiramate-treated TAC mice, compared with vehicle. Bicuculline-treated TAC mice had a variance effect. In addition, Masson trichrome and Sirius Red staining showed that areas of interstitial or perivascular fibrosis and I type collagen ratio after TAC were significantly increased by topiramate treatment (Figures 2D, E, G–I). However, cardiac fibrosis was significantly less prominent in mice treated with bicuculline after TAC than in vehicle. Furthermore, the GABAA receptor agonist or antagonist had no effect on the structure of the aorta, mesenteric arteries, and kidneys (Supplementary 1). GAPDH-F CAGTGGCAAAGTGGAGATTGTTG GAPDH- R TCGCTCCTGGAAGATGGTGAT GAPDH-F CAGTGGCAAAGTGGAGATTGTTG GAPDH- R TCGCTCCTGGAAGATGGTGAT GAPDH-F CAGTGGCAAAGTGGAGATTGTTG GAPDH- R TCGCTCCTGGAAGATGGTGAT C c l 2 - F C A G G T C C C T G T C A T G C T T C T C c l 2 - R CCCATTCCTTCTTGGGGTCA Cxcl 12-F CCTTCAGATTGTTGCACGGC Cxcl 12- R TTACAAAGCGCCAGAGCAGA osteopontin-F TTGCTTGGGTTTGCAGTCTTC osteopontin- R TATAGGATCTGGGTGCAGGCT osteopontin-F TTGCTTGGGTTTGCAGTCTTC osteopontin- R TATAGGATCTGGGTGCAGGCT TGF-B1-F AGGGCTACCATGCCAACTTC TGF-B1- R CCACGTAGTAGACGATGGGC Activation or Blockade of the GABAA Receptor Selectively Increases or Reduces Accumulation of Ly6Clow Macrophages in the Heart p g To assess the effect of the GABAA receptor on macrophage polarization after TAC, we analyzed macrophage subpopulations in the heart by FCM at days 1, 3, 7, 14, 21, and 28 post-TAC. We used a Ly-6C marker to identify two subsets of macrophages with distinct functions, namely Ly6Chigh and Ly6Clow macrophages. As shown in Figures 3A, B, compared with vehicle, topiramate resulted in the expansion of CD45+CD11b+F4/80+Ly6Clow macrophages (hereafter referred to as Ly6Clow macrophages) in the myocardium at days 3, 7, 14, 21, and 28 post-TAC, whereas bicuculline treatment exhibited opposite effects. Mice treated with either topiramate or bicuculline after TAC did not have significantly altered CD45+CD11b+F4/80+Ly6Chigh macrophage populations (hereafter referred to as Ly6Chigh macrophages), compared with vehicle at days 1, 3, 7, 14, and 21 post-TAC (Figures 3A, B and Supplementary 2). However, topiramate treatment markedly induced the expansion of Ly6Chigh macrophages at day 28 post-TAC (late-phase POH) compared to vehicle (Figures 3A, B). RNA Extraction and Quantitative Real-Time PCR (RT-PCR) Analysis ( ) y The total RNA of the LV were isolated using TRIzol®Reagent, and then, cDNA was synthesized using Prime Script™RT May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 3 Bu et al. The GABAA Receptor and Macrophage was significantly higher in bicuculline-treated than that in topiramate-treated TAC mice and vehicle (Figure 1H). was significantly higher in bicuculline-treated than that in topiramate-treated TAC mice and vehicle (Figure 1H). Master Mix(Takara, Japan). The internal control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All reactions were performed in a 10-ml volume-containing 1 ml cDNA, 5 ml SYBR-Green reaction mix, 0.2 ml sense primer, 0.2 ml anti-sense primer (both from TSINGKE), 0.4 ml ROX, and 3.2 ml ddH2O twice. All reactions were performed in a Applied Biosystems. Relative changes were determined using the 2−DDCt method. The primer sequences were as follows: Activation or Blockade of the GABAA Receptor Aggravates or Attenuates Pressure Overload-Induced Cardiac Dysfunction To assess the effect of the GABAA receptor on TAC-induced cardiac function, echocardiography was performed in topiramate- or bicuculline-treated TAC mice. Four weeks after TAC, the transition from concentric to dilated hypertrophy in the LV was accelerated by topiramate, while the development of HF was attenuated by bicuculline. At day 28 post-TAC, topiramate-treated TAC mice showed significantly reduced cardiac systolic function, expressed as fractional shortening (FS) or ejection fraction (EF) in comparison to vehicle. By contrast, cardiac systolic function in bicuculline-treated TAC mice was significantly improved compared with vehicle (Figures 1B, C and Supplementary Table 1). Four weeks after pressure overload, cardiac hypertrophy parameters including end- diastolic and end-systolic left ventricular internal diameter (LVIDd and LVIDs), LV mass, and heart-to-body weight, were significantly aggravated by topiramate, and attenuated by bicuculline (Figures 1A, D–G and Supplementary Table 1). The survival rate was observed up to 30 days post-TAC, which Recent studies have shown that cardiac Ly6Clow macrophages were independent of monocyte infiltration in early-phase POH. We hypothesized that activation of the GABAA receptor increased levels of Ly6Clow macrophages by stimulating local proliferation in early-phase POH. Indeed, we found that activation of the GABAA receptor led to the local proliferation of macrophages, as demonstrated by 5-ethynyl-2′-deoxyuridine (EdU) staining at day 7, but not day 28 post-TAC (Figure 3C and Supplementary 2). In parallel, activation of the GABAA receptor significantly induced the expression of the Ki-67 protein, May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org The GABAA Receptor and Macrophage Bu et al. A B D E F G H C FIGURE 1 | Activation or blockade of the GABAA receptor aggravates or attenuates cardiac dysfunction in pressure-overload hypertrophy mice. Mice underwent sham or transverse aortic constriction (TAC) operation; mice in the TAC group were treated with intraperitoneal injections once a day of NaCl, topiramate or bicuculline for 3 weeks. (A) Representative left ventricular echocardiographic recordings at day 28 post-TAC. Short-axis M-mode (left) and long-axis B-mode (right). Arrows and lines marked (left ventricle internal dimension) LVID in systole (dashed) and diastole (firm). (B) Ejection fraction (%EF), (C) fractional shortening (%FS), (D) left-ventricular mass (LV mass), (E) left ventricle internal dimension in systole (LVIDs), and (F) in diastole (LVIDd) in TAC- and sham-operated mice at day 28 post- TAC. (G) Heart weight/body weight (HW/BW) in indicated groups at day 28 post-TAC. Activation or Blockade of the GABAA Receptor Aggravates or Attenuates Pressure Overload-Induced Cardiac Dysfunction Data showed the mean ± SEM of %FS, %EF, LV mass, LVIDd, LVIDs and HW/BW for each experimental group, by one-way ANOVA with Bonferroni’s multiple comparison test. (H) Compared with vehicle, the survival rate increased in bicuculline-treated TAC group during 30 days follow-up post-TAC (n = 20 in each group). For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. NaCl-treated TAC mice (for simplicity, vehicle). (n = 4–6 in sham group, n = 10 in TAC-operated group). D E F G H FIGURE 1 | Activation or blockade of the GABAA receptor aggravates or attenuates cardiac dysfunction in pressure-overload hypertrophy mice. Mice underwent sham or transverse aortic constriction (TAC) operation; mice in the TAC group were treated with intraperitoneal injections once a day of NaCl, topiramate or bicuculline for 3 weeks. (A) Representative left ventricular echocardiographic recordings at day 28 post-TAC. Short-axis M-mode (left) and long-axis B-mode (right). Arrows and lines marked (left ventricle internal dimension) LVID in systole (dashed) and diastole (firm). (B) Ejection fraction (%EF), (C) fractional shortening (%FS), (D) left-ventricular mass (LV mass), (E) left ventricle internal dimension in systole (LVIDs), and (F) in diastole (LVIDd) in TAC- and sham-operated mice at day 28 post- TAC. (G) Heart weight/body weight (HW/BW) in indicated groups at day 28 post-TAC. Data showed the mean ± SEM of %FS, %EF, LV mass, LVIDd, LVIDs and HW/BW for each experimental group, by one-way ANOVA with Bonferroni’s multiple comparison test. (H) Compared with vehicle, the survival rate increased in bicuculline-treated TAC group during 30 days follow-up post-TAC (n = 20 in each group). For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. NaCl-treated TAC mice (for simplicity, vehicle). (n = 4–6 in sham group, n = 10 in TAC-operated group). of monocyte subsets (CD45+Ly6G-CD11b+Ly6Chigh or CD45+Ly6G-CD11b+Ly6Clow) in the peripheral blood and spleen, and monocytes (CD45+Ly6G-CD11b+Ly6C+) in marrow bone by FCM at days 1, 7, 14, 21, and 28. The results showed that the GABAA receptor exerted no influence on monocytes in the peripheral blood, spleen, and marrow bone at days 1, 7, 14, and 21 post-TAC (Figures 4D–F and Supplementary 3–5). However, treatment with either topiramate or bicuculline markedly affected Ly6Chigh monocyte counts in the peripheral blood and spleen not in marrow bone at day 28 post-TAC (late-phase POH) (Figures 4A–F). Activation or Blockade of the GABAA Receptor Aggravates or Attenuates Pressure Overload-Induced Cardiac Dysfunction For example, topiramate treatment resulted in Ly6Chigh monocyte a nuclear proliferation marker, in Ly6Clow macrophages at day 7 post-TAC (Figures 3D, E). Collectively, these results suggest that activation of the GABAA receptor increases the number of Ly6Clow macrophages by stimulating local proliferation of macrophages in early-phase POH. Activation or Blockade of the GABAA Receptor Promotes or Reduces Monocyte Recruitment in Late-Phase POH To further explore the effect of the GABAA receptor on systemic innate immunity in mice after TAC, we analyzed the number May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org The GABAA Receptor and Macrophage Bu et al. Bu et al. A B D E F G I H C FIGURE 2 | Activation or blockade of the GABAA receptor promotes or alleviates myocardial remodeling in pressure-overload hypertrophy mice. (A) Representative visible images of hearts (top) and Masson trichrome staining of heart vertical section (bottom) at day 28 post-TAC. Scale bar = 2.5 mm. (B, C) Cardiomyocytes by wheat germ agglutinin (WGA) staining (B), and Masson trichrome staining (C) at day 28 post-TAC. scale bar = 50 mm. (D, E) Cardiac fibrosis by Masson trichrome staining (D), and Sirius Red staining (E) at day 28 post-TAC. (top, perivascular fibrosis; bottom, interstitial fibrosis). (F–I) Quantified analysis of cardiomyocytes size (F), the perivascular collagen area relative to the vessel area (G), the percentage of interstitial fibrosis area in each picture (H), and the ratio of type I collagen fibrosis to total collagen-stained area (I). Sections were analyzed with Image-Pro Plus 6.3 software. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6–10). D D A B E E C C C I I H G G F H G FIGURE 2 | Activation or blockade of the GABAA receptor promotes or alleviates myocardial remodeling in pressure-overload hypertrophy mice. (A) Representative visible images of hearts (top) and Masson trichrome staining of heart vertical section (bottom) at day 28 post-TAC. Scale bar = 2.5 mm. (B, C) Cardiomyocytes by wheat germ agglutinin (WGA) staining (B), and Masson trichrome staining (C) at day 28 post-TAC. scale bar = 50 mm. (D, E) Cardiac fibrosis by Masson trichrome staining (D), and Sirius Red staining (E) at day 28 post-TAC. (top, perivascular fibrosis; bottom, interstitial fibrosis). (F–I) Quantified analysis of cardiomyocytes size (F), the perivascular collagen area relative to the vessel area (G), the percentage of interstitial fibrosis area in each picture (H), and the ratio of type I collagen fibrosis to total collagen-stained area (I). Sections were analyzed with Image-Pro Plus 6.3 software. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. Activation or Blockade of the GABAA Receptor Promotes or Reduces Monocyte Recruitment in Late-Phase POH For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6–10). TAC (Supplementary 7). However, in topiramate/bicuculline- treated TAC mice, chemokine C-X-C motif ligand-12 (Cxcl12) levels were unaltered at days 7 and 28 post-TAC, which aligned well with unaltered blood neutrophil densities (Figure 4H and Supplementary 6). These data demonstrate that activation of the GABAA receptor induces circulating Ly6Chigh classic monocyte infiltration in late-phase POH via CCL2/CCR2-dependent monocyte migration. counts in the peripheral blood and spleen to increase from 0.81 ± 0.07 × 104 to 1.78 ± 0.37 × 104, and 0.01 ± 0.01 × 104 to 0.03 ± 0.01 × 104, respectively. Furthermore, blood neutrophil counts were evaluated at days 7 and 28 post-TAC, revealing that counts in topiramate/bicuculline-treated TAC mice did not differ from those in vehicle (Supplementary 6). To determine whether the accumulation of myocardial macrophages depends on the recruitment of monocytes from blood in late-phase POH, we measured chemokine expression in myocardial tissues at days 7 and 28 post-TAC. Intriguingly, chemokine C-C motif ligand-2 (CCL2) levels were elevated in topiramate-treated TAC mice versus vehicle at day 28, but not day 7 post-TAC (Figure 4G), whereas CCL2 levels in bicuculline-treated TAC mice were lower than those in vehicle. We also found that topiramate treatment increased the percentage of CCR2+ macrophages in the heart at day 28 post- Frontiers in Immunology | www.frontiersin.org GABAA Receptor-Mediated Myocardial Hypertrophy Is Associated With Areg- Induced Akt/mTOR Signaling To elucidate the mechanism by which the GABAA receptor affects cardiac macrophages and thus, induces myocardial hypertrophy, May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 6 The GABAA Receptor and Macrophage Bu et al. B B A B D E C FIGURE 3 | Activation or blockade of the GABAA receptor selectively increases or reduces the number of Ly6Clow macrophages in the hearts of pressure-overload hypertrophy mice. Macrophage (CD45+F4/80+CD11b+) subpopulations were respectively defined as Ly6Chigh or Ly6Clow macrophages according to Ly-6C expression levels. (A) Representative images of Ly6Chigh and Ly6Clow macrophages at days 7, 14, and 28 post-TAC. The representative images of 1, 3, and 21 days after TAC were shown in supplementary materials. (B) The number of Ly6Clow macrophages or Ly6Chigh macrophages (per mg heart tissue) among the total number of live cells isolated from hearts at the indicated time points after TAC. (C) The number of CD45+CD11b+F4/80+EdU+cell (per mg heart tissue) among the total number of live cells isolated from hearts at the indicated time points after TAC. (D) Representative images of Ki-67+ expression in Ly6Clow macrophages at day 7 post-TAC. (E) The percentage of Ki-67+ expression in Ly6Clow macrophages. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for all other groups). A B C C C C D E E D E E FIGURE 3 | Activation or blockade of the GABAA receptor selectively increases or reduces the number of Ly6Clow macrophages in the hearts of pressure-overload hypertrophy mice. Macrophage (CD45+F4/80+CD11b+) subpopulations were respectively defined as Ly6Chigh or Ly6Clow macrophages according to Ly-6C expression levels. (A) Representative images of Ly6Chigh and Ly6Clow macrophages at days 7, 14, and 28 post-TAC. The representative images of 1, 3, and 21 days after TAC were shown in supplementary materials. (B) The number of Ly6Clow macrophages or Ly6Chigh macrophages (per mg heart tissue) among the total number of live cells isolated from hearts at the indicated time points after TAC. (C) The number of CD45+CD11b+F4/80+EdU+cell (per mg heart tissue) among the total number of live cells isolated from hearts at the indicated time points after TAC. (D) Representative images of Ki-67+ expression in Ly6Clow macrophages at day 7 post-TAC. GABAA Receptor-Mediated Myocardial Hypertrophy Is Associated With Areg- Induced Akt/mTOR Signaling (E) The percentage of Ki-67+ expression in Ly6Clow macrophages. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for all other groups). The GABAA Receptor Affects Fibrosis by Regulating MHCIIlow/MHCIIhigh Ratios in Subpopulations of Ly6Clow Macrophages To understand how the GABAA receptor affects fibrosis, we assessed the crosstalk that occurred between macrophages and fibroblasts. As reported, Ly6Clow macrophages can be further classified into two subpopulations based on expression levels of major histocompatibility complex (MHC) class II proteins. We found that topiramate treatment reduced MHCIIlow/MHCIIhigh ratios in subpopulations of Ly6Clow macrophages, and bicuculline increased the ratios of MHCIIlow/MHCIIhigh at days 7 and 28 post- TAC, compared with vehicle (Figures 7A, B). Furthermore, RT- PCR revealed that topiramate treatment increased osteopontin (OPN) and TGF-b transcript levels at days 7 and 28 post-TAC, whereas bicuculline treatment reduced their expression levels, compared with vehicle (Figures 7C, D). These data suggest that activation of the GABAA receptor polarizes macrophages toward we tested whether humoral factors produced from macrophages affected cardiomyocytes. Areg has recently been shown to induce cardiomyocyte hypertrophy (22). We found that topiramate increased the areas of regions that co-stained positive for Areg and F4/80 and upregulated Areg protein levels in the heart. Conversely, bicuculline treatment significantly reduced the area of co-stained regions and levels of Areg protein expression (Figures 5A–C). Furthermore, increased cardiomyocyte size in topiramate-treated mice was accompanied by increasing levels of phosphorylation of AKT and mTOR, while bicuculline reduced phosphorylation levels of both proteins relative to those which were observed in vehicle (Figures 5D–F and Supplementary 8). The capability of the GABAA receptor activation to induce Akt and mTOR phosphorylation was blocked by a neutralizing antibody specific for Areg (Figure 6). These data suggest that Areg-induced Akt/mTOR signaling pathway may be essential for GABAA receptor-mediated cardiac hypertrophy. we tested whether humoral factors produced from macrophages affected cardiomyocytes. Areg has recently been shown to induce cardiomyocyte hypertrophy (22). We found that topiramate increased the areas of regions that co-stained positive for Areg and F4/80 and upregulated Areg protein levels in the heart. Conversely, bicuculline treatment significantly reduced the area of co-stained regions and levels of Areg protein expression (Figures 5A–C). GABAA Receptor-Mediated Myocardial Hypertrophy Is Associated With Areg- Induced Akt/mTOR Signaling Furthermore, increased cardiomyocyte size in topiramate-treated mice was accompanied by increasing levels of phosphorylation of AKT and mTOR, while bicuculline reduced phosphorylation levels of both proteins relative to those which were observed in vehicle (Figures 5D–F and Supplementary 8). The capability of the GABAA receptor activation to induce Akt and mTOR phosphorylation was blocked by a neutralizing antibody specific for Areg (Figure 6). These data suggest that Areg-induced Akt/mTOR signaling pathway may be essential for GABAA receptor-mediated cardiac hypertrophy. May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 7 The GABAA Receptor and Macrophage Bu et al. A B D E F G H C FIGURE 4 | Activation or blockade of the GABAA receptor promotes or reduces monocyte recruitment in pressure-overload hypertrophy mice. (A, B) Representative images for flow cytometric analysis of CD45+Ly6G-CD11b+Ly6Chigh and CD45+Ly6G-CD11b+Ly6Clow monocytes in the blood (A), and spleen (B) at day 28 post-TAC. (C) Representative images for flow cytometric analysis of monocytes (CD45+Ly6G-CD11b+Ly6C+) in bone marrow at day 28 post-TAC. The representative images of other time points are shown in supplementary materials. (D) The number of Ly6Chigh or Ly6Clow monocytes (per ml blood) among the total number of live cells isolated from blood at the indicated time points. (E) The number of Ly6Chigh or Ly6Clow monocytes (per mg spleen) among the total number of live cells isolated from spleen at the indicated time points. (F) The number of monocytes (per mg marrow bone) among the total number of live cells isolated from marrow bone at the indicated time points. (G, H) Expression of Ccl2 and Cxcl12 marker genes at days 7 and 28 post-TAC. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for all other groups). D D D A B E E E C F H F G H G G FIGURE 4 | Activation or blockade of the GABAA receptor promotes or reduces monocyte recruitment in pressure-overload hypertrophy mice. (A, B) Representative images for flow cytometric analysis of CD45+Ly6G-CD11b+Ly6Chigh and CD45+Ly6G-CD11b+Ly6Clow monocytes in the blood (A), and spleen (B) at day 28 post-TAC. (C) Representative images for flow cytometric analysis of monocytes (CD45+Ly6G-CD11b+Ly6C+) in bone marrow at day 28 post-TAC. GABAA Receptor-Mediated Myocardial Hypertrophy Is Associated With Areg- Induced Akt/mTOR Signaling The representative images of other time points are shown in supplementary materials. (D) The number of Ly6Chigh or Ly6Clow monocytes (per ml blood) among the total number of live cells isolated from blood at the indicated time points. (E) The number of Ly6Chigh or Ly6Clow monocytes (per mg spleen) among the total number of live cells isolated from spleen at the indicated time points. (F) The number of monocytes (per mg marrow bone) among the total number of live cells isolated from marrow bone at the indicated time points. (G, H) Expression of Ccl2 and Cxcl12 marker genes at days 7 and 28 post-TAC. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for all other groups). Ly6ClowMHCIIhigh subpopulations after pressure overload in the heart. which were identified as Ly6Clow macrophages during early-phase POH, and promoted bloodborne monocyte recruitment and macrophage polarization in late-phase POH. We also found that topiramate promoted the polarization of Ly6Clow macrophages labeled with MHCIIhigh. As a consequence of Ly6Clow macrophage polarization by topiramate treatment, the Areg-induced Akt/ mTOR signaling pathway was activated, which resulted in myocardial hypertrophy. With a decrease in the ratios of MHCIIlow/MHCIIhigh subpopulations of Ly6Clow macrophages, OPN and TGF-B levels increased, thereby contributing to fibrosis. As the antagonist, bicuculline exhibited the opposite effect. Frontiers in Immunology | www.frontiersin.org DISCUSSION In this study, we identified the role of GABAA receptors in pressure overload-induced HF. Using the GABAA-specific receptor agonist or antagonist, we found that activation of the GABAA receptor by topiramate resulted in cardiac dysfunction, as shown by worsened FS & EF, enlarged LVIDd & LVIDs, and aggravation of cardiac hypertrophy and fibrosis, in comparison to vehicle. In contrast, blockade of the GABAA receptor by bicuculline reversed this effect in mice with POH, protecting them against cardiac dysfunction and hypertrophy. Mechanistically, topiramate stimulated the local proliferation of cardiac resident macrophages, In this study, we identified the role of GABAA receptors in pressure overload-induced HF. Using the GABAA-specific receptor agonist or antagonist, we found that activation of the GABAA receptor by topiramate resulted in cardiac dysfunction, as shown by worsened FS & EF, enlarged LVIDd & LVIDs, and aggravation of cardiac hypertrophy and fibrosis, in comparison to vehicle. In contrast, blockade of the GABAA receptor by bicuculline reversed this effect in mice with POH, protecting them against cardiac dysfunction and hypertrophy. Mechanistically, topiramate stimulated the local proliferation of cardiac resident macrophages, TAC is a classic model that presents dynamic changes of pressure overload-induced hypertrophy and HF (23). In response to TAC, as it is commonly held, the compensatory cardiac hypertrophy occurs within the initial 2 weeks for the sake of preserved contractile function (24). In a period ranging from 2 to 4 weeks after TAC, decompensation and HF manifest (9). Here, we May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 8 The GABAA Receptor and Macrophage Bu et al. Bu et al. A B D E F C FIGURE 5 | Areg expression and Akt/mTOR signaling are involved in the GABAA receptor-mediated cardiac hypertrophy. (A) Representative micrographs showing immunofluorescent staining of Areg (red) and a macrophage marker, F4/80 (green) in a sample of heart tissue. Nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. (B) Representative images of Areg protein at day 7 post-TAC in heart. (D) Representative images of P-Akt and P-mTOR protein at day 7 post-TAC in heart. (C, E, F) Quantitative analysis of Areg protein (C), P-Akt protein (E), and P-mTOR protein (F) at day 7 post-TAC in heart. GAPDH mAb (middle) was used as a loading control. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. DISCUSSION For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6). A B C B A B C C E F D D E FIGURE 5 | Areg expression and Akt/mTOR signaling are involved in the GABAA receptor-mediated cardiac hypertrophy. (A) Representative micrographs showing immunofluorescent staining of Areg (red) and a macrophage marker, F4/80 (green) in a sample of heart tissue. Nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. (B) Representative images of Areg protein at day 7 post-TAC in heart. (D) Representative images of P-Akt and P-mTOR protein at day 7 post-TAC in heart. (C, E, F) Quantitative analysis of Areg protein (C), P-Akt protein (E), and P-mTOR protein (F) at day 7 post-TAC in heart. GAPDH mAb (middle) was used as a loading control. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6). activation of the GABAA receptor A3 subunit has been shown to impair colon barrier function by upregulating macrophage activity in early-life stress mice (16). In comparison, inhibition of the GABAA receptor reduced neuroinflammation in the hippocampus and CNS (26, 27). These diverse effects might be due to GABAA receptor activation in different pathological situations. present findings indicating that macrophages are a potential pharmacological target against pressure overload-induced HF. Intriguingly, bicuculline, a GABAA receptor antagonist that is clinically used as an analgesic, produced novel effect and emerged as a chemical candidate for the reversal of cardiac dysfunction due to pressure overload. It exerted a significant influence on myocardial hypertrophy as well as fibrosis, contrasted by topiramate. By affecting both the pathological changes underlying pressure overload-induced HF, the process of POH can be modulated. Therefore, during an initial or early phase, blockade of the GABAA receptor made it possible to manipulate cardiac resident macrophages to achieve the goal of reversing POH. p p g In early-phase POH, GABAA receptor activation or blockade did not affect neutrophils and monocytes, whereas it promoted or suppressed local proliferation of cardiac resident macrophages marked with Ly6Clow as shown in Figures 3 and 4. These results are in accordance with those of Liao X et al. who showed that cardiac residence in macrophages is a mechanism of self- renewal, which occurred within early-phase POH. Frontiers in Immunology | www.frontiersin.org DISCUSSION Increasing evidences support that GABAergic drugs can cause CNS or myocardial pathologic changes through activation or blockade of the GABAA receptor in macrophages independent of CNS (14, 26). A recent study shows that Ly6Clow macrophages can induce a hypertrophic response in cardiomyocytes for myocardial adaptive response to pressure overload (22). Evidence indicates that cardiac resident macrophages have tissue-repairing capacities after injuries (28). In mice with myocardial Recently, increasing evidence has indicated that the GABAA receptor is enriched in immune cells, where it exhibits anti- or pro- inflammatory effects. Previous findings showed that activation of the GABAA receptor could potently inhibit post-infarction inflammation by acting upon bloodborne or cardiac resident macrophages (14). Moreover, Kim et al. suggested that GABAergic activation promoted macrophage antimicrobial responses against mycobacterial infection (25). However, May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 9 The GABAA Receptor and Macrophage Bu et al. Bu et al. A B D C FIGURE 6 | Blocking of Areg activity suppresses topiramate-mediated effect on Akt/mTOR signaling. (A) Representative images of Areg, P-Akt, and P-mTOR proteins at day 7 post-TAC in heart. T: topiramate; I: IgG; A: Anti-Areg Ab + IgG + topiramate. (B–D) Quantitative analysis of Areg protein (B), P-Akt protein (C), and P-mTOR protein (D) at day 7 post-TAC in heart. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05 vs. TAC + Topiramate+ IgG. (each group, n = 6). A B D C C D FIGURE 6 | Blocking of Areg activity suppresses topiramate-mediated effect on Akt/mTOR signaling. (A) Representative images of Areg, P-Akt, and P-mTOR proteins at day 7 post-TAC in heart. T: topiramate; I: IgG; A: Anti-Areg Ab + IgG + topiramate. (B–D) Quantitative analysis of Areg protein (B), P-Akt protein (C), and P-mTOR protein (D) at day 7 post-TAC in heart. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05 vs. TAC + Topiramate+ IgG. (each group, n = 6). macrophages may induce cardiac fibrosis, which leads to diastolic dysfunction in mice with hypertension or advanced age (31). Our data showed that activation of the GABAA receptor increased the ratios of MHCIIhigh/MHCIIlow. Due to the shift toward MHCII high macrophages, TGF-B or OPN expression was upregulated, thereby aggravating cardiac fibrosis. Frontiers in Immunology | www.frontiersin.org DISCUSSION infarction (MI), proliferation of cardiac resident macrophages together with monocyte-derived Ly6Clow macrophages activated by topiramate attenuated ventricular remodeling by promoting neovascularization and fibrosis instead of infarcts. However, activation of the GABAA receptor compelled cardiac resident macrophages to play a new role in ventricular remodeling in response to pressure overload. These players contribute to cardiac hypertrophy and fibrosis by producing effectors. For instance, Areg, a humoral factor specifically produced by Ly6Clow macrophages, induces a hypertrophic response and activates AKT/mTOR signaling which is proportional to rate of eccentric hypertrophy progression under pressure overload (29, 30). In late-phase POH, which was accompanied by EF reduction and enlargement of LV, Ly6Chigh monocytes infiltrated the myocardium and were derived into macrophages, which participated in myocardial injury and repair responses. Thus, macrophages acted similar to MI. The mechanism of GABAergic drugs on cultured peritoneal macrophages and bone marrow-derived macrophages was ever investigated (13, 25). However, cardiac resident macrophages emerge in recent years with little knowledge of their nature in vivo or vitro and their subtypes (7, 32). Thus, it is imperative to gain more knowledge in future. Similar to monocyte-derived Ly6Clow macrophages, cardiac resident macrophages can also promote fibrosis. As reported recently, these Ly6Clow macrophages can be further classified into MHCIIhigh or MHCIIlow macrophages based on MHCII expression levels (28). MHCIIlow macrophages favor matrix breakdown, whereas MHCIIhigh macrophages display enrichment in a gene set that regulates fibrosis. The high ratios of MHCIIhigh/MHCIIlow In conclusion, our study provides novel insights into the role of cardiac resident macrophages and GABAA receptor in pressure overload-induced HF. The pros and cons of our results May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 10 The GABAA Receptor and Macrophage Bu et al. B A B D C FIGURE 7 | The GABAA receptor affects fibrosis by regulating MHCIIlow/MHCIIhigh ratios in subpopulations of Ly6Clow macrophages. Ly6Clow macrophages could be further classified into two subpopulations based on MHC II proteins. (A) Representative images of MHC II high and MHC II low macrophages at days 7 and 28 post-TAC in heart. (B) Quantified analysis of the percentage of MHC II low and MHC II high macrophages at days 7 and 28 post-TAC. (C, D) Relative mRNA expression of OPN (C), and TGF-B marker genes (D) at days 7 and 28 post-TAC in the heart. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. DISCUSSION For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6). A C D C D D FIGURE 7 | The GABAA receptor affects fibrosis by regulating MHCIIlow/MHCIIhigh ratios in subpopulations of Ly6Clow macrophages. Ly6Clow macrophages could be further classified into two subpopulations based on MHC II proteins. (A) Representative images of MHC II high and MHC II low macrophages at days 7 and 28 post-TAC in heart. (B) Quantified analysis of the percentage of MHC II low and MHC II high macrophages at days 7 and 28 post-TAC. (C, D) Relative mRNA expression of OPN (C), and TGF-B marker genes (D) at days 7 and 28 post-TAC in the heart. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6). AUTHOR CONTRIBUTIONS show that cardiac resident macrophages can probably act as key players in the progression from HFpEF to HFrEF. Thus, targeting the GABAA receptor in cardiac resident macrophages will likely benefit the early treatment of HFpEF. And the antagonist or blocker emerges as a promising therapeutical chemical. JB and ZW conceived the study. JB, YY, TX, HL, and JW performed the experiments and data analyses. KL and SH provided intellectual inputs. JB and KL wrote the manuscript. All authors edited and approved the manuscript. All authors contributed to the article and approved the submitted version. JB and ZW conceived the study. JB, YY, TX, HL, and JW performed the experiments and data analyses. KL and SH provided intellectual inputs. JB and KL wrote the manuscript. All authors edited and approved the manuscript. All authors contributed to the article and approved the submitted version. FUNDING This work was supported by the National Natural Science Foundation of China (81901429 to SH and 82070514 to KL). DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. REFERENCES Inflammation in Mice. Hypertension (2017) 70(4):759–69. doi: 10.1161/ HYPERTENSIONAHA.117.09262 Inflammation in Mice. Hypertension (2017) 70(4):759–69. doi: 10.1161/ HYPERTENSIONAHA.117.09262 1. Braunwald E. The War Against Heart Failure: The Lancet Lecture. Lancet (2015) 385(9970):812–24. doi: 10.1016/s0140-6736(14)61889-4 19. Song L, Wang L, Li F, Yukht A, Qin M, Ruther H, et al. 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Kallikourdis M, Martini E, Carullo P, Sardi C, Roselli G, Greco CM, et al. T Cell Costimulation Blockade Blunts Pressure Overload-Induced Heart Failure. Nat Commun (2017) 8:14680. doi: 10.1038/ncomms14680 24. Yoo J, Chepurko V, Hajjar RJ, Jeong D. Conventional Method of Transverse Aortic Constriction in Mice. Methods Mol Biol (2018) 1816:183–93. doi: 10.1007/978-1-4939-8597-5_14 7. Kierdorf K, Prinz M, Geissmann F, Gomez Perdiguero E. Development and Function of Tissue Resident Macrophages in Mice. REFERENCES Semin Immunol (2015) 27 (6):369–78. doi: 10.1016/j.smim.2016.03.017 25. Kim JK, Kim YS, Lee HM, Jin HS, Neupane C, Kim S, et al. Gabaergic Signaling Linked to Autophagy Enhances Host Protection Against Intracellular Bacterial Infections. Nat Commun (2018) 9(1):4184. doi: 10.1038/s41467-018-06487-5 8. Epelman S, Lavine KJ, Beaudin AE, Sojka DK, Carrero JA, Calderon B, et al. 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Shimizu I, Minamino T, Toko H, Okada S, Ikeda H, Yasuda N, et al. Excessive Cardiac Insulin Signaling Exacerbates Systolic Dysfunction Induced by Pressure Overload in Rodents. J Clin Invest (2010) 120(5):1506–14. doi: 10.1172/JCI40096 12. Kain D, Amit U, Yagil C, Landa N, Naftali-Shani N, Molotski N, et al. Macrophages Dictate the Progression and Manifestation of Hypertensive Heart Disease. Int J Cardiol (2016) 203:381–95. doi: 10.1016/j.ijcard.2015.10.126 30. Sciarretta S, Forte M, Frati G, Sadoshima J. New Insights Into the Role of Mtor Signaling in the Cardiovascular System. Circ Res (2018) 122(3):489–505. doi: 10.1161/CIRCRESAHA.117.311147 13. SUPPLEMENTARY MATERIAL The animal study was reviewed and approved by Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from the owners for the participation of their animals in this study. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. 670153/full#supplementary-material May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org The GABAA Receptor and Macrophage Bu et al. REFERENCES Bhat R, Axtell R, Mitra A, Miranda M, Lock C, Tsien RW, et al. Inhibitory Role for GABA in Autoimmune Inflammation. Proc Natl Acad Sci USA (2010) 107(6):2580–5. doi: 10.1073/pnas.0915139107 14. Wang Z, Huang S, Sheng Y, Peng X, Liu H, Jin N, et al. Topiramate Modulates Post-Infarction Inflammation Primarily by Targeting Monocytes or Macrophages. Cardiovasc Res (2017) 113(5):475–87. doi: 10.1093/cvr/cvx027 31. Hulsmans M, Sager HB, Roh JD, Valero-Munoz M, Houstis NE, Iwamoto Y, et al. Cardiac Macrophages Promote Diastolic Dysfunction. J Exp Med (2018) 215(2):423–40. doi: 10.1084/jem.20171274 32. Nicolas-Avila JA, Hidalgo A, Ballesteros I. Specialized Functions of Resident Macrophages in Brain and Heart. J Leukoc Biol (2018) 104(4):743–56. doi: 10.1002/JLB.6MR0118-041R 15. Jiang SH, Zhu LL, Zhang M, Li RK, Yang Q, Yan JY, et al. GABRP Regulates Chemokine Signalling, Macrophage Recruitment and Tumour Progression in Pancreatic Cancer Through Tuning KCNN4-Mediated Ca(2+) Signalling in a GABA-independent Manner. Gut (2019) 68(11):1994–2006. doi: 10.1136/ gutjnl-2018-317479 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 16. SeifiM, Rodaway S, Rudolph U, Swinny JD. Gabaa Receptor Subtypes Regulate Stress-Induced Colon Inflammation in Mice. Gastroenterology (2018) 155(3):852–64.e3. doi: 10.1053/j.gastro.2018.05.033 17. Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, et al. Segregation of Atrial-Specific and Inducible Expression of an Atrial Natriuretic Factor Transgene in an In Vivo Murine Model of Cardiac Hypertrophy. Proc Natl Acad Sci USA (1991) 88(18):8277–81. doi: 10.1073/ pnas.88.18.8277 Copyright © 2021 Bu, Huang, Wang, Xia, Liu, You, Wang and Liu. 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. 18. Wu L, Zhao F, Dai M, Li H, Chen C, Nie J, et al. P2y12 Receptor Promotes Pressure Overload-Induced Cardiac Remodeling Via Platelet-Driven May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 12
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Edge Intelligence in Private Mobile Networks for Next-Generation Railway Systems
Frontiers in communications and networks
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Edge Intelligence in Private Mobile Networks for Next-Generation Railway Systems Syed Muhammad Asad 1,2*, Ahsen Tahir 3, Rao Naveed Bin Rais 4, Shuja Ansari 1, Attai Ibrahim Abubakar 1, Sajjad Hussain 1, Qammer H. Abbasi 1 and Muhammad Ali Imran 1,5 1James Watt School of Engineering, University of Glasgow, Glasgow, United Kingdom, 2Transport for London (TfL), London, United Kingdom, 3Electrical Engineering, University of Engineering and Technology, Lahore, Pakistan, 4Electrical and Computer Engineering, Ajman University, Ajman, United Arab Emirates, 5Artificial Intelligence Research Center (AIRC), Ajman University, Ajman, United Arab Emirates The integration of Private Mobile Networks (PMN) with edge intelligence is expected to play an instrumental role in realizing the next generation of industry applications. This combination collectively termed Intelligent Private Networks (IPN) deployed within the scope of specific industries such as transport systems can unlock several use cases and critical applications that in turn can address rising business demands. This article presents a conceptual IPN that hosts intelligence at the network edge employing emerging technologies that satisfy a number of Next-Generation Railway System (NGRS) applications. NGRS use cases along with their applications and respective beyond 5G (B5G) enabling technologies have been discussed along with possible future research and development directions that will allow these promising technologies to be used and implemented widely. Edited by: Akram Alomainy, Queen Mary University of London, United Kingdom Edited by: Akram Alomainy, Queen Mary University of London, United Kingdom Reviewed by: Syed Aziz Shah, Coventry University, United Kingdom Xiaodong Yang, Xidian University, China Aifeng Ren, Xidian University, China Reviewed by: Syed Aziz Shah, Coventry University, United Kingdom Xiaodong Yang, Xidian University, China Aifeng Ren, Xidian University, China Keywords: B5G, edge intelligence, NB-IoT, intelligent transport systems, intelligent private network, next generation transport systems, 6G *Correspondence: Syed Muhammad Asad s.asad.1@research.gla.ac.uk *Correspondence: Syed Muhammad Asad s.asad.1@research.gla.ac.uk 1 INTRODUCTION Connectivity is now found in almost all facets of modern lives. What started with the simple provision of connecting humans paved the way for connected processes and developments that changed the outlook of what now is a global economy. Over the past two decades, the number of connected devices and subsequent processes have risen immensely. The advantages this connectivity brings into our lives are not only limited to convenience and globalization but also provide the basis of use cases and applications that substantially contribute toward the socioeconomic welfare of our society. Specialty section: This article was submitted to IoT and Sensor Networks, a section of the journal Frontiers in Communications and Networks Received: 01 September 2021 Accepted: 20 September 2021 Published: 14 October 2021 Citation: Asad SM, Tahir A, Rais RNB, Ansari S, Abubakar AI, Hussain S, Abbasi QH and Imran MA (2021) Edge Intelligence in Private Mobile Networks for Next- Generation Railway Systems. Front. Comms. Net 2:769299. doi: 10.3389/frcmn.2021.769299 Specialty section: This article was submitted to IoT and Sensor Networks, a section of the journal Frontiers in Communications and Networks Received: 01 September 2021 Accepted: 20 September 2021 Published: 14 October 2021 Specialty section: This article was submitted to IoT and Sensor Networks, a section of the journal Frontiers in Communications and Networks Due to the rise in capacity, security, and critical communication needs Asad et al. (2019), major industries are now seeking to establish their own Private Mobile Networks (PMN). These PMN are private wide-area multiple access wireless networks that are highly scalable, provide an adequate range, are and consistent Quality of Service (QoS) in comparison to license-exempt solutions such as WiFi. The privatization of mobile networks was made possible with the advent of Long-Term Evolution (LTE) due to its simplicity and an all-IP nature. Such networks consist of an Evolved Packet Core (EPC) usually integrated with IP Multimedia System (IPMS) for handling voice calls and a typical Radio Access Network (RAN) (Katsalis et al., 2017). Another key enabler for PMN is the virtualization of computing and network resources where an entire core network and parts of access network can be hosted in the cloud while the cognitive radio at the edge of the network can make ORIGINAL RESEARCH published: 14 October 2021 doi: 10.3389/frcmn.2021.769299 Citation: Asad SM, Tahir A, Rais RNB, Ansari S, Abubakar AI, Hussain S, Abbasi QH and Imran MA (2021) Edge Intelligence in Private Mobile Networks for Next- Generation Railway Systems. Front. Comms. Net 2:769299. doi: 10.3389/frcmn.2021.769299 October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org Asad et al. IPN Architecture efficient use of unused spectrum to provide seamless end-to-end connectivity. Improvements in the LTE technologies in terms of added intelligence and improved capacity, both in core and access networks, have paved the way for 5G mobile networks (Zhang, 2020). We perceive that these improvements along with application-centric intelligence can further shape PMN for individual industries. Current railway systems are facing many challenges due to the deficiency of advanced 5G functions and capabilities to support various use cases, services, and scenarios (Dat et al., 2016). The 5G networking model use cases that support railway systems architecture are amazing speed; great service in a crowd; ubiquitous device communications; super real- time and reliable connections; and the best user experience. All of these use cases are challenges to railway networks which in turn become a necessity due to high rise data rates and capacity needs. In this article, we present IPN for the Next-Generation Railway System (NGRS) and outline enabling technologies and frameworks that will be the key enablers in the scope of intelligent mobility. This article is structured in such a way that we start by outlining edge intelligence in the context of mobile networks in Section 2 and then a discussion on key challenges in NGRS is included in Section 3. Section 4 then includes mobile edge technologies and use cases that can potentially address the challenges outlined in Section 3. Section 5 presents the IPN architecture used to integrate edge intelligence and PMN in the context of addressing NGRS challenges via 5G or B5G technologies. Finally, Section 6 presents the conclusion and some future research directions. management, and service provisioning management. In Zhang (2020), several Internet-of-Things (IoT) and 5G sensor networks in railway communication have been exploited along with the previous industry challenges and their forthcoming resolutions. In Zoha et al. (2020), a summary of self-healing block in self- organizing 5G networks which presents a framework for autonomous outage detection and coverage optimization in an LTE has been presented. Hussain et al. 2.1 User-Centric Edge Intelligence 2.1 User Centric Edge Intelligence The QoS of any network is a key metric in evaluating its performance as it correlates to the users’ level of satisfaction. In a similar pursuit, added intelligence in the network make them more user-centric by including users’ experience in the feedback loop. Beyond 5G (B5G) networks will be able to assess the users’ demands by running various distributed federated learning algorithms at the edge of the network. With the inference capability derived from federated learning in hand, the edge nodes performing user sentiment analysis can subsequently inform the network of required changes (Wang et al., 2019). 2 EDGE INTELLIGENCE IN MOBILE NETWORKS The exponential increase in computing resources and substantial improvements in Artificial Intelligence (AI) algorithms over the past decade has paved the way for intelligence to be integrated into the day-to-day operations of mobile networks. Several studies, Niyato (2020), Li et al. (2017), Zhang (2020), Zoha et al. (2020), and Hussain et al. (2020), have presented the use of AI-based frameworks to enable the imminent and future demands of ever-expanding mobile networks. For instance, in Niyato (2020), artificial intelligence- (AI-) based applications and services and their challenges in a comprehensive overview have been provided. It also highlights AI services and their mechanism as to how they are being applied to the network edge near the data sources and demonstrates how AI and edge computing can be mutually beneficial. To do so, it introduces and discusses edge intelligence and intelligent edge and their implementation methods and enabling technologies, namely, AI training and inference in the customized edge-computing framework. In Li et al. (2017), the standardization of 5G cellular networks is being expedited to provide insight into the candidate techniques as a whole and examine the design philosophy behind them. It highlights one of the most fundamental features among the revolutionary techniques in the 5G era; i.e., there emerges initial intelligence in nearly every important aspect of cellular networks, including radio resource management, mobility Citation: (2020) presented a split in the 5G network into several 100-cell regions, each monitored by an edge server, and proposed a framework that preprocesses raw call detail records having user activities to create an image-like volume, fed to a machine learning (ML) model. Yang et al. (2019) presented the application of tactile Internet in healthcare by considering the diabetic ketoacidosis (DKA) as a 5G use case to monitor life-threatening complications of diabetes mellitus by using the C-Band sensing technique. Reducing the computational distribution and load in a dense deployment will require edge intelligence. With edge intelligence and dense deployment of base stations, the applications will be numerous. To explain the intelligence presented in future IPN for NGRS, we classify these operations into user-centric, network-centric, and application-centric edge intelligence. Although these will operate hand in hand in utter harmony, it is important to understand the perspective of intelligence from these different angles as shown in Figure 1. efficient use of unused spectrum to provide seamless end-to-end connectivity. Improvements in the LTE technologies in terms of added intelligence and improved capacity, both in core and access networks, have paved the way for 5G mobile networks (Zhang, 2020). We perceive that these improvements along with application-centric intelligence can further shape PMN for individual industries. Current railway systems are facing many challenges due to the deficiency of advanced 5G functions and capabilities to support various use cases, services, and scenarios (Dat et al., 2016). The 5G networking model use cases that support railway systems architecture are amazing speed; great service in a crowd; ubiquitous device communications; super real- time and reliable connections; and the best user experience. All of these use cases are challenges to railway networks which in turn become a necessity due to high rise data rates and capacity needs. In this article, we present IPN for the Next-Generation Railway System (NGRS) and outline enabling technologies and frameworks that will be the key enablers in the scope of intelligent mobility. This article is structured in such a way that we start by outlining edge intelligence in the context of mobile networks in Section 2 and then a discussion on key challenges in NGRS is included in Section 3. Section 4 then includes mobile edge technologies and use cases that can potentially address the challenges outlined in Section 3. Citation: Section 5 presents the IPN architecture used to integrate edge intelligence and PMN in the context of addressing NGRS challenges via 5G or B5G technologies. Finally, Section 6 presents the conclusion and some future research directions. 2.2 Network-Centric Edge Intelligence 2.2 Network Centric Edge Intelligence The efficient utilization of network resources can make the network more sustainable in terms of lower Operational and Capital Expenditure (OPEX/CAPEX). With that in mind, self-organizing and self-optimizing algorithms deployed at the network edge can enable timely inference and quick decisions for effective radio resource management, user mobility management, network orchestration, and service provisioning. 2.3 Application-Centric Edge Intelligence 2.3 Application-Centric Edge Intelligence In B5G networks, several applications are expected to be hosted at the edge of the network. This will require virtualization of application containers and related computational resources at the base station. Since applications have varying demands, the application intelligence employed along with the deployment strategy will vary from case to case; for example, AI algorithms used in train automation will have a different October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 2 Asad et al. IPN Architecture FIGURE 1 | Intelligent Private Network (IPN) with user, network, and application-centric edge intelligence for Next-Generation Railway Systems (NGRSs). FIGURE 1 | Intelligent Private Network (IPN) with user, network, and application-centric edge intelligence for Next-Generation Railway Systems (NGRSs). deployment strategy compared to AI campaigns that elevate passenger travel experience. that would address all the outstanding issues. This lack of literature motivates this article where we have gathered evidence to identify the challenges and opportunities faced within the current railway transport networks. This will help us understand how the edge intelligence in B5G private networks holds significant importance while prioritizing relevant solutions. 3 ANTICIPATED CHALLENGES IN NGRS The advancement in wireless networks along with the technical developments in the transport sector has acquainted the industry with an entirely new approach to public safety, travel efficiency, and information dissemination. Intelligent Transport Systems (ITS) refer to a new set of information and communication technologies that allow transport entities to exchange data with each other or the infrastructure to support several transport-related applications. However, predominantly, the focus within ITS has been toward road users; hence in this article, we move that focus to intelligent railway systems. In the context of IPN, we discuss the key challenges and their use cases enabled by edge intelligence in railway transport networks. Frontiers in Communications and Networks | www.frontiersin.org 3.1 Safety and Security Public panic situations in the current underground train network can become severely adverse if prompt monitoring and response are not provided. Safety and security measures are highly essential for diffusing such situations in the interest of public safety. There are also concerns about crime and disorder that act as a barrier to passengers’ travel. An evolving focus on such issues is critical to reduce crime and improve passengers’ confidence. Public safety applications utilizing intelligent solutions at the network edge can help in the predicament of daily travel against the threat of terrorism, financial constraints, and monitoring passenger commutes. Currently, the underground transportation system is facing key challenges with the passengers’ movement information and pathways, bidirectional communication between passenger flows on transit and information centers, and collection and processing of “big data” coming from a range of Internet-of-Things (IoT) devices. These challenges are mainly due to the maintenance, reliability, connectivity, and logistics issues while innovation management, cost management, deployment of IoT devices, durability, and sustainability of these devices also pose concerns. The methodologies prevailing in ITS where railway networks are concerned have not attained the level of research 3.2 Traffic and Crowd Management Congestion due to traffic rise causes impatience, anger, and frustration within passengers which in turn influence their travel behavior causing the journeys to become slow, unpredictable, and stagnant (Asad et al., 2019). For transport businesses, any form of disruption or congestion in their services directly or indirectly affects their CAPEX and OPEX while causing harm not only to the business operations but also to the reputation of the company. If these travel delay issues are not October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 3 Asad et al. IPN Architecture addressed, on average, a passenger can waste up to two and a half days per year waiting for a congested train. Identification of passenger movement in underground train networks is also a persisting challenge that contributes to high energy consumption and subsequent failure of effective optimization measures for mobile networks (Asad et al., 2020a; Asad et al., 2020b). AI can fuel numerous traffic management applications residing at the edge of the PMN while keeping a closer look at the traffic patterns and inferring decisions in smarter ways. climate. The transport system must be resilient to extreme weather conditions and must have the capability of self- healing against climate adversities. 3.1 Safety and Security Disruption to the trains and overall transport network could be caused by either rainfall, heatwaves, high winds, or snow. With a distributed private network employing intelligence at many facets, it can provide useful information to the control center in identifying weather patterns with future predictions allowing informed decision-making. 4.1.2 Contact-Tracing System The novel Coronavirus 2019 (COVID-19) proved its viral infectious nature and consequently forced the entire world into a lockdown. For transport operators, such an outbreak placed an enormous challenge in continuing their services in a safe manner (Asad et al., 2020c). Train passengers are liable to face viral health issues while traveling in congested trains where all the travelers are in proximity to one another without social 4.1 Use Cases and Applications Evolution toward B5G networks for transport systems aims to address challenges in essentially all businesses that come under the umbrella of ITS. Edge intelligence is expected to remodel the existing trends in terrestrial networks to accommodate the mobile nature of sensor networks, train signaling, security, and overall logistics within underground train networks. Although there are numerous use cases, we enlist a few that we believe will address the current challenges around underground railway environments. 3.4 Latency Real-time mission-critical applications and quick emergency response operations require the satisfaction of specific latency criteria without considering existing operational technology for train networks. However, the current wideband radio spectrum shared among various services pose challenges and obstacles in effectively serving the critical transport entity operations. Similarly, anomalies from existing passenger traffic flows are difficult to predict for such a demand critical consortium. For instance, if a criminal has to be put into custody by chasing them within a massive traffic flow, the real-time crowd information can be highly vital in providing the emergency services (ES) a safe and less disrupting passage to the criminal. 4.1.1 Intelligent Train-to-Train (IT2T) Communications The wireless industry is developing processes and methods that will achieve sustainable solutions to support IT2T Communications. Most of the approaches and state-of-the-art works regarding direct communications fall short in determining the low-level requirements, use cases, and their applications to the best of our knowledge. IT2T communications will be able to offer low-latency intelligent train networks, autonomous trains, fast adaptive management of congestion control, onboard Internet and infotainment services, broadcasting of real-time fault incidents, real-time fault detection and recovery, and teleoperated trains ensuring fast and safe control against hazards. Some of these use cases are now possible by utilizing small-sized antenna arrays within underground trains and tunnels operating in the sub-6 GHz and/or mmWave band. The vulnerability due to shadowing and poor isotropic propagation loss in the underground environment would need to be controlled and managed effectively within these use cases (Ford et al., 2017). 4 ADDRESSING NGRS CHALLENGES WITH IPN EDGE INTELLIGENCE Common transport systems are designed based on the principle of “reaction for any action.” While this has proven to work in many cases, with the current increase in quick response requirements, other modes of reactive operations are now being sought. With the reactive approach, actions operate after an event has occurred. For time-critical applications, this results in a waste of resources and user QoS. Therefore, there is a need to address these issues with proactiveness that can administer the dynamics of the transport specifically in an underground train network where the conditions are rapidly changing. This proactive nature can be achieved by having intelligence deployed at the edge of the network where the application can predict the future network state well in advance. In the following subsections, we first discuss the use cases of NGRS, and then we describe IPN technologies that we believe will play an instrumental role in the realization of NGRS. 3.5 Planning and Costing Existing technologies within underground train networks are extremely beneficial; however, they bring in deployment and sustainability-related costs. Several strategies and techniques have been developed and deployed for the planning and costing of densely populated networks and energy-efficient systems, but they fail to address many challenges. Planning regulations are required to be in compliance with the active development procedures and regulations. We believe that network intelligence at the edge can provide a scalable and cost-effective solution in the shape of a real-time informed decision-making framework suitable for planning and costing related activities of underground train networks. 4.1.7 Intelligent Wireless Audio Today, emergency services in the underground environment use wireless audio links through existing technologies that offer highly specialized RF communication solutions. Due to the scarce spectrum and shared resources, in-use emergency audio devices are facing challenges within the dynamic underground environment. Besides this fact, the multioperational emergency services are demanding an improvement in the quality (increment in quantity) of their radio spectrum in order to provide effective safety to their staff and passengers against antisocial behaviors. An innovative edge intelligence agent employed within the IPN can address such needs with the use of ultrareliable low-latency communications (URLLC). Integration of URLLC in IWA can be seen in current automation industries (Pilz et al., 2018) where there are high demands on communication for automation-related use cases but it has scarcely been used as a use case in the application of the transport industry specifically underground train networks where high reliability and low-latency services would benefit the IWA infrastructure. 4.1.4 Transport Hub Crowd Management p g Predicting and managing major transport hubs’ capacity in both businesses as usual (BAU) and exceptional circumstances, e.g., rush hour, travel disruption, and planned events by using Machine Learning (ML), is one of the challenging aspects for Transport Hub Crowd Management (THCM). Inspired by fixed monitoring systems, THCM could employ fixed sensors (Asad et al., 2020a; Ansari et al., 2018) or 5G mobile networks (Li et al., 2017) to cope with the limitations it currently faces. This is stressed out by the exponential spread of uncertainties enabled by a high passenger density and frequent footprint in transport hubs. Sensors in the hubs would use a triangulation method to detect the location of passengers that would be echoed from their 5G handsets, called echolocation. Digital tools such as Real-Time Location Systems (RTLS) or Intelligent Positioning Systems (IPS) hosted at the network edge will be important for precise echolocation gathering of real-time crowd information. 4.1.3 Intelligent Drones System 4.1.3 Intelligent Drones System Encouraged by the promising 5G capabilities, we envisage surveying, information capturing, and live surveillance streaming through Intelligent Drones System (IDS). The functional application of the IDS will be a key enabler to address overcrowding situations in places such as train terminals. The other two functions for IDS can be the provisioning of communication services through network surveillance using low bandwidth control signals and/or streaming live camera surveillance using high bandwidth data signals. The operation of IDS is expected to be a mixture of multiple technologies to achieve highly versatile services. Drones within IDS will be able to communicate with the IPN utilizing their respective network slice in order to collate multiple communication services into one back-end cloud network (Solomitckii et al., 2018). This is achieved by integrating a Centralized Intelligent Multiplexer (CIM) at the edge of the network that receives multiple signals from different drones and combines them to establish the accurate content (multipath propagation) for data analytics (Solomitckii et al., 2018; Paulo Klaine, 2018). 4.1.6 Intelligent Monitoring Services The rapid growth of connected wireless devices drives the purpose of the Internet of Things (IoT) through mMTC within 5G and beyond networks. IoT within the underground environment requires the capability of interconnecting diverse services via the wireless network utilizing narrow bandwidth and consuming low power while providing maximum penetration in the train tunnels. In a complex underground ecology, the number of applications for such a use case suited to Narrow Band IoT (NB-IoT) (Li et al., 2017) is, but not limited to, underground tunnels gas and water metering, pressurized fuel detection, smoke alarms detection, fire alarms monitoring, and event detectors. AI within IPN is a key enabler for many isolated and distributed devices to function under one centralized cloud. Additionally, with such an IPN framework, broadcasting to and from all the connected devices at one time is also an essential component. For the implementation of broadcasting, it would require Intelligent B5G-Xcast (IBX) by leveraging the findings of point to point (PTP), point to multipoint (PTM), and 5G-Xcast project (Banchs et al., 2019). The use of laser-based IoT devices to monitor and subsequently recommend safe distances from the toxic gases found in underground confined areas also has utmost importance for safety purposes. 3.6 Resilience The impact of weather on the transport network is another main challenge that requires daily/hourly weather forecasting and understanding of the anticipated impact of change in the October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 4 Asad et al. IPN Architecture distancing measures. Contact-tracing and passenger distancing will be highly necessary and will act as one of the crucial types of mediation to avoid further spread. In such circumstances, an intelligent Contact-Tracing technique potentially hosted at the edge of the network integrated within the transport infrastructure will become an essential need for transport operators. able to provide robust remote connectivity from the location of the fault within underground ecology to where the experts are. In this case, a central control center with highly skilled workers would be available to support multiple remote operations simultaneously without physically going to the fault location. In other words, within the NGRS, the staff will be empowered to complete tasks by remotely connecting with skilled specialists while supporting a “paper-free” maintenance log, moving to fully digital infrastructure for secure instructions. The use of Intelligent Extended Reality (IXR) will be able to enhance a mixture of Augmented Reality (AR), Virtual Reality (VR), and Tactile Services (TS) for enhanced remote operations. Use cases Use cases Intelligent Train-to-Train (IT2T) Communication Contact-Tracing System (CTS) Intelligent Drones System (IDS) Transport Hub Crowd Management (THCM) Disaster Scenario Response (DSR) Intelligent Monitoring Services (IMS) Intelligent Wireless Audio (IWA) Emergency Services (ES) Intelligent Depots 4.0 (ID 4.0) Train signaling, train-t-train communication, driverless trains, etc. Passengers/staff who tested positive against the COVID-19 would be tracked (on a high level) by using 5G network and 5G enabled devices Overcrowding stations monitoring 5G drones Positioning, resource allocation, and management RTLS, IPS, cMTC, ICCTV, and 5G cellular and 5G private network 5G Intelligent Extended Reality (IXR) portable devices with HD cameras Remote monitoring, detection, and providing suggestions for onsite incidents team equipped with visualization cameras to see faults and hidden services Sensor-based cMTC, smartphone, gadget monitoring application, and LPWAN technology such as NB-IoT. Laser-based 5G technology to monitor toxic gases 5G handsets with URLLC connectivity Sensor-based cMTC, smartphone, gadget monitoring application, and LPWAN technology such as NB-IoT. Laser-based 5G technology to monitor toxic gases 5G handsets with URLLC connectivity Underground tunnels gas metering, water metering, pressurized fuel detection, smoke alarms detection, fire alarms monitoring, and other alarms and event detectors 5G handsets with MCPTT Sensor-based cMTC, gadget monitoring application, CAI, and LPWAN technology such as NB-IoT Sensor-based cMTC, gadget monitoring application, CAI, and LPWAN technology such as NB-IoT Sensor-based cMTC, gadget monitoring application, CAI, and LPWAN technology such as NB-IoT Diverse bandwidth and wireless automation optimizing logistics. Furthermore, train parking, monitoring trains movement by Train Access Controller (TAC) through Radio-frequency identification (RFID) sensors, identification of electrified and nonelectrified tracks by using AR devices, self-automated track components between the running rails, use of Conversational AI (CAI) between moving trains and TAC when accessing live tracks, intelligence in infringing over the platform edge, automation in stopping a train in an emergency through the use of specialized low-latency devices, and real-time visibility of occupied and unoccupied train depot tracks are the hallmarks of Industry 4.0 that will feed into the ID 4.0. 4.2 IPN Technologies for NGRS 4.2 IPN Technologies for NGRS ITS-focused AI is a key enabler that can offer a solution to the complex train network in the form of an IPN in proximity to an operational environment. The demand for ubiquity, privacy, low latency, low-cost computing, and resource management requires an integration of heterogeneous systems and edge compatible applications into one smart network, presented as an IPN. With such a use case, isolated and disparate edge services will provide sufficient security by having multiple Virtual Private Networks (VPN) to safeguard against malicious attacks. The motivation of IPN comes from a combination of private mobile networks and capabilities of B5G networks in lieu of ITS and more specifically future railway systems which are summarized in 4.1.5 Disaster Scenario Response Edge intelligence within B5G networks will introduce an assortment of services that will operate on high throughput and low latency to address critical events that require a timely response. In the same way, the underground train network is a highly deserving environment to adopt such technology into portable devices leveraging the benefits of Massive Machine- Type Communications (mMTC) (Zhang, 2020). New portable lightweight devices equipped with intelligent computing applications and storage enabled via the IPN cloud would be October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 5 IPN Architecture Asad et al. TABLE 1 | Summary of NGRS use cases and applications. Use cases Applications Enabling technologies Research challenges Intelligent Train-to-Train (IT2T) Communication Train signaling, train-t-train communication, driverless trains, etc. 5G NR, mmWave, and Ultrareliable Low-Latency Communication (URLLC) High bandwidth and low latency Contact-Tracing System (CTS) Passengers/staff who tested positive against the COVID-19 would be tracked (on a high level) by using 5G network and 5G enabled devices 5G UWB (ultrawideband) monitoring short-range devices Data security and privacy and low- power communication Intelligent Drones System (IDS) Overcrowding stations monitoring 5G drones Real-time low-latency communication and high-resolution image processing Transport Hub Crowd Management (THCM) Positioning, resource allocation, and management RTLS, IPS, cMTC, ICCTV, and 5G cellular and 5G private network Diverse bandwidth and low latency Disaster Scenario Response (DSR) Remote monitoring, detection, and providing suggestions for onsite incidents team equipped with visualization cameras to see faults and hidden services 5G Intelligent Extended Reality (IXR) portable devices with HD cameras High bandwidth, latency, and reliability Intelligent Monitoring Services (IMS) Underground tunnels gas metering, water metering, pressurized fuel detection, smoke alarms detection, fire alarms monitoring, and other alarms and event detectors Sensor-based cMTC, smartphone, gadget monitoring application, and LPWAN technology such as NB-IoT. Laser-based 5G technology to monitor toxic gases Longer battery life and low latency for critical alarms Intelligent Wireless Audio (IWA) Emergency departments such as police, detectives, special forces, and Accident and Emergency (A&E) 5G handsets with URLLC connectivity Low latency and reliability Emergency Services (ES) Ambulance, firefighters, first-aid staff, mission- critical fault detection teams, paramedic services, etc. 5G handsets with MCPTT Diverse bandwidth, low latency, and reliability Intelligent Depots 4.0 (ID 4.0) Depot services Sensor-based cMTC, gadget monitoring application, CAI, and LPWAN technology such as NB-IoT Diverse bandwidth and wireless automation TABLE 1 | Summary of NGRS use cases and applications. 4.1.8 Emergency Services One of the great achievements the Third-Generation Partnership Project (3GPP) has obtained is standardizing the Mission-Critical Push-To-Talk (MCPTT) (Sanchoyerto et al., 2019) service as a use case. This ES use case can be used to act against anything abnormal such as fare evasion, suicide attempts, and incidents. MCPTT empowered with Enhanced Mobile Broadband (eMBB) can be an essential focus in ITS when considering the underground train environment. This will require an application that employs high bandwidth internet access, video streaming, and virtual reality with latency-sensitive devices. At the same time, utilizing network and radio slicing techniques, several high bandwidth sessions for separate emergency departments such as ambulance, firefighters, first-aid staff, mission-critical fault detection teams, or other paramedic services can be established within the IPN. 4.1.9 Intelligent Depots 4.0 (ID 4.0) AI-based industrial automation powered by IPN is making a tremendous revolution toward B5G technologies. In the field of supply chain, this transposes toward the edge intelligence of logistics 4.0 that advances beyond process automation by using intelligent collaboration and interconnection between connected systems providing transformative applications (Wang et al., 2017). ID in the train network is an important use case that will push communications to another level where train depots will use automated coordination between objects and autonomous self- October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 6 Asad et al. IPN Architecture offerings from 410 to 7,125 MHz. The latter one introduces frequency bands from 24.25 to 52.6 GHz with offerings of even higher bandwidth than the first ranges. An important aim in the development of the 5G NR within NGRS has been to ensure dynamic bandwidth communications depending on the requirement. In the presented IPN, NR is introduced at the interface of trains communication to the centralized smart architecture ensuring a high degree of forward compatibility in the radio interface design. In the context of NGRS, either IT2T communication or Train Signaling radio interface allows for substantial future evolution in terms of introducing new technology and enabling new services. For IT2T communication or Train Signaling radio interface, NR would support a massive number (e.g., ≥64) of steerable antenna elements for both transmission and reception. At higher frequency bands, the large number of antenna elements are primarily used for beamforming to achieve coverage, while at lower frequency bands they enable full- dimensional multiple-input multiple-output (MIMO) and interference avoidance by spatial filtering (Parkvall et al., 2017). Table 1 along with their applications, enabling technologies, and challenges. The idea of running several applications within a single network environment consolidates with IPN’s scalability, security, flexibility, and coverage expansion. This will evolve the existing private mobile networks toward smart transport digital facilities with the use of Machine Learning (ML) (Asad et al., 2020a; Zhang, 2020; Klaine et al., 2017; Liu et al., 2018; Ren et al., 2020; Haider et al., 2019; Fioranelli et al., 2019), AI, and cognitive analytics that is fully primed for existence at the edge establishing ground toward B5G networks. IPN main features are as follows: • Scalability: massive connectivity and capacity in a cost- effective manner for business expansion and growth. 1https://www.nokia.com/networks/portfolio/mobile-transport/. 4.2.3 Mission-Critical Push to Talk 4.2.3 Mission-Critical Push to Talk MCPTT originates from old-fashioned Push-to-talk Over Cellular (POC) services that integrate with mobile phone networks with a world of opportunities. The service supports pairs and group users for communication in the specific time slotting procedure. Each user will be able to converse both with audio (using low bandwidth) and with video (using higher bandwidth). MCPTT system allows participants to request a token to speak. A priority mechanism to arbitrate the participation of the users is provided in the communication of critical services (Sanchoyerto et al., 2019). Traditionally, the choice of which user requests to depend on their token time frames and numbers when using the service simultaneously. IPN presents intelligent MCPTT on the edge devices where simultaneous users on their critical missions would be addressed without delays. IPN-MCPTT connectivity works on a principle of separated Control and Data Architecture (CDSA) where mission-critical intelligence is handled on the edge without consulting IPN in every communication leaving IPN to monitor activities that each MCPTT device performs. Following are the components that we envisage will enable the IPN framework. 4.2.4 Intelligent CCTVs g Intelligent CCTVs integrated with edge intelligence will guard assets and processes in real-time by employing AI and ML algorithms for quick detection (Akbar and Azhar, 2018). The high-quality video stream from the CCTV multiplexed with edge intelligence will even allow the prediction of hazardous or emergency situations. The system can also grant access, monitor, and log people’s entry/exit into a building allowing a much-centralized control of railway systems. ICCTVs hold the local cache to record real-time events on the edge without the need of IPN core which makes the system intelligent, locally 4.2.1 Artificially Intelligent Anyhaul y g y Due to the overwhelming data growth experienced by mobile network operators, they are compelled to further densify, centralize, and/or cloudify their networks. This has led to the provisioning of centralized streams and edge intelligent IoT/M2M devices used in the NGRS which are the key drivers to provide quick and robust responses. Through the use of AIA, transport network would be cost-effectively driven by reaching edge intelligent devices in no time from the centralized core1. Centralized IPN would only be consulted for control signaling when required, leaving edge devices to perform faster responses on their own in their locally present intelligent compute storage. With this approach, the IPN architecture presented would be exploited for delivering high capacity, ultralow latency, and highly accurate synchronization distribution needed to boost the overall performance of IPN while taking advantage of virtualized edge intelligent resources (Hilt, 2019). 4.1.9 Intelligent Depots 4.0 (ID 4.0) • Security: block unauthorized access, maintain end-to-end data confidentiality, and provide encryption with a complete on-demand solution. • Flexibility: adaptive tethering and real-time adjustment with the environmental processes. • Reliability: stable continuous connectivity and safe service continuity for business-critical operations. • Evolution to B5G: future proof intelligence to support existing deployed solutions with investment opportunities to support additional use cases with stricter requirements. 2Ultra-Wideband (UWB) Ofcom document can be found at: https://www.ofcom. org.uk/__data/assets/pdf_file/0015/25152/uwb.pdf. 4.2.7 Critical Machine-Type Communication The cMTC is the interaction of machines for the actuation of data collection for necessary and time-critical actions (Zhang, 2020). The network of machines such as sensors, RFID tags, and collection of smart antennas in massive numbers with no intervention of humans would be secure, robust, reliable, and consistently scalable to cope with high capacity and low- latency demands. The cMTC can also function by using multiple varying frequency bands where NGRS have been postulated as a potential solution offering a single IPN platform for the “ecosystem” to several connected IoT devices. cMTC provides coverage in challenging environments such as underground train tunnels where coverage penetration is difficult. It also employs faster responses through the use of faster data throughput, without limitations on the usage. The presented architecture 4.2.2 New Radio 4.2.2 New Radio The 3GPP standard for the 5G radio access technologies (RAT) known as 5G New Radio (NR) supports a substantial expansion unlike traditional technologies with two new ranges of frequency bands, FR1 and FR2 (Pilz et al., 2018). The first range includes sub-6 GHz in addition to bandwidth October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 7 Asad et al. IPN Architecture has cMTC driven connected devices employing hybrid configuration with a control center to provide centralized control through IPN with edge intelligence for the connected devices. administrative, and less overhead intensive with only required frames per second to be sent to the centralized IPN in every recording. 4.2.8 Conversational Artificial Intelligence UAVs, more commonly known as drones, have a controller, usually ground-based with a strong communication link between the two. UAVs flight have various degrees of autonomy operation (Solomitckii et al., 2018; Sambo et al., 2019) either under remote control by a human operator or autonomously by onboard computers. Drones can extend network coverage and capacity to reach remote or highly crowded areas. Either individually or as a swarm controlled by IDS, they can also function as robotic equipment that offers detection, traction, interference mitigation, and/or optimization of systems avoiding costly human interventions. UAVs can be utilized to extend network coverage in highly crowded areas such as train terminus. In addition, UAVs can also be utilized in emergency coverage in situations of local infrastructure malfunction or disaster. Intelligent UAVs have local automation to take important decisions at the edge with control functions to be monitored from IPN. This network-centric intelligence provides a bridge that integrates UAVs into the IPN infrastructure as access points. 4.2.8 Conversational Artificial Intelligence CAI is an AI variant that enables people to communicate with machines such as applications and devices with the functionality of human-like natural language via voice, text, touch, or gesture. It helps in achieving tasks with proactiveness and personalized engagement (Dashtipour, 2016). CAI integrated with the rest of the network entities will feature the IPN as a living entity that interacts with all its users employing several social AI features hosted at the network edge. At first, when a user tries to say something, Natural Language Understanding (NLU) deciphers its meaning in the user’s understandable words regardless of how words have been stated. Even among user’s grammatical mistakes, the CAI is able to understand the user’s intent with NLU. It even works with user’s idiosyncrasies and shortcuts and remembers the context from one statement to another, comprehending what is being said throughout the conversation. At the second stage, CAI determines the right response based on its intelligence using ML and by understanding the user’s intent. Finally, using natural language generation, the CAI generates a user-friendly response for easy comprehension. 4.2.9 Ultrawideband 4.2.9 Ultrawideband UWB is a radio technology that uses a wide frequency spectrum to locate and track people in real-time when short-range links are paramount. UWB has an unlicensed frequency range from 3.1 to 10.6 GHz. Private networks utilizing these bands within their premises can reduce the risk of interference among their applications or technologies. UWB is a potential alternative to other local area wireless technologies, such as WiFi, Bluetooth, and other WLAN technologies. The principal advantage of UWB which distinguishes its features from others is its offering of faster data transfer rates (100 Mbits/s up to 1 Gbits/s) in comparison. Extended battery and durability give UWB an edge over prominent wireless technologies deployed to date2. 4.2.6 Intelligent Network Management The automation technology designed for the management of automated planning, automated configuration, automated healing, and automated optimization is called Intelligent Network Management (INM). The functionality and behavior of the INM are standardized by the 3GPP and NGMNs (Next-Generation Mobile Networks) consortium while undergoing continuous improvements. AI will play an instrumental role in making the private networks sustainable as it requires almost none physical or remote interventions. INM supports a real-time reconfigurable managed ecosystem with virtualization for fast and ultrafast services between centralized IPN and devices on the edge through AIA. 5.2 Edge Caching technologies provided at the edge under the light of network, application, and user-centric intelligence as shown in Figure 2. The presented architecture would also exploit service continuity as a PMN where an edge-computing solution will handle passenger’s mobility. IPN link is used for backup and restore operation and for major reporting purposes. Intelligent actions are done at the edge with locally captured data. g g Under edge intelligence, edge caching is a simplistic method to effectively improve the performance of the IPN where it administers the incoming distributed data coming toward the edge devices from end-users and its surrounding environment (Zhang et al., 2018; Deng et al., 2020). This property exploits edge caching with virtually no setup overhead apart from sending control signals in the form of reporting to the IPN. Along with this property, the data generated by edge devices itself fall under edge caching. In our presented IPN, the data generated in IT2T communication, CTS, IDS, gadgets used in DSR, IMS, IWA, and MCPTT in ES is stored locally without the need to send every instance data back to centralized IPN. Also, the sensors-based cMTC such as RFID, NB-IoT, CAI, and mobile (accelerometer, gyroscope, and magnetometer) gather environment data which is processed and stored at reasonable places and is used by AI algorithms to provide services to users. 5.1 IPN Connectivity y The presented IPN is envisaged to have numerous edge devices in its network with profound connectivity links. It integrates edge devices and edge compatible applications into one smart network, thus forming a heterogeneous system environment (Niyato, 2020). IPN controls the functions of all connected links and devices by preprocessing through software-defined tools. The training of edge devices is a lightweight task that mainly relies on the edge servers performed by IPN in the initial setup. Once edge devices are trained, they take their intelligent decisions locally without IPN consultation on every step which reduces the overhead, time, and cost. IPN also allows direct connectivity to local cloud resource and offload major tasks to an on-premise server. The on-premise server then takes care of synchronization with the centralized cloud for reporting purposes. 5 IPN-DRIVEN EDGE INTELLIGENT NGRS ARCHITECTURE In the following subsections, we discuss IPN-driven edge intelligence architecture comprised of centralized IPN connectivity and local edge decisions for NGRS (Niyato, 2020; Deng et al., 2020; Zhou et al., 2019; Dey et al., 2019; Dai et al., 2020). This integrates our use cases with the October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 8 Asad et al. IPN Architecture FIGURE 2 | IPN-driven edge intelligent NGRS architecture. FIGURE 2 | IPN-driven edge intelligent NGRS architecture. 3Narrowband-IoT Vodafone document can be found at: https://www.vodafone. com/business/iot/managed-iot-connectivity/nb-iot. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author. • Low-latency communication: an important parameter for use cases like IT2T communication, Intelligent Drone 5.4 Edge Inference • Low-power communication: for use cases like Contact-Tracing Systems and Intelligent Monitoring Services (approx. 15 years of battery life3), it will be crucial to establish low-power communications for sustainable NGRS solutions. Edge Inference is a process that holds responsibility for evaluating performance once the ML model is trained by datasets based on learnt computational outputs on edge devices (Niyato, 2020). The ML model is constructed and trained by powerful Central Processing Units (CPUs) and Graphical Processing Units (GPUs). These units support the fast processing of numerous functions such as face image recognition and remote assistance with data-hungry devices. Once deployed on the edge devices, they maintain their performance and resilience when intelligently designed to accelerate edge inference. • Reliability: to ensure communication reliability in scenarios like Disaster Scenario Response and Emergency Services, there would be an important future research direction in the context of NGRS. Furthermore, as we move toward a net zero carbon footprint, research on energy-efficient Heterogeneous Networks (HetNets) will also be required to address the energy consumption of NGRS IPN. Other challenges that would need to be addressed in order to make IPN open up for general public use will be to enable neutral hosting, security measures through blockchain, and dynamic spectrum sharing among mobile network operators for adaptive capacity protocols. 5.5 Edge Offloading Under edge intelligence, edge offloading is another critical feature that actively offloads its tasks to other edge devices in a network (Dey et al., 2019). Similarly, the presented IPN uses edge offloading characteristics in its edge devices for offloading necessary tasks like edge training, edge caching, or edge inference in a distributed computing paradigm. This property exploits an intelligent ecosystem created by edge devices. Edge offloading is of utmost importance due to its resilient-like strategy by independent edge devices compared to edge caching, edge training, and edge inference. Effective implementation of IPN gives edge intelligence a structured way to available resources in the edge environment. 5.3 Edge Training Traditionally the training of AI models works on a principle of centralized deployment architecture which carries heavy traffic in both communication directions (Kukreja et al., 2019; Zhou et al., 2019), eventually creating overhead. In our presented IPN, ML- driven federated learning technique also known as collaborative learning is used to train the AI model with an aim to port onto edge devices. The AI model runs on a powerful central server using its October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 9 Asad et al. IPN Architecture Systems, and Intelligent Wireless Audio. The role of URLLC and mmWave will be critical toward the realization of low-latency communications. compatible edge software tools and runtime environment. With this approach, controlling, retraining, and deployment of models to edge devices using IPN cloud connectivity proves to be the most reliable and effective method for edge training. Once all the edge devices are intelligently trained, the communication link to the IPN would only be used for backup and restore operation or for reporting purposes where edge devices take their own decisions locally. • Communication with Diverse Bandwidth Requirements: to meet the challenges of variable data rate services like in the case of Transport Hub Crowd Management, Emergency Services, and Intelligent Depots, it will be important to develop systems that are capable of providing diverse data rates while meeting task completion deadlines. AUTHOR CONTRIBUTIONS The NGRS will play an active role with edge intelligence integrated within the IPN domain. NGRS should be constructed using a variety of technologies and network configurations discussed in this article and other related researches and will also depend on geographic areas, deployment models, and application scenarios in the underground train environments. We presented several use cases, applications, required technologies, capacity needs, and measures in this article that we believe will provide opportunities for transport businesses to put B5G mobile networks on their private trail. Most of the modern researches have addressed or are currently investigating the traditional challenges of ITS. However, there seems to be a gap between 5G/B5G use cases and their applications for ITS. It is believed that these research gaps will drive future research to unlock the true potentials of ITS through edge intelligence. Below we list few critical parameters that will be important research themes in the context of NGRS realization through edge intelligent IPN. 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Edge Intelligence: Paving the Last Mile of Artificial Intelligence with Edge Computing. Proc. IEEE 107, 1738–1762. doi:10.1109/JPROC.2019.2918951 Frontiers in Communications and Networks | www.frontiersin.org October 2021 | Volume 2 | Article 769299 REFERENCES IPN Architecture Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Zhou, Z., Chen, X., Li, E., Zeng, L., Luo, K., and Zhang, J. (2019). Edge Intelligence: Paving the Last Mile of Artificial Intelligence with Edge Computing. Proc. IEEE 107, 1738–1762. doi:10.1109/JPROC.2019.2918951 Zoha, A., Abbasi, Q. H., and Imran, M. A. (2020). Anomaly Detection and Self- Healing in Industrial Wireless Networks. John Wiley & Sons, 109–139. doi:10.1002/9781119552635.ch6 Copyright © 2021 Asad, Tahir, Rais, Ansari, Abubakar, Hussain, Abbasi and Imran. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Conflict of Interest: The author SMA is employed by the company Transport for London (TfL) as a Senior Network and Telecoms Architect. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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0–3 éves korú csecsemők és kisdedek táplálkozási szokásainak felmérése, különös tekintettel a makro- és mikronutriens-bevitelre
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Erdélyi-Sipos Alíz1 ■ Badacsonyiné dr. Kassai Krisztina1 ■ Kubányi Jolán1 Szűcs Zsuzsanna1 ■ Biró Lajos dr.2 ■ Raposa L. Bence dr.3 p Következtetés: Bár a makro- és mikronutrienseknek a WHO/FAO/UNU által 2007-ben megadott beviteli értékein alapuló, 2013-as EFSA-ajánlás meghatározza az ajánlott beviteli mennyiségeket, a mintában felmért eredmények annak teljes körű megbízhatóságát nem támasztották alá. Orv Hetil 2019; 160(50): 1990–1998 Kulcsszavak: csecsemő, táplálkozás, gyermek, fehérjebevitel, testtömegindex Erdélyi-Sipos Alíz1 ■ Badacsonyiné dr. Kassai Krisztina1 ■ Kubányi Jolán1 Szűcs Zsuzsanna1 ■ Biró Lajos dr.2 ■ Raposa L. Bence dr.3 1Magyar Dietetikusok Országos Szövetsége, Budapest 2NutriComp Bt., Budapest 3Pécsi Tudományegyetem, Egészségtudományi Kar, Ápolástudományi, Alapozó Egészségtudományi és Védőnői Intézet, Pécs Bevezetés: Az elmúlt időszakban megjelent kutatási eredmények alátámasztják azt a feltételezést, hogy a felnőttkori krónikus betegségek kialakulását jelentősen befolyásolja a méhen belüli tápanyag-ellátottság és a kisdedkorban folyta­ tott táplálás és táplálkozás. p p Célkitűzés: A vizsgálat célja a hazai 0–3 éves csecsemők, kisdedek táplálkozási szokásainak felmérése, illetve a csecse­ mő- és kisdedkorú gyermekek táplálkozását a leginkább jellemző problémák feltárása, a táplálási/táplálkozási ajánlá­ sok módosításához, korszerűsítéséhez szükséges adatok megismerése, szolgáltatása volt. Módszer: A vizsgálat a Magyar Dietetikusok Országos Szövetségének szakmai koordinálásával, iparági kutatás kereté­ ben valósult meg 2015. június és augusztus között, Budapest, Debrecen, Győr, Szeged és Pécs városainak 0–3 éves populációja körében. A felmérés antropometriai mérésekkel és validált háromnapos táplálkozási naplóval történt. Módszer: A vizsgálat a Magyar Dietetikusok Országos Szövetségének szakmai koordinálásával, iparági kutatás kereté­ ben valósult meg 2015. június és augusztus között, Budapest, Debrecen, Győr, Szeged és Pécs városainak 0–3 éves populációja körében. A felmérés antropometriai mérésekkel és validált háromnapos táplálkozási naplóval történt. Eredmények: A 4–12 hónapos (n = 220) csecsemők 18,6%-a 10 percentilis alatti értékkel rendelkezett, 10% 85–97 ili kö é í 3% h l d 97 ili A 12 24 hó ( 227) k k 15% 10 Módszer: A vizsgálat a Magyar Dietetikusok Országos Szövetségének szakmai koordinálásával, iparági kutatás kereté­ ben valósult meg 2015. június és augusztus között, Budapest, Debrecen, Győr, Szeged és Pécs városainak 0–3 éves populációja körében. A felmérés antropometriai mérésekkel és validált háromnapos táplálkozási naplóval történt. Eredmények: A 4–12 hónapos (n = 220) csecsemők 18,6%-a 10 percentilis alatti értékkel rendelkezett, 10% 85–97 percentilis közé esett, míg 3% meghaladta a 97 percentilist. A 12–24 hónapos (n = 227) gyermekek 15%-a 10 percen­ tilis alatti testtömegindexszel (BMI) rendelkezett (sovány), 14% BMI-je 85–97 percentilis közé esett (túlsúlyos), és 2,6% a 97 percentilist meghaladó BMI-vel rendelkezett (elhízott). A 25–36 hónapos gyermekek (n = 184) 70%-ának normál-BMI-je volt, 4%-uk túlsúlyos, 2%-uk elhízott, 24%-uk sovány volt. Míg a hazai referenciaérték alapján a fel­ mérésben részt vevő 4–12 hónapos gyerekek 10,9%-a, az 1–2 évesek 20%-a, a 2–3 évesek 47%-a a magas fehérjebe­ viteli tartományba esett, addig a 2013-ban kiadott EFSA- (Európai Élelmiszerbiztonsági Hatóság) ajánlás referencia­ értékeihez viszonyítva viszont a vizsgált gyerekek 100%-a a magas fehérjebeviteli csoportba tartozik minden korcsoportban. EREDETI KÖZLEMÉNY EREDETI KÖZLEMÉNY 0–3 éves korú csecsemők és kisdedek táplálkozási szokásainak felmérése, különös tekintettel a makro- és mikronutriens-bevitelre Erdélyi-Sipos Alíz1 ■ Badacsonyiné dr. Kassai Krisztina1 ■ Kubányi Jolán1 Szűcs Zsuzsanna1 ■ Biró Lajos dr.2 ■ Raposa L. Bence dr.3 Rövidítések kezményének tekinthetők. Az említett jelenség ugyanak­ kor bármely életkorban számos lehetőséget ad a beavat­ kozásra [4, 5]. BMI = (body mass index) testtömegindex; EFSA = (European Food Safety Authority) Európai Élelmiszerbiztonsági Hatóság; FAO = (Food and Agriculture Organization of the United ­Nations) Élelmezésügyi és Mezőgazdasági Világszervezet; ICP = (infancy childhood puberty) csecsemő-, gyermek-, ser­ dülőkori modell; IGF1 = (insulin-like growth factor 1) inzulin­ szerű növekedési faktor-1; RÉ = referenciaérték; TGI = (Target Group Index) Célcsoport Index; UNU = (United Nations University) Egyesült Nemzetek Egyeteme; UVB = (ultraviolet B) ibolyántúli B, közepes hullámhosszú sugárzás; WHO = (World Health Organization) Egészségügyi Világszervezet [ , ] A gyermek születési testtömege, illetve hossza megha­ tározó nemcsak a pillanatnyi morbiditás, mortalitás szempontjából, de hosszú távon is befolyásolja az egész­ ségügyi kilátásokat. Példaként említhető az intrauterin növekedés elmaradása (kis súlyú magzat) és a szívkoszo­ rúér-betegségek, a stroke, a diabetes és a magas vérnyo­ más magasabb rizikója közötti összefüggés. Ugyanígy a magas születési testtömeg (ún. macrosomia), valamint a diabetes, illetve a szív- és érrendszeri betegségek, az emlő- és egyéb daganatok emelkedett kockázata közötti korreláció is megfigyelhető [6–11]. A felnőttkori króni­ kus betegségek kialakulását igen jelentősen befolyásolja az élet fogantatástól számított első 1000 nap időszaká­ ban a táplálás módja, annak mennyisége és minősége [12]. Ennek ismeretében a táplálás kiemelt jelentőséggel kezelendő már a magzati élet kezdetétől. A tudományos figyelem korábban a méhen belüli alultápláltság és a csökkent születési testtömeg káros hatásaira irányult. Az elhízás járványszerű terjedésével azonban napjainkban egyre inkább a várandósság és a szoptatás alatt bekövet­ kező anyai túltáplálkozásnak az utód hosszú távú egész­ ségi állapotát érintő hatására tevődik át a hangsúly [13]. Az étrend minősége minden életszakaszban meghatáro­ zó eleme az egyén egészségi állapotának. Szerepe a kró­ nikus, nem fertőző betegségek kialakulásában ma már kellően bizonyított, éppen ezért a táplálkozás területén megvalósított intervenció nélkülözhetetlen eleme a pre­ venciónak. A krónikus betegségek előfordulása napjaink­ ban világszerte egyre szélesebb tömegeket érint. Ma már azt is tudjuk, hogy ezen kórállapotok többsége ugyan­ azon kockázati tényezőkre, köztük a helytelen táplálko­ zásra vezethető vissza. Felmerül ugyanakkor, ha a meg­ betegedések önmagukban nem fertőzőek is, mégis annak tekinthetők a kockázati tényezők szintjén, hiszen az élet­ módszokások öröklődnek egyik generációról, populáció­ ról a másikra, illetve a globalizációnak köszönhetően sokszor az országhatárokat is átlépik [1–3]. A kora csecsemőkori élet környezetének jelentősége is egyre jobban felértékelődik, hiszen a korai gyors súlygya­ rapodáshoz későbbi elhízás társulhat, a méhen belüli gyarapodástól függetlenül. Nutrition assessment of 0–3-year-old infants and toddlers with particular focus on macro- and micronutrient intake Introduction: Recent research findings support the assumption that the development of chronic diseases in adults is greatly influenced by the supply of nutrients in the uterus and the nutrition, nourishment of the early, toddler ages. Aim: The aim of the present study was to evaluate the nutritional habits of infants and toddlers aged 0–3 in Hungary, and to identify the most typical problems of their nutrition, to get to know and provide the necessary data for the modification and modernization of feeding/nutrition recommendations for infants and young children in Hungary. Method: The study was carried out with the professional coordination of the Hungarian Dietetic Association ­(MDOSZ) in the framework of industry research between June and August 2015, in the 0–3-year-old population, in 2019 ■ 160. évfolyam, 50. szám ■ 1990–1998. 1990 DOI: 10.1556/650.2019.31585 ■ © Szerző(k) EREDETI KÖZLEMÉNY the cities Budapest, Debrecen, Győr, Szeged and Pécs. The survey was conducted with anthropometric measure­ ments and validated by three-day dietary log templates. cities Budapest, Debrecen, Győr, Szeged and Pécs. The survey was conducted with anthropometric measure ts and validated by three-day dietary log templates. the cities Budapest, Debrecen, Győr, Szeged and Pécs. The survey was conducted with anthropometric measure­ ments and validated by three-day dietary log templates. Results: 18.6% of infants aged 4 to 12 months (n = 220) had values below 10th percentile, 10% were between 85– 97th percentiles and 3% were above 97th percentile. 15% of children aged 12–24 months (n = 227) had a body mass index (BMI) below 10th percentile (underweight), 14% were between 85–97th percentile (overweight) and 2.6% had BMI over the 97th percentile (obese). 70% of 25–36-month-old children (n = 184) had normal BMI, 4% were overweight, 2% obese, 24% underweight. Based on the Hungarian reference value, 10.9% of the 4–12-month-old children, 20% of the 1–2-year-olds, 47% of the 2–3-year-olds were in high protein intake group. However, compared to the 2013’s reference values of the EFSA (European Food Safety Authority) recommendation, 100% of the chil­ dren belong to the high protein intake group in all age groups. y y y g p Results: 18.6% of infants aged 4 to 12 months (n = 220) had values below 10th percentile, 10% were between 85– 97th percentiles and 3% were above 97th percentile. Nutrition assessment of 0–3-year-old infants and toddlers with particular focus on macro- and micronutrient intake 15% of children aged 12–24 months (n = 227) had a body mass index (BMI) below 10th percentile (underweight), 14% were between 85–97th percentile (overweight) and 2.6% had BMI over the 97th percentile (obese). 70% of 25–36-month-old children (n = 184) had normal BMI, 4% were overweight, 2% obese, 24% underweight. Based on the Hungarian reference value, 10.9% of the 4–12-month-old children, 20% of the 1–2-year-olds, 47% of the 2–3-year-olds were in high protein intake group. However, compared to the 2013’s reference values of the EFSA (European Food Safety Authority) recommendation, 100% of the chil­ dren belong to the high protein intake group in all age groups. Conclusion: Although the EFSA recommendation – based on the WHO/FAO/UNU macro- and micronutrient intake values in 2007 – defines the recommended intake quantities, the results in the sample did not support its overall reliability. Keywords: infant, nutrition, child, protein intake, body mass index Erdélyi-Sipos A, Badacsonyiné Kassai K, Kubányi J, Szűcs Zs, Biró L, Raposa LB. [Nutrition assessment of 0–3-year- old infants and toddlers with particular focus on macro- and micronutrient intake]. Orv Hetil. 2019; 160(50): 1990–1998. (Beérkezett: 2019. július 18.; elfogadva: 2019. augusztus 20.) Rövidítések Meglátása szerint a gyermek növekedése három jól elkülöníthető szakaszban zajlik, melyek folyamán három szabályozó tényező egymást átfedő hatásokat fejt ki. Gyermekkorban a növekedést a növekedési hormon sza­ bályozza, melynek hatását az inzulinszerű növekedési faktor, az IGF1 termelése közvetíti. A serdülőkori növe­ kedési megugrást a nemi hormonok hozzátevődő ana­ bolikus hatása okozza. A csecsemőkori gyors növekedést ezzel ellentétben azoknak a tápanyagoknak a bevitele serkenti, amelyek a növekedési faktor, az inzulin és az IGF1 termelését stimulálják. Ez lehet a magyarázata an­ nak, hogy az anyatejjel táplált csecsemőkhöz viszonyítva a tápszerrel tápláltaknál előálló magasabb fehérjebevitel megemelheti a vérplazmában és a szövetekben az inzu­ lin- és IGF1-szintet, ilyen módon fokozva a korai súly­ gyarapodást és később az elhízást [16]. A csecsemők fe­ hérjebevitelével foglalkozó kutatás és klinikai érdeklődés középpontjában korábban a hiány kockázatai, ezek meg­ előzése és kezelése állt. Mivel a szükséglettől elmaradó fehérjebevitel egyértelműen károsan érinti a növekedést, fejlődést, csecsemőkorban hagyományosan bőséges fe­ hérjebevitelt szorgalmaznak [17]. A koraifehérje-hipoté­ zis azonban, vagyis annak a feltevésnek a tudományos megfogalmazása, miszerint az első életévben nyújtott feleslegesen nagy fehérjebevitelhez a csecsemő gyors súlygyarapodása társul, ami a későbbi életkorokban az elhízás és a vele rokon állapotok kockázatának növekedé­ sével is jár, ezt az irányvonalat kérdőjelezi meg [18, 19]. Ezen folyamat legjobb megelőzési eszköze az anyatejes táplálás, hisz tudjuk, hogy az anyatejjel táplált csecsemők körében a túlsúly vagy elhízás előfordulása alacsonyabb, mint tápszeres társaiknál. Amennyiben nem lehetséges a szoptatás, vagy nem áll rendelkezésre elegendő anyatej, akkor viszont kiemelt jelentőségű, hogy az anyatej fehér­ je-összetételéhez legközelebb álló tápszerrel történjen a táplálás [20]. g Kutatásunkban feldolgozásra kerültek az egészséges, időre született, 0–6 hónapos, nem kizárólag anyatejjel táplált (anyatej, anyatej és/vagy tápszer) csecsemőknek (n = 97), a 4–12 hónapos, a hozzátáplálást megkezdett csecsemőknek (n = 220) és a 12–36 hónapos kisdedek­ nek (n = 411) a táplálkozására, tápláltságára vonatkozó adatok egyaránt. A kérdőívek (n = 728) 1,2%-a kizárásra került azonosítási problémák, valamint a szélsőségesen csekély vagy nagy energiafelvétel miatt, biztosítva ezzel a szélsőértékek kizárását. A jelen cikkben a 0–6 hónapos korosztályra vonatkozó mérési eredményeket nem mu­ tatjuk be, a kizárólagos anyatejes táplálás preferálása ­miatt. A felmérés ,,háromnapos táplálkozási napló” és kérdő­ ív segítségével történt, a nemzetközi ajánlásnak megfele­ lően, majd a visszakérdezett és pontosított adatokat die­ tetikus szakemberek rögzítették a ,,NutriComp Étrend” szoftverben, amellyel energiatartalom- és tápanyagszá­ mítás történt. Módszer A nagyvárosi reprezentativitás biztosítása érdekében több területről (Budapest, Debrecen, Szeged, Pécs, Győr) kerültek bevonásra résztvevők a mintába. A min­ tavétel alapját egyrészről a TNS Hofmann Kft. által biz­ tosított Target Group Index (TGI) nagymintás kutatása, másrészről a célzott adatfelvétel adta. A reprezentativitás biztosítása érdekében a TGI kutatási mintájából megha­ tározásra került a célcsoport nagysága, valamint a minta demográfiai háttere, összetétele (korcsoport, lakóhely, iskolai végzettség) egyaránt. A vizsgálatban részt vevő édesanyák megkeresése kérdezőbiztos által történt, vé­ letlenszerű, random mintaválasztással és ,,snow-balling” módszerrel 2015. június és augusztus között. Kizárási kritérium: az önkéntesség feltételén kívül, a normál, szá­ jon át való táplálást akadályozó betegség, valamint a lá­ zas állapot, fertőző betegség. A validált kérdőívek, táplál­ kozási naplók adatainak statisztikai feldolgozását az Rövidítések Az energia- és tápanyagfelvételi adatokat a hazai aján­ lások referenciaértékei (RÉ) alapján kategóriákba sorol­ tuk (kritikus határérték alatti – a RÉ <70%-a; megfelelő felvétel – a RÉ 70–130%-a; határérték feletti – a RÉ >130%-a ), majd összehasonlítottuk. A szoftver adatbázi­ sa lehetővé tette a speciális elemzéseket, mivel a korcso­ port-specifikus, csecsemők és kisgyermekek számára ké­ szített élelmiszereket, ételeket, valamint az esetlegesen alkalmazott étrend-kiegészítőket is tartalmazta. A kutatásban részt vevő gyermekek antropometriai paraméterei – mint a testtömeg, a testhossz és a testma­ gasság – validált műszerekkel kerültek felmérésre. A mért adatokból testtömegindexet (BMI) számoltunk, majd a Cole-féle módszer alapján kategóriákat alkot­ tunk; s mivel a gyermekek kétéves kora alatt nem áll ren­ delkezésre megbízható BMI-határérték-táblázat, szük­ ségszerűen a hazai BMI-percentilis-táblázatok határérté­ kei alapján meghatározott kategóriák kerültek kialakí­ tásra [21–24]. Rövidítések A megfigyeléseken alapuló tanulmányok után most ennek a területnek a kutatása a programozás hátterében álló molekuláris mechanizmu­ sok jobb megismerésére törekszik [14, 15]. Többek kö­ zött ezért is kiemelt jelentőségű a csecsemőtáplálás. Az 1980-as években Karlberg az emberi növekedés modulá­ torainak modelljét terjesztette elő, a csecsemő-, gyer­ Szintén bizonyítottnak tekinthető, hogy a krónikus megbetegedések rizikója már a magzati életben megjele­ nik és az időskorig elkíséri az egyént. Ennélfogva a fel­ nőttkorban manifesztálódó betegségek az egész életen át tartó környezeti hatások (fizikai és szociális), valamint életmódszokások (például táplálkozás) halmazati követ­ 1991 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP EREDETI KÖZLEMÉNY Excel 2016 (Microsoft Corporation, Redmond, WA, Amerikai Egyesült Államok [USA]), valamint az SPSS 24.0 (IBM Corporation, Armonk, NY, USA) progra­ mokkal végeztük el. mek-, serdülőkori modellt (infancy childhood puberty, ICP). Meglátása szerint a gyermek növekedése három jól elkülöníthető szakaszban zajlik, melyek folyamán három szabályozó tényező egymást átfedő hatásokat fejt ki. Gyermekkorban a növekedést a növekedési hormon sza­ bályozza, melynek hatását az inzulinszerű növekedési faktor, az IGF1 termelése közvetíti. A serdülőkori növe­ kedési megugrást a nemi hormonok hozzátevődő ana­ bolikus hatása okozza. A csecsemőkori gyors növekedést ezzel ellentétben azoknak a tápanyagoknak a bevitele serkenti, amelyek a növekedési faktor, az inzulin és az IGF1 termelését stimulálják. Ez lehet a magyarázata an­ nak, hogy az anyatejjel táplált csecsemőkhöz viszonyítva a tápszerrel tápláltaknál előálló magasabb fehérjebevitel megemelheti a vérplazmában és a szövetekben az inzu­ lin- és IGF1-szintet, ilyen módon fokozva a korai súly­ gyarapodást és később az elhízást [16]. A csecsemők fe­ hérjebevitelével foglalkozó kutatás és klinikai érdeklődés középpontjában korábban a hiány kockázatai, ezek meg­ előzése és kezelése állt. Mivel a szükséglettől elmaradó fehérjebevitel egyértelműen károsan érinti a növekedést, fejlődést, csecsemőkorban hagyományosan bőséges fe­ hérjebevitelt szorgalmaznak [17]. A koraifehérje-hipoté­ zis azonban, vagyis annak a feltevésnek a tudományos megfogalmazása, miszerint az első életévben nyújtott feleslegesen nagy fehérjebevitelhez a csecsemő gyors súlygyarapodása társul, ami a későbbi életkorokban az elhízás és a vele rokon állapotok kockázatának növekedé­ sével is jár, ezt az irányvonalat kérdőjelezi meg [18, 19]. Ezen folyamat legjobb megelőzési eszköze az anyatejes táplálás, hisz tudjuk, hogy az anyatejjel táplált csecsemők körében a túlsúly vagy elhízás előfordulása alacsonyabb, mint tápszeres társaiknál. Amennyiben nem lehetséges a szoptatás, vagy nem áll rendelkezésre elegendő anyatej, akkor viszont kiemelt jelentőségű, hogy az anyatej fehér­ je-összetételéhez legközelebb álló tápszerrel történjen a táplálás [20]. mek-, serdülőkori modellt (infancy childhood puberty, ICP). 2019 ■ 160. évfolyam, 50. szám Tápláltsági állapot, energiabevitel táblázat A 4–36 hónapos korosztály tápláltsági állapota Tápláltsági állapot, energiabevitel Az összes energiabevitelt megvizsgálva elmondható, hogy a 4–12 hónapos korcsoport fele a határérték feletti, magas energiabevitellel rendelkezik. Ez az arány az 1–2 évesek körében közel 85%, míg a 2–3 éves kisdedek ese­ tében megközelíti a 76%-ot. A kutatásban felmért 4–36 hónapos korosztály energiabeviteli adatainak megoszlá­ sát az 1. ábra szemlélteti. Megemlítendő továbbá, hogy az energiabeviteli ada­ tok mellett a testtömegadatok kiértékelésekor is körülte­ kintésre volt szükség. Az adatok helyes interpretációjá­ 1992 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP EREDETI KÖZLEMÉNY 51 15 24 49 85 76 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas Energiabevitel 1. ábra A 4–36 hónapos korcsoport napi energiabevitele a referencia-beviteliérték arányában EREDETI KÖZLEMÉNY 51 15 24 49 85 76 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas Energiabevitel 1. ábra A 4–36 hónapos korcsoport napi energiabevitele a referencia-beviteliérték arányában EREDETI KÖZLEMÉNY ónapos korcsoport napi energiabevitele a referencia-beviteliérték arányáb 1. ábra A 4–36 hónapos korcsoport napi energiabevitele a referencia-beviteliérték arányában 1. ábra delkezik, potenciálisan sovány, 10–14,1%-uk potenciáli­ san túlsúlyos (BMI: 85–97 percentilis), 2,6–3,5%-uk po­ tenciálisan elhízott (BMI >97 percentilis) (1. táblázat) [23]. hoz a hazai és a nemzetközi ajánlások felnőttkori BMI-táblázatai mellett figyelembe kellett vennünk és mérlegelnünk a vizsgált csoportok egyedi sajátosságait is. Ennek ismeretében elmondható, hogy a túlsúly és az el­ hízás a 2–3 éves korosztály 6,5%-át érintette. A mért ada­ tokat az 1. táblázat prezentálja. Ezen adatok ismeretében fontos felhívnunk a szakem­ berek és a lakosság figyelmét, hogy egészséges gyermekek esetében az első két évben (0–24 hó) nem kívánatos a gyors testtömeg-gyarapodás. A percentilisek határán el­ helyezkedő BMI pedig célzott beavatkozást igényel [25]. A hazai BMI-percentilis-táblázatok alapján (korcso­ portok szerint szeparálva) a kétéves vagy annál fiatalabb gyermekek 15–18,6%-a 10 percentilis alatti BMI-vel ren­ 1. táblázat A 4–36 hónapos korosztály tápláltsági állapota Hazai percentilisértékek, kategóriák BMI-átlag (kg/m2) <10 pct 10–84,9 pct 85–96,9 pct ≥97 pct 4–12 hó 16,5 18,6 68,2 10,0 3,2 12–24 hó 16,5 15,0 68,3 14,1 2,6 Cole-kategóriák „Sovány” „Normál” „Túlsúlyos” „Elhízott” 25–36 hó 15,8 23,9 69,6 4,3 2,2 1. táblázat A 4–36 hónapos korosztály tápláltsági állapota 1. táblázat A 4–36 hónapos korosztály tápláltsági állapota 1. Fehérjebevitel A testtömegkilogrammra meghatározott fehérjebevitel a 4–12 hónaposak esetében 3,1 g/ttkg (EFSA-ajánlás: 1,48–1,65 g/ttkg), míg a 12–36 hónapos gyermekeké 4,1 g/ttkg (EFSA-ajánlás: 1,13–1,26 g/ttkg) volt. Fél– egy éves kor körüli időszaktól kezdődően a gyermekek fehérjebevitele jelentősen meghaladja a testtömeg­ arányos ajánlásokat. A mért fehérjebeviteli értékeket a 2. ábra mutatja be. A hazai BMI-percentilis-táblázatok alapján (korcso­ portok szerint szeparálva) a kétéves vagy annál fiatalabb gyermekek 15–18,6%-a 10 percentilis alatti BMI-vel ren­ A hazai BMI-percentilis-táblázatok alapján (korcso­ portok szerint szeparálva) a kétéves vagy annál fiatalabb gyermekek 15–18,6%-a 10 percentilis alatti BMI-vel ren­ 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas 2 98 100 100 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Fehérjebevitel 2. ábra A 4–36 hónapos korcsoport fehérjebevitele 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas 2 98 100 100 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Fehérjebevitel 2. ábra A 4–36 hónapos korcsoport fehérjebevitele 2019 ■ 160. évfolyam, 50. szám 1993 ORVOSI HETILAP EREDETI KÖZLEMÉNY 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas 45 65 68 43 27 24 12 8 8 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Zsírbevitel 3 áb A 4 36 hó k í b i l EREDETI KÖZLEMÉNY 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas 45 65 68 43 27 24 12 8 8 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Zsírbevitel 3. ábra A 4–36 hónapos korcsoport zsírbevitele EREDETI KÖZLEMÉNY 3. ábra A 4–36 hónapos korcsoport zsírbevitele Zsírbevitel A 4–12 hónapos (hozzátáplált) csecsemők jelentős há­ nyada kedvezőtlenül magas bevitelt mutat koleszterin (40,9%) esetén. Ez igaz az 1–3 éves gyermekek felére is (50,1%). A koleszterinbevitel szempontjából pozitív ös�­ szefüggés van a tejek, tejtermékek, tojás, húsok, majd a hús- és májkészítmények esetében. A zsírok felvételének tekintetében e korcsoportban kevés­ bé jellemző a határérték feletti bevitel. A 4–12 hónaposak 43%-ának, az 1–2 évesek 27%-ának és a 2–3 éves kisdedek 24%-ának megfelelő a zsírbevitele. Az alacsony zsír­ fogyasztás a leginkább az 1 éves feletti korcsoportot jel­ lemzi (12–24 hónaposak: 65%, 25–36 hónaposak: 68%) (3. ábra). Amennyiben a hazai referenciaérték-tarto­ mányhoz (Rodler Imre [szerk.] Új tápanyagtáblázat) vi­ szonyítjuk, akkor nagyobb százalékban szerepelnek a cse­ csemők és a kisdedek a normáltartományban, és kevesebb az alacsony zsírbevitelűek aránya. Az egyes tápanyagok élelmiszercsoportonként való megoszlásának (százalékos tápanyagprofil) közvetlen elemzésére nem volt lehetőség, azonban vizsgálható az egyes tápanyagok bevitele és az élelmiszercsoportok fogyasztásának összefüggése. A 4–12 hónapos gyermekek (n = 220) napi átlagos zsírfo­ gyasztása 30,6 g/fő, amelyből állati eredetű 14,6 g, nö­ vényi eredetű 6,7 g. 12–24 hónaposaknál (n = 259) a napi zsírfogyasztáson (45,9 g) belül ez az arány 25,3 g és 16,6 g, 25–36 hónapos korban (n = 152) a napi 53,9 g zsírbevitelből 30,5 g állati eredetű, míg 22,7 g növényi. Szénhidrát és hozzáadott cukor bevitele A 4–12 hónapos korcsoport közel felének határérték fe­ letti, míg az 1–3 évesek többségének megfelelő (12–24 hónaposak: 77%, 25–36 hónaposak: 82%) a szénhidrát­ bevitele (4. ábra). A hozzáadott cukor 4–12 hónapos korban fordított összefüggést mutat a tej és tejtermékek fogyasztásával, pozitív összefüggést pedig a kész és félkész ételek, bébi­ ételek fogyasztásával. A hozzáadott cukor napi bevitele a 4–12 hónapos korcsoportban 11,5 g, az 1–2 éveseké 27,5 g, és a 2–3 éveseké 45,3 g. A szénhidrátokon belül a hozzáadott cukorból származó energia a 12–36 hóna­ pos gyermekek esetében közel 10%, csecsemők esetén 3–5% körüli. 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP 1994 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas 5 11 9 47 77 82 48 12 9 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Szénhidrátbevitel 4. ábra A 4–36 hónapos korcsoport napi szénhidrátbevitele 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) 4–12 hónaposak (n = 220) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 Alacsony Normál Magas 5 11 9 47 77 82 48 12 9 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Szénhidrátbevitel ORVOSI HETILAP 1994 2019 ■ 160. évfolyam, 50. szám EREDETI KÖZLEMÉNY 2. táblázat Ásványi anyagok átlagos napi bevitele (4–36 hónapos gyerme­ kek körében) Korcsoport (hó) Nátrium (mg) Kálium (mg) Kalcium (mg) Magnézium (mg) Vas (mg) 4–12 (n = 220)   623 1378,7 465,7 128,71 6 12–36 (n = 411) 1236 1949,9 632,57 228,62 7,4 12–24 (n = 259) 1704 1823,4 590,2 210,1 7,19 25–36 (n = 152) 2198,5 2165,4 704,6 260 7,76 2. táblázat Ásványi anyagok átlagos napi bevitele (4–36 hónapos gyerme­ kek körében) húskészítményt (átlagosan 33,6 g/fő/nap) és 18%-uk belsőséget (átlagosan 9,1 g/fő/nap). A 12–36 hónapo­ sak 99%-a fogyaszt húst és húskészítményeket (átlagosan 71,2 g/fő/nap), 33%-uk belsőséget (átlagosan 9,7 g/ fő/nap). / p) A kalciumbevitel a 4–12 hónapos (hozzátáplált) cse­ csemők 22,1%-ánál és az 1–3 éves gyermekek 41,8%- ánál hiányosnak tekinthető. Az életkor növekedésével jelentősen nő az alacsony kalciumbevitellel rendelkezők száma. A 4–12 hónapos csecsemők 79%-a kap tejet és tejterméket (átlagosan 362,1 g/fő/nap). A 12–36 hó­ naposak 100%-a kap ebből az élelmiszercsoportból (átla­ gosan 320,7 g/fő/nap), vagyis az életkor előrehalad­ tával csökken a tej és tejtermékek fogyasztásának mennyisége. Ásványi anyagok és vitaminok bevitele Ásványi anyagok és vitaminok bevitele A 4–12 hónapos (hozzátáplált) csecsemők jelentős há­ nyadának (71%) kedvezőtlenül magas a nátriumbevitele, míg az egyéb ásványi anyagok és vitaminok többségének felvétele jónak mondható; ugyanakkor fontos megemlí­ teni a jelentős prevalenciájú (22,1%) elégtelen kalcium­ felvételt, valamint a nagyarányú vas- (43%) és D-vitamin- hiányt (79%). Az 1–3 éves gyermekek száz százaléka magas bevitelt mutat a nátrium esetén, és foszforból ez az arány 67%; hiányosnak tekinthető a kalcium (41,8%), a vas (25%) és a D-vitamin (87,1%) bevitele. E korcso­ port kevésbé használ D-vitaminos étrend-kiegészítőt, így étrend-kiegészítővel ez az arány csak 65%-ra csökken. A korcsoportokra lebontott ásványianyag-bevitelek átla­ gát a 2. táblázat tartalmazza. Szénhidrát és hozzáadott cukor bevitele Az eredményt tovább rontja, hogy az 1–3 éves korosztály 31,6%-a kedvezőtlenül magas bevitelt mutat a foszfor ­tekintetében, így még inkább eltolódik az ajánlott 1,4 : 1 – 1,9 : 1 kalcium : foszfor arány. Ez az intenzív csontfejlődés szakaszában igen nagy problémát jelent. Az adatok a későbbi életkorban sem alakulnak kedvezően [26]. Ásványi anyagok és vitaminok bevitele Vitaminok bevitele A tudományos publikáció formai követelményei nem te­ szik lehetővé a teljes vizsgálati anyag részletes ismerteté­ sét, ugyanakkor az egyik legfontosabb vitamin beviteli értékei mellett nem mehetünk el szó nélkül, annak kor­ csoport-specifikus fontossága miatt. p pi g A D-vitamin-bevitel az elvárt szintnél alacsonyabb ­értékeket mutatott mind a 12–24 hónapos kisdedek (83%), mind a 25–36 hónapos gyermekek esetében (94%). Étrend-kiegészítőket is figyelembe véve ezek az értékek 58%-ra, illetve 76%-ra csökkentek. A táplálkozási naplók elemzésekor az érintettek táplálékkal, illetve ét­ rend-kiegészítővel történő D-vitamin-felvételének vizs­ gálatára volt elsősorban lehetőségünk. Ugyanakkor a vizsgálat során kitöltött kérdőívből kiderült, hogy a gyer­ mek növekedésével egyenes arányban nő a szabad leve­ gőn töltött idő hossza. Az 1 éves kor alatti gyermekekkel tipikusan 1–2 órát, míg az idősebbekkel a legtöbben 2–4 órát töltenek a szabadban. Ez a bőrt érő napsugárzásnak, illetve a napsugarak UVB-spektrumának az endogén-D- vitamin-szintézisben betöltött szerepe miatt nagy jelen­ tőségű [27]. A többlet-nátriumbevitel a 4–12 hónapos és az 1–3 éves korcsoportban elsősorban a kenyérfélék, a húské­ szítmények és az ízesítőanyagok, fűszerek, egyéb élelmi­ szercsoportok fogyasztásából származik. Az ételkészítési szokásokkal és az élelmiszer-választásban megfigyelhető sajátosságokkal magyarázható a kedvezőtlenül magas nátriumbevitel. A vas nélkülözhetetlen a pszichomotoros és kognitív funkciók fejlődéséhez, hiányában ezekben a funkciókban zavarok keletkezhetnek. A kor előrehaladtával javuló tendenciát mutat az elégtelen vasbevitel, hiszen a 4–12 hónapos (hozzátáplált) csecsemőknél talált nagyarányú vashiány 42,3%-ról 1–3 éves gyermekeknél 25,3%-ra csökkent. Fontos kiemelni, hogy megfelelő hozzátáplá­ lással, étkeztetéssel ezek az arányok jelentősen javíthatók lennének. A 4–12 hónapos csecsemők 70%-a kap húst és A felmért, részletes vitaminbeviteli átlagokat a 3. táb­ lázat tartalmazza. 1995 ORVOSI HETILAP 2019 ■ 160. évfolyam, 50. szám 3. táblázat Vitaminok átlagos napi bevitele (4–36 hónapos gyermekek körében) Korcsoport (hó) A-vitamin (µg) β-Karotin (mg) B1-vitamin (µg) B2-vitamin (µg) B6-vitamin (µg) B12-vitamin (µg) C-vitamin (mg) D-vitamin (µg) Folsav (µg) 4–12 (n = 220) 574,8 3,76 643,15   682,7   775,4 1,19 80,8 4,35   72,8 12–36 (n = 411) 419,9 2,47 726 1020,9 1115,5 2,51 86,37 3,36 109,1 12–24 (n = 259) 435,3 2,53 711,9   947,76 1048,7 2,00 78,9 3,93 102,4 25–36 (n = 152) 393,6 2,37 750,2 1145,7 1229,4 3,31 99 2,4 120,5 3. táblázat Vitaminok átlagos napi bevitele (4–36 hónapos gyermekek körében) 2019 ■ 160. évfolyam, 50. Egyéb tápanyagok g g Vegyes táplálkozás mellett a 12–24 hónapos korcsoport felének megfelelő az élelmirost-bevitele, csupán 4%-uk­ nak alacsonyabb a referenciaértékhez hasonlítva. Az 1–3 évesek 59%-ára magasabb rostbevitel jellemző, 35%-uk megfelelő mennyiségű élelmi rostot fogyaszt, és csak 6%- nak elégtelen az élelmirost-bevitele (5. ábra). Vitaminok bevitele szám ORVOSI HETILAP EREDETI KÖZLEMÉNY 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 (12 hónapos kor alatt Alacsony Normál Magas 4 6 51 35 45 59 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Élelmirost-bevitel EREDETI KÖZLEMÉNY 25–36 hónaposak (n = 152) 12–24 hónaposak (n = 259) A referenciaérték forrása: EFSA-ajánlás, EFSA Journal 2013 (12 hónapos kor alatt nincs beviteli ajánlás) Alacsony Normál Magas 4 6 51 35 45 59 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Élelmirost-bevitel 5. ábra A 12–36 hónapos korcsoport élelmirost-bevitele 5. ábra A 12–36 hónapos korcsoport élelmirost-bevitele 5. ábra A 12–36 hónapos korcsoport élelmirost-bevitele Az EFSA által meghatározott, 0–3 éves gyermekekre vonatkozó referencia-beviteliértékek szerint magas ener­ giabevitelű a 12–24 hónapos gyermekek 84,6%-a; 25–36 hónapos korban ez az érték 75,7%. Míg a hazai referen­ ciaérték alapján a felmérésben a 4–12 hónapos gyerekek 10,9%-a, az 1–2 évesek 20%-a, a 2–3 évesek 47%-a a ma­ gas fehérjebeviteli tartományba esett, addig a 2013-ban kiadott – a makro- és mikronutrienseknek a WHO/ FAO/UNU által 2007-ben megadott beviteli értékein alapuló – EFSA-ajánlás referenciaértékeihez viszonyítva a vizsgált gyerekek 100%-a a magas fehérjebeviteli cso­ portba tartozik minden korcsoportban [31–32]. A Ma­ gyar Gyermekorvosok Társaságának koordinálásában 2010-ben az 1–3 éves gyermekek komplex táplálkozási vizsgálata történt meg hasonló módszerekkel, igaz, ki­ sebb elemszámmal (92 fő 1–2 éves és 102 fő 2–3 éves) [29]. Ezért a jelen vizsgálat néhány értékét összevetettük az öt évvel ezelőtt kapott adatokkal is. Az 1–3 éves gyer­ mekek esetében az energiabevitel szempontjából jelen­ tős különbség nincs a két vizsgálat (2010 vs. 2015) ered­ ménye között, de a magas energiatartományba mintegy 6%-kal több gyermek tartozik 2015-ben. A makrotáp­ anyagok tekintetében közel megháromszorozódott a magas zsírbevitelű gyermekek aránya (8,2% vs. 24,1%). Vizsgálatunkban az energiát szolgáltató makrotápanya­ gok (zsír és szénhidrát) a 4–12 hónapos korcsoportban közel normálmegoszlást mutattak, a kor növekedésével azonban az EFSA-ajánlás szerint magas az alacsony zsír­ bevitellel rendelkezők aránya (mintegy 65%) – a gyakor­ latilag megfelelő, többségében normál- (mintegy 80%) szénhidrátbevitel mellett [32]. 4–12 hónapos korban pozitív összefüggés van a BMI és a napi energiabevitel, valamint a makrotápanyagok (fehérje, zsír, szénhidrát) napi bevitt mennyisége között. Az energiamegoszláson belül a zsírból származó energiával pozitív, a szénhidrát­ energia-%-kal negatív összefüggés mutatható ki. 12–36 hónapos korban a táplálkozás szerkezetének jelentős vál­ tozása, valamint a szomatikus fejlődés dinamizmusa mi­ att a csoport nem tekinthető homogénnek; ez megmu­ Irodalom [1]  Kimokoti RW, Millen BE. Nutrition for the prevention of chron­ ic diseases. Med Clin North Am. 2016; 100: 1185–1198. [2]  Chizuru N, Ricardo U, Shiriki K, et al. The Joint WHO/FAO Expert Consultation on diet, nutrition and the prevention of chronic diseases: process, product and policy implications. Public Health Nutr. 2004; 7: 245–250. Egy 2014-ben 4–10 éves budapesti és kecskeméti gyermekekre reprezentatív táplálkozási felmérésben (n = 799) a kalcium átlagos napi felvétele korcsoportonként kissé változó mértékben, de szintén elmaradt az ajánlott napi 800 mg értéktől, és a gyermekek 35%-a kritikus, határérték alatti mennyiségben fogyasztotta ezt az ásvá­ nyi anyagot. Összességében a 4–12 hónapos gyermekek 22%-ánál, a 12–36 hónaposak 42%-ánál volt detektálha­ tó az elégtelen kalciumbevitel. Itt is megfigyelhető, hogy a napi foszforfelvétel átlaga meghaladja az ajánlott 620 mg értéket, így kedvezőtlen a kalcium és a foszfor hánya­ dosa is. Az elégtelen kalciumbevitelhez vezető egyik té­ nyező, hogy a kalcium fő élelmiszerforrásául szolgáló tej és tejtermék fogyasztása a 4–6 éveseknél összességében 314 g/nap, míg a 7–10 éveseké 290 g/nap, s ez elmarad a kívánatos mintegy 450–500 g/nap mennyiségtől [33]. [3]  Brennan P, Perola M, Jan van Ommen G, et al. Chronic disease research in Europe and the need for integrated population co­ horts. Eur J Epidemiol. 2017; 32: 741–749. [4]  Gluckman PD, Hanson MA, Cooper C, et al. Effect of in utero and early-life conditions on adult health and disease. N Engl J Med. 2008; 359: 61–73. [5]  Barker DJ. In utero programming of chronic disease. Clin Sci (Lond). 1998; 95: 115–128. [6]  Osmond C, Barker DJ, Winter PD, et al. Early growth and death from cardiovascular disease in women. BMJ 1993; 307: 1519– 1524. [7]  Hales CN, Barker DJ. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 1992; 35: 595–601. [8]  Cooper C, Fall C, Egger P, et al. Growth in infancy and bone mass in later life. Ann Rheum Dis. 1997; 56: 17–21. [9]  Rich-Edwards JW, Stampfer MJ, Manson JE, et al. Birth weight and risk of cardiovascular disease in a cohort of women followed up since 1976. BMJ 1997; 315: 396–400. A nátriumbevitel szempontjából is aggasztóak az ered­ mények. Egy hazai vizsgálat megállapításai szerint 6 hó­ napos kor felett minden korcsoportban magasabb a nát­ riumbevitel: 400 mg [34]. [10]  Charalambous M, da Rocha ST, Ferguson-Smith AC. Genomic imprinting, growth control and the allocation of nutritional re­ sources: consequences for postnatal life. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Megbeszélés 2015-ben végzett kutatásunk célcsoportját 0–3 éves kor közötti csecsemők és kisdedek képezték. A vizsgálati mintát önként jelentkező édesanyák és gyermekeik al­ kották. A csecsemő- és kisdedkorban jellemző etetési és táplálkozási szokások felmérése az esetleges intervenciók tervezése, megvalósítása szempontjából ideális időszak, mivel a helyes étkezési mintákat ekkor sajátíthatja el a gyermek, és a hibák korrekciójára még lehetősége van a családnak. Az epigenetika, a korai anyagcsere-programozás rávi­ lágít a legfontosabb környezeti tényezők – kiemelten a táplálkozás – hatására, a csecsemők hosszú távú egészsé­ gére vagy éppen betegségeik kialakulásának kockázatára, vagyis a csecsemő és kisded táplálása nemcsak az adott időszakban befolyásolja a gyermek fejlődését, hanem a felnőttkori nem fertőző betegségek kialakulására is ha­ tással van [28]. A társadalom egészségfejlesztését, vala­ mint a civilizációs betegségek prevencióját ennek okán már gyermekkorban szükséges megkezdeni, ugyanis egy 2010-ben végzett felmérés szerint a vizsgált 1–3 éves korcsoportú gyermekek táplálkozásában, illetve egy 2014-ben 4–10 éves gyermekek körében végzett vizsgá­ lat adatainak elemzése alapján a felnőtt lakosságra is jel­ lemző táplálkozási hibák nyomai már felfedezhetők [29, 30]. A csecsemő- vagy kisdedkorú gyermekek fejlődésük azon periódusában vannak, amikor érdemi beavatkozás tehető a kedvező táplálkozási szokások és preferenciák kialakításáért, melyek a felnőttkori egészségi állapotot is befolyásolhatják [12]. 1996 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP EREDETI KÖZLEMÉNY Szerzői munkamegosztás: K. J., B. K. K.: A vizsgálat ter­ vezése és lefolytatása. Sz. Zs., E.-S. A., R. L. B.: Iroda­ lomkutatás. B. L.: Statisztikai elemzések. E.-S. A., B. K. K., Sz. Zs., K. J., R. L. B.: Az eredmények interpretá­ ciója. E.-S. A., B. K. K., Sz. Zs., R. L. B.: A kézirat meg­ szövegezése. A cikk végleges változatát valamennyi szer­ ző elolvasta és jóváhagyta. tatkozik abban is, hogy a nagy mintaszám ellenére sem mutatható ki összefüggés a tápanyagkomponensek és a BMI között. A mikrotápanyagok tekintetében kedvezőtlen változás következett be: nőtt az alacsony kalciumbevitelűek ará­ nya, javult azonban a megfelelő vas- és cinkbevitelűek hányada. A 2010-ben 1–3 éves gyerekek (n = 211) között vég­ zett felméréshez képest pozitívnak mondható, hogy 2015-re 10%-kal csökkent az alacsony, 3%-kal nőtt a nor­ mál- és 7%-kal a magas vasbevitellel rendelkező 1–3 éves gyermekek (n = 411) száma, de az 1–3 éves korosztály 25%-a még mindig nem jut megfelelő mennyiségű vas­ hoz [29]. Megbeszélés A 2010-ben 1–3 éves gyerekek (n = 211) között vég­ zett felméréshez képest pozitívnak mondható, hogy 2015-re 10%-kal csökkent az alacsony, 3%-kal nőtt a nor­ mál- és 7%-kal a magas vasbevitellel rendelkező 1–3 éves gyermekek (n = 411) száma, de az 1–3 éves korosztály 25%-a még mindig nem jut megfelelő mennyiségű vas­ hoz [29]. Irodalom Nutrition in early life and the programming of adult disease: a review. J Hum Nutr Diet. 2015; 28: 1–14. [29]  Biró L, Szabó L. Complex nutritional examination of 1–3-year- old children. [1–3 éves gyermekek komplex táplálkozási vizsgála­ ta.] Gyermekgyógyászat 2011; 62: 80–85. [Hungarian] [20]  Weber M, Grote V, Closa-Monasterolo R, et al. Lower protein content in infant formula reduces BMI and obesity risk at school age: follow-up of a randomized trial. Am J Clin Nutr. 2014; 99: 1041–1051. [30]  Kiss-Tóth B. The relationship between the nutritional habits of Hungarian children and adults according to the surveys. [A ma­ gyar gyermekek és felnőttek táplálkozási szokásainak összefüg­ gései a felmérések tükrében.] Új Diéta 2015; 24(5): 2–4. [Hun­ garian] [21]  Cole TJ, Flegal KM, Nicholls D, et al. Body mass index cut offs to define thinness in children and adolescents: international sur­ vey. BMJ 2007; 335: 194. [31]  World Health Organization. Report of a Joint WHO/FAO/ UNU Expert Consultation. Protein and amino acid require­ ments in human nutrition, 2007. WHO, Technical Report Series 935, Geneva. Available from: https://apps.who.int/iris/bit­ stream/handle/ 10665/43411/WHO_TRS_935_eng.pdf;jsess ionid=7293079636537FAA3BE27687025B728E?sequence=1 [accessed: October 6. 2019]. [22]  Joubert K, Ujhelyi G. Growth and nutrition assessment in in­ fants, children and adolescents (BMI) program. [Növekedés és a tápláltsági állapot értékelése csecsemő-, gyermek- és serdülő­ korban (BMI) program.] Available from: https://mave.hu/up­ loads/file/BMI/Nov.taplaltsag-BMI-85.P.gyakorlati_utmutato. ppt [accessed: October 6, 2019]. [23]  Cole TJ, Bellizzi MC, Flegal KM, et al. Establishing a standard definition for child overweight and obesity worldwide: interna­ tional survey. BMJ 2000; 320: 1240–1243. [32]  EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies). Scientific opinion on nutrient requirements and dietary intakes of infants and young children in the European Union. EFSA Journal 2013; 11: 340. [24]  Cameron N. Body mass index cut offs to define thinness in chil­ dren and adolescents: international survey. BMJ 2007; 335: 166–167. [33]  Kiss-Tóth B. Assessment of nutrition and physical activity of four-to-ten-year-old children – Part 2. [Négy–tíz éves gyermekek táplálkozásának és fizikai aktivitásának felmérése – 2. rész.] Új Diéta 2014; 23(5): 5–7. [Hungarian] [25]  Fleddermann M, Demmelmair H, Grote V, et al. Infant formula composition affects energetic efficiency for growth: the BeMIM study, a randomized controlled trial. Clin Nutr. 2014; 33: 588– 595. [34]  Szűcs Zs, Pálfi E, Badacsonyiné Kassai K. Nutrition habits of do­ mestic children in the context of a comparative analysis. [A hazai gyermekek táplálkozási szokásai egy összehasonlító elemzés tükrében.] Új Diéta 2019; 28(füzet): 2–5. Irodalom Curr Opin Endocrinol Diabetes Obes. 2007; 14: 3–12. Vizsgálatunkban ,,fény derült” arra a tényre is, hogy 1 éves kor felett a gyermekek 61%-a ugyanazt eszi, mint a család felnőtt tagjai, így a helytelen táplálkozási minta már ekkor megmutatkozik – többek között – a maga­ sabb nátriumfogyasztásban. [11]  Dos Santos Silva I, De Stavola BL, Hardy RJ, et al. Is the asso­ ciation of birth weight with premenopausal breast cancer risk mediated through childhood growth? Br J Cancer 2004; 91: 519–524. [12]  Koletzko B, Brands B, Poston L, et al. Early nutrition program­ ming of long term health. Proc Nutr Soc. 2012; 71: 371–378. A vizsgálati eredményekből látható, hogy mind a ge­ netikai heterogenitás, mind pedig az egyes nemzetekre jellemző táplálkozás nagyban befolyásolja a beviteli refe­ renciák alkalmazhatóságát. Tekintettel arra, hogy a táp­ láltsági állapotnak, valamint az egyes makro- és mikro­ nutriensek bevitelének a megítélése kiemelten fontos ebben a korosztályban, így célszerű lenne egy hazai kon­ szenzus megalkotása, amelynek kialakításában az összes hazai, érintett szakmai társaság részt venne. Ezzel sor kerülhetne a kutatásban felmerült anomáliák feloldására, a gyakorlati megvalósíthatóság figyelembevételével. [13]  Schellong K, Schulz S, Harder T, et al. Birth weight and long- term overweight risk: systematic review and a meta-analysis in­ cluding 643,902 persons from 66 studies and 26 countries glob­ ally. PLoS ONE 2012; 7: e47776. [14]  Mühlhäusler BS, Adam CL, McMillen IC. Maternal nutrition and the programming of obesity: the brain. Organogenesis 2008; 4: 144–152. [15]  Wu G, Imhoff-Kunsch B, Girard AW. Biological mechanisms for nutritional regulation of maternal health and fetal development. Paediatr Perinat Epidemiol. 2012; 26(Suppl 1): 4–26. [16]  Socha P, Grote V, Gruszfeld D, et al. Milk protein intake, the metabolic-endocrine response, and growth in infancy: data from a randomized clinical trial. Am J Clin Nutr. 2011; 94: 1776S- 1784S. [17]  Scrimshaw SN. Malnutrition, brain development, learning, and behavior. Nutr Res. 1998; 18: 351–379. Anyagi támogatás: A vizsgálat lefolytatását a Nestlé Hungária Kft. és a Numil Kft. finanszírozta, ugyanakkor a közlemény megírása anyagi támogatásban nem része­ sült. [18]  Koletzko B, von Kries R, Closa-Monasterolo R, et al. Can infant feeding choices modulate later obesity risk? Am J Clin Nutr. 2009; 89: 1502S–1508S. 1997 ORVOSI HETILAP ■ 160. évfolyam, 50. szám 2019 EREDETI KÖZLEMÉNY [19]  Embleton ND, Cooke RJ. Protein requirements in preterm in­ fants: effect of different levels of protein intake on growth and body composition. Pediat Res. 2005; 58: 855–860. [28]  Langley-Evans SC. A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) Irodalom [Hungarian] [26]  EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies). Scientific opinion on dietary reference values for phosphorus. EFSA Journal 2015; 13: 4185. (Raposa L. Bence dr., Pécs, Pollack M. u. 5., 7633 e-mail: raposa.bence@gmail.com; bence.raposa@etk.pte.hu) [27]  Takács I, Benkő I, Toldy E, et al. Domestic consensus on the role of vitamin D in disease prevention and treatment. [Hazai kon­ szenzus a D-vitamin szerepéről a betegségek megelőzésében és kezelésében.] Orv Hetil. 2012; 153: 5–26. [Hungarian] A rendezvények és kongresszusok híranyagának leadása a lap megjelenése előtt legalább 40 nappal lehetséges, a 6 hetes nyomdai átfutás miatt. Kérjük megrendelőink szíves megértését. A híranyagokat a következő címre kérjük: Orvosi Hetilap titkársága: edit.budai@akademiai.hu Akadémiai Kiadó Zrt. A rendezvények és kongresszusok híranyagának leadása a lap megjelenése előtt legalább 40 nappal lehetséges, a 6 hetes nyomdai átfutás miatt. Kérjük megrendelőink szíves megértését. A híranyagokat a következő címre kérjük: Orvosi Hetilap titkársága: edit.budai@akademiai.hu Akadémiai Kiadó Zrt. A rendezvények és kongresszusok híranyagának leadása A híranyagokat a következő címre kérjük: Orvosi Hetilap titkársága: edit.budai@akademiai.hu Akadémiai Kiadó Zrt. A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) 1998 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP
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Organization of Lifelong Learning in the System of Professional Training of Teachers
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Organization of Lifelong Learning in the System of Professional Training of Teachers Burlakova*(a), Anastasiya S. Budnik (b), Elizaveta S.Burlakova (c) (a) Moscow State University of Psychology and Education, Moscow, Russia (b) Moscow City Pedagogical University, Moscow, Russia (c) Academy of Social Management, Moscow, Russia * Corresponding author. E-mail address: iiburlakova@mail.ru © 2019 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published by Kazan Federal University and peer-reviewed under responsibility of IFTE-2019 (V International Forum on Teacher Education) Proceedings IFTE-2019, 57-67 doi:10.3897/ap.1.e0051 V International Forum on Teacher Education V International Forum on Teacher Education Organization of Lifelong Learning in the System of Professional Training of Teachers Irina I. Burlakova*(a), Anastasiya S. Budnik (b), Elizaveta S.Burlakova (c) (a) Moscow State University of Psychology and Education, Moscow, Russia (b) Moscow City Pedagogical University, Moscow, Russia (c) Academy of Social Management, Moscow, Russia Introduction Development of continuous education in EU countries In the context of globalization, most countries have reformed their professional training systems in line with the “Lisbon strategy” adopted by the leaders of EU states, the goal of which was to create the most competitive and dynamic knowledge-based economy in the world by 2010 (Kok, 2004). The European Commission, assessing the results of educational reforms in 2008, pointed to the need to develop national strategies for lifelong learning. The principles of reorganization of the existing educational systems and construction of lifelong learning are universal. The most significant of them are: - orientation of the educational system to the individual, unique personality and basic needs, among which the leading place is the need for lifelong self-improvement, self-development and self-realization; - democratization of the educational system, accessibility and openness of any level and form of education to each individual, regardless of social status, nationality, race, physical condition; - flexibility of the educational system, rapid response to the educational needs of the population, special interests, styles and rates of potential students learning; - variability of educational services and the realization of the right of every person to choose their own strategy for further education after graduation; - integration of formal and non-formal types of education, creation of a holistic educational segment that turns the adult population of the country into “students”; - use of information technologies for educational purposes at any stage of their life (home, work, recreation, transportation, etc.); - development of an integrated system of information support; - development of an integrated system of information support; - the existence of an extensive and constantly updated legal and regulatory framework for lifelong learning. In European countries, lifelong learning is essential for two reasons: In European countries, lifelong learning is essential for two reasons: 1) the population of the European Union lives in a complex socio-political and economic environment, where the full development of the person is impossible without mastering the ability to adapt quickly to cultural, linguistic and ethnic diversity; 2) knowledge-centrality is the basis of modern European society. This means that the knowledge, information and motivation to update it constantly and the skills, necessary to do this, become decisive factors for the competitiveness of the individual and the effectiveness of the human potential in a market economy (Commission of the European Communities, 2000). Abstract The relevance of the present issue is caused by the need for carrying out lifelong learning of teachers as an essential condition of their professional development. The aim of the article is to prove the necessity of integrating formal and informal professional and optional development of educators. The main methods of the research are analysis and synthesis of the data obtained. The authors hold an analysis of the lifelong learning development in the EU countries and in Russia, which can be formal and informal. The latest represents an entirely new phenomenon in the social and educational practice. Its content bases on their own principles, carries out specific functions and solves existing problems in an innovative way. The article also views the strategies and models of lifelong professional education of teachers being under a significant pressure of informational and communicational technologies and relying on informal mechanisms of giving knowledge and forming competences. In the research part of the article the objective and subjective difficulties of teachers and their professional needs are revealed. Lifelong learning is bound to overcome these difficulties and satisfy the necessity of forming and developing new professional competences. The analysis of the documents of educational institutions shows the dominance of informal education. It proves the need for defining the status of informal education as a constituent part of professional development of an educator. Keywords: lifelong learning of teachers, informal education, personality-oriented trajectory of continuous education, models and strategies of lifelong learning, professional development of an educator. © 2019 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published by Kazan Federal University and peer-reviewed under responsibility of IFTE-2019 (V International Forum on Teacher Education) * Corresponding author. E-mail address: iiburlakova@mail.ru 58 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 Introduction In such circumstances, education is designed to help people to cope successfully with adaptation to new conditions of life. These reasons for the development of lifelong learning are closely interrelated and determine the two main goals: the active position of citizens and the competitiveness of a professional in the labor market. It must be noted that the governments of European countries have come to a common understanding of the need to develop lifelong learning as a combination of formal, non-formal and informal education. Taking into account this factor, the modern national systems of lifelong learning develop nowadays in the European Union. The concept of “lifelong learning” appeared in science in 1929, and in 1968 – in the materials of UNESCO. In the terminology of UNESCO from 1997 lifelong learning is defined as “formal, non-formal, non-institutional (informal)”, i.e. it is possible in any organizational form (UNESCO, 1995-2012). The scientific literature uses several concepts of lifelong learning, differing from - education as lifelong learning (life-long education); Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 59 - lifelong education for adults; - lifelong education for adults; - lifelong professional education. - lifelong professional education. The European Union defines formal education in the 2000 Memorandum as the process leading to the issuance of a state diploma or certificate (Commission of the European Communities, 2000). According to the International standard classification (2004), formal education is provided through formal institutions (College / University). Formal education is a system of hierarchically structured and inherent to the majority of States including Russia (International Labour Organization). Non-formal education is a qualitatively new phenomenon in social and educational practice, the content of which is based on its own principles, performs certain functions, solving many old problems in a new way. Non-formal education is a flexible educational system focused on specific needs and interests of students. Non-formal education has the characteristic of «complementarity» of the acquired knowledge in relation to the professional education and experience. A study of non-formal education shows that it is neither a simple addition to or continuation of traditional education nor an alternative to traditional education. According to Klimov, non-formal education, located between the poles of formal and informal education, is conscious in contrast to the latest, organized and managed, focused on the specific educational needs of different professional groups (Klimov, 1998). Introduction In 1987, the Decision of the CPSU Central Committee and the USSR Council of Ministers “On measures to improve the training and use of scientific-pedagogical and scientific personnel” sets the task of restructuring the training of scientific-pedagogical and scientific personnel, presenting it as an integral and mandatory part of the unified system of lifelong education in the country. In 1989, and then in 2010, the concepts of lifelong learning were discussed and adopted, but they were not implemented. A new stage in the development of lifelong learning began with the adoption of a new Federal Law “Оn education in the Russian Federation”. This law introduces such concepts as: “lifelong learning”, “e- learning”, “modular principle of presenting the content of programs and building curricula”, “network forms of program implementation”. The drafters of the education act define different ways of getting education: - through the organization of credit-modular system of educational process; - through networking in the implementation of educational programs; - through the use of distance learning technologies in the educational process; - through the organization of training with the help of integrated educational programs; - through the use of information and educational resources in the educational process, etc. (Ministry of Education and Science, 2012). The priority direction of the state policy in the sphere of education is the development of lifelong learning, which includes various forms of education throughout the life of a person. In the Federal Program of education development for 2016-2020 years, it is noted that due to the increase in the requirements for teaching staff in connection with the adoption of the professional standard of the teacher, a fundamental change in the socio-cultural educational environment, the need for highly qualified teaching staff capable of effectively solving the problems of education modernization at all levels gets stronger (Ministry of Education and Science of the Russian Federation, 2015). The government has set a difficult task to design a new image of the integrated education system as a branch of the national economy, creating favorable conditions for both personal and professional development of employees. In June 2015, the Ministry of Education and Science of the Russian Federation presented for discussion the Concept of lifelong learning development of adults in the Russian Federation for the period up to 2025. Introduction McConnell and Brue (1992) highlighted the general characteristics of all types of non-formal education: - continues throughout life; - focuses on active participation in the training of students; - emphasizes the importance of the problems and needs of people as the fundamental points for the organization of training; - carried out within certain communities (professional communities); - uses the methods and contexts of non-formal education, the impact of direct life experience - the absence of coercive based on other people's motivation; - high personal meaning of learning; - high personal meaning of learning; - internal responsibility of students for the result of educational activities; - flexibility in organization and methods of teaching; - high level of activity of students; - self-assessment of students' results on the basis of their relevant criteria. The increasing role of the teacher in the modernization of education has led to the fact that in developed European countries the concept of “lifelong professional development of teachers” appeared. M. Fullan, a researcher of the effects of school reforms carried out in the United States and Canada at the end of the twentieth century, believes that the professional development of teachers – a set of formal and non-formal education throughout the career (Fullan, 2010). Based on the design of the educational program, it is possible to trace the difference between formal and non-formal education. In this sense, formal education can be seen as “top-down” education. Accordingly, almost all teacher-training programmers fall into this category. In turn, non-formal education is organized exclusively on request and for the benefit of students, and students themselves participate in the planning of programs, i.e. such education is carried out, largely, “from the bottom to up”. Lifelong learning in the normative acts of the Russian Federation The concept of “lifelong learning” appeared in the USSR in 1986 in the Decision of the CPSU Central Committee and the USSR Council of Ministers “On measures to improve the quality of training and use of Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 60 specialists with higher education in the national economy”. qualified personnel. The answer to the ever-increasing need for updating knowledge and skills (competencies) is the active introduction of modern technologies into the lifelong learning process, including e-learning, open education, remote technologies. The Concept of development of lifelong learning in the Russian Federation for the period up to 2025 is based on the following principles: The Concept of development of lifelong learning in the Russian Federation for the period up to 2025 is based on the following principles: - the right of every citizen to lifelong learning; esponsibility of the citizen for his / her own professional growth and personal development; - individual character of educational trajectories and variability of forms and methods of education; - internationalization of adult lifelong learning; - information openness of the sphere of lifelong learning. The main directions of the Concept implementation are: 1. Ensuring the implementation of individual educational trajectories (routes). 2. Development of infrastructure and mechanisms for independent assessment and recognition of non- formal learning qualifications and outcomes. 3. Creation of conditions for on-the-job training, formation of corporate training systems 4. Development and updating of educational programs in accordance with the requirements of professional standards, taking into account the best national and international experience. 5. Formation of the system of quality assurance of lifelong learning for adults. 6. Development of personnel potential of the system of lifelong learning for adults. Educational organizations and organizations engaged in educational activities, implementing basic and additional educational programs develop programs for the development of lifelong learning, taking into account the principles of this Concept. As a result, of the Concept implementation the following effects will be provided: - strengthening of social stability of Russian society through the development of mechanisms for recognition of qualifications and results of non-formal education, socialization and integration of citizens; - improving the competitiveness of the teaching staff of educational organizations through the formation of professional competencies to meet the requirements of the modern labor market; - ensuring high quality and constant updating and variability of programs of continuous education through the creation of a competitive environment; - attracting qualified personnel, combination of state control and independent quality assessment tools, self-regulation of lifelong learning; - development of effective mechanisms to stimulate and support the lifelong professional development of teaching and management personnel in the field of lifelong learning for adults (Ministry of Education and Science of the Russian Federation, 2015). Introduction The Concept considers the priorities of state policy, principles and content that provide conditions for the realization of the right of the adult population to receive education throughout life. Adult education is offered in the following areas: - formal education (development of educational programs in organizations engaged in educational activities); - non-formal education (training outside organizations engaged in educational activities, at the place of work (in the form of mentoring, training, coaching, training, exchange of experience, etc.), education in public and socially oriented organizations); - information (spontaneous education through the organization of individual cognitive activity or self- education) (Ministry of Education and Science, 2015). It should be noted that the role of “corporate education” institutions is currently being strengthened through the activities of personnel services of organizations that organize on-the-job training with the help of tutors, mentors, instructors, organize professional conferences and seminars to exchange experience. In general, their activities are aimed at the implementation of comprehensive training programs for highly Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 61 qualified personnel. qualified personnel. qualified personnel. Models and strategies of lifelong professional education of teachers Dynamic changes in all spheres of life and numerous educational reforms introduced in European countries and the USA at the end of the 20th century have led to the appearance of a new model of professional development in education called ‘post-technocratic’. It is an alternative of the existing model of discrete formal education or advanced training. The post-technocratic model includes non-formal and informal education and provides continuous professional development of teachers. The model implies: - constant professional educational needs; - including ability of professional development in plans of developing educational institutions; 62 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 - combining needs for personal development with needs of educational institution; - regular monitoring and assessment of professional competences of teachers and heads of educational institutions (Chechel, 2014). - regular monitoring and assessment of professional competences of teachers and heads of educational institutions (Chechel, 2014). The teacher’s work in terms of reformation and constant modernization of the educational system leads to the increase of tension and shortage of competences. Consequently, there is need for organizing proper education in order to develop necessary knowledge and skills. P. Jackson called it the ‘model of deficit’. Its point is that teachers are to be provided with knowledge and skills that are missing at the moment, but become essential in specific educational conditions. This model has long remained dominant in many countries (including Russia) which were members of the Organization of Economic Cooperation and Development (OECD). The author of the model assumed that the teacher’s professionalism increases in a discreet way – from one deficit to another, from one level of education to the next one, from one advanced training course to another (The World Bank, 2003). This thesis is connected to such usual formal practice of teachers’ work as the demand to take advanced training course at least once in three years. Although everyone understands that daily teacher’s experience and abilities to master new forms of professional activities are the main components of the teacher’s professionalism, this approach is seldom used in the practice of teachers’ advanced training. A wider application in the sphere of professional education and advanced teachers’ training has been given to the idea of ‘mentoring’ – consulting by more experienced colleagues or experts during the working process. qualified personnel. This idea has features of traditional tutoring, but it means structural and informative collaboration in order to develop professionalism. The accent is put on additional training of specialists according to the specific character of their activity and without interfering with their work. The idea also implies further constant access to informational resources of continuous professional development including: - programs of preparation and discussing the needs of young teachers who have Bachelor’s degree to continue their education on the next level; - creating conditions for continuous professional development and further statement of acquired competences during non-formal and informal education; - availability of equal opportunities of continuous professional development of teachers from all regions (Gracheva, 2012). There are also models of lineal, parallel and simultaneous inclusion of non-formal education into formal one. These models are realized through such educational technologies as online discussions, webinars, net interaction, scientifically methodological and practice-oriented seminars, attendance of authorities and educational institutions, retraining, conferences, corporative training in the form of tutoring, internship, experience exchange, etc. (Korshunov & Gaponova, 2017). In England there are so called ‘lighthouse’ schools which have been created to spread advanced experience in the fields of methodology, technologies, pedagogy and psychology. Fixed competences preceding professional pedagogical career are replaced with teachers’ immersion into the area of constantly changing competences (Dachkovskaya, 2001). Non-formal and informal education is often realized through professional associations of teachers. The main task of such societies is the exchange of practical experience gained during professional educational activity. Fullan calls this process ‘interactive professional development’ singling it out as a key source of realizing educational reforms (Fullan, 1991). Network interaction provides with such advantages as constant professional development of teachers, decrease of isolation, finding of collective solutions of Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 63 common professional problems and experience exchange. An association is a platform for informal communication which is created for meetings of professionals, connection with international professional societies, discussion of project ideas, assessment of their results and spread of experience and results both inside and outside the region. The infrastructure of such networks is worked out by the virtual variant of a methodological study, which has become the meeting point of project groups, informal communication of teachers, storage of methodological works of teachers and a reading hall at the same time. qualified personnel. This informative resource centre is aimed at creating conditions for non-formal and informal education of future teachers. The symbiosis of formal (getting education, advanced training courses), non-formal (societies, associations) and informal (informal professional communication) sides holds wide educational opportunities (Tyunnikov & Maznichenko, 2014). Thus, the structure of professional development contains the networks of formal, non-formal and informal education represented in the form of Euler diagram. Education is realized in terms of combining formal (lectures, presentations, texts) and informal communication which is followed by discussions, reflection and experimental part. Unfortunately, it must be stated that in Russia not a great number of teachers are members of professional societies. However, a significant amount of useful information motivates local subject methodological communities to form teachers’ associations. This practice in Russia does not have either normative base or traditions. In terms of modernization of higher education there is a widespread practice of teaching at schools when students and young teachers observe, assist and teach under the guidance of senior mentor. The problem is that the mechanism of mentoring isn’t worked out properly and only a few teachers are ready to have students during their practice period. However, in several regions there are networks of schools of teachers’ professional development, which provide interschool advanced training courses for teachers. One of the innovations of education is group work of students and teachers. Classes in pedagogical colleges, universities and advanced training courses often contain collective (group) forms of work. In modern conditions the accent is made on projecting and realizing of professional development not only of an individual teacher, but the whole group including teachers of a difficult form, the same school or those working on a specific problem. It is necessary to state that the change of professional education strategies and development is influenced by informational and communicative technologies. They add such innovative elements as new network services and extra informational resources to professional education and advanced training of teachers. During the development of informational communicative technologies the system of education has absorbed them in full rate. For example, network libraries formed outside education are used actively by students and teachers. Recently a wide spread in the educational process have been received by interactive technologies which are an alternative of traditional pedagogical interaction. (Burlakova, 2014). The leading role of the teacher in interactive methods is a facilitator, a helper in organizing mental activity, creativity and reflexive activity of students. The activeness of students is gained by combining common educative forms of pedagogical interaction: frontal, group, pair work and individual. Practically each interactive method implies interconnection and combination of all these forms (Romashina & Mayer, 2011). It is essential to cover the experience of European countries while reforming school inspections into agencies of professional development. In fact, inspection gradually turns into a kind of consulting which in its turn is closely connected to the teachers’ professional development. There is an interesting example of Scottish inspectional agency, which declares not to assess schools, but to teach them self-assessment. Modern strategies of teachers’ professional development like traditional approaches face the problem of assessing the results of professional education. In the past the dominant role was given to fixating the fact of education itself when students or trainees got certificates of finishing the advanced training courses or took exams. It was considered that this way could show the degree of acquiring knowledge and skills in the context of the course taken, but not in the real educational environment. Such assessments lead to formalization of curriculums. Nowadays the solution of this problem is realized in two directions: on the one hand, by improving the ways of laboratory certification and testing of teachers who are not linked to specific courses, but they put certain demands to those who project the courses. During such examination procedures an attempt is made to imitate real contexts of teacher’s actions. On the other hand, there has been a wide spread of authentic assessment, which implies teachers’ observing how students solves tasks in real situations. An instrument of the authentic assessment can also be realized in the form of teacher’s portfolio, which presents data of their pedagogical activity results. (Tyunnikov & Maznichenko, 2014). In general, it can be stated that new strategies of teachers’ professional development rely on non-formal mechanisms of giving knowledge and forming competences. qualified personnel. The priority of interactive pedagogical process is given to such characteristics as procedural forms, activity, communication, dialogue, opportunity of self- expression, creativity, reflection, etc. Traditional pedagogical impact as an attribute of the authoritarian pedagogical process is aimed, first of all, at formal implementation of compulsory curriculum. During the realization of interactive methods it is learning, not teaching dominates. The teacher in this method is not a translator of readymade information, but they organize autonomous activity of students directed at producing knowledge about the reality, they encourage the search, research of phenomena and processes and autonomous solution of problems. A teacher is merely one of the sources of information. Their function is to create conditions for students’ activeness, initiative in activities, primarily the cognitive one 64 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 (Burlakova, 2014). Issue study In the process of research the following methods were used: theoretical (analysis; synthesis; concretization; generalization); diagnostic (questioning; interviewing); empirical (study of the experience of educational organizations, normative and educational documentation; pedagogical observation). The subject of this research is revealing students’ needs for continuing their professional education. The concept of lifelong learning is based not on the offers from the system of professional education, but on the demands of the labour market. Thus, it is crucial to state and analyze the needs of educational institutions and their employees for the programs of further education and development. Some researchers from the Institute of Foreign Languages of Moscow City Teachers’ Training University have conducted a research to reveal professional difficulties and needs of teachers. The research was aimed at analyzing and preventing difficulties of future teachers in terms of working out various modules of Master programs. During the research in 2017-2018 the following subjective professional difficulties were revealed: - creation of curriculums connected to forming students’ universal educational skills; - use of new pedagogical technologies; - methodology of using ICT in educational process; - creation of upbringing and socialization programs for students; 65 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 - forming students’ interdisciplinary skills; - working with talented students; - criterial assessment of students’ educational achievements; - organization of extra-curricular classes; - working with poor students; - planning and predicting final results of education; - motivating students to master a subject and prepare successfully for the state exams. Objective difficulties: - insufficient technical equipment of educational process, lack of computers, activity books, smart boards; - difficulties in creating of school development program, the basic educational program of the educational organization and subject programs; - not all teachers understand the ideology of new time and use old-fashioned soviet metho - incompetence in organizing lessons with more than 35 students. - incompetence in organizing lessons with more than 35 students. - unwillingness and inability to work with students with special physical and mental deve - unwillingness and inability to work with students with special physical and mental development; During the research there has been singled out a wide range of teachers’ needs which can be divided into basic and specific. During the research there has been singled out a wide range of teachers’ needs which can be divided nto basic and specific. Issue study Basic needs include: Basic needs include: - enhancing the level of professional competence; - effective use of modern interactive pedagogical technologies; - mastering methodologies of organizing students’ project and research activities; - studying methods of alternative education; - knowledge of peculiarities of group work technology; - studying personality-oriented and operational approaches and their possible use in educational process; - learning about methodology of giving integrative and binary lessons; - use of different forms of lessons, methods, techniques and means of education; - ability to form students’ motivation in mastering a subject; - getting information about projecting modern lessons; - organizing extra-curricular classes; - organizing pay educational services in order to raise non-budget resources for the educational institution. Specific needs of teachers of different subjects are the following: Specific needs of teachers of different subjects are the following: - knowledge of monitoring students’ skills in various lessons; - mastering technology based on volunteering (equal educates equal); enhancing professional competence; - mastering technology based on volunteering (equal educates equal); - enhancing professional competence; - getting information about projecting modern lessons; - understanding the structure of school reforms; - ability to hold double lessons, massive events in various types of school curriculum Analysis of teacher’s needs for continuing their professional education of optional education shows the contradiction between the things teachers know and can do and those they have to know and be able to do. Continuing professional education on the next level or optional professional education is not to be regarded as correcting mistakes. Education implies revealing and satisfying the needs for forming and developing Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 66 new competences essential to conduct professional functions on a qualitatively new level. Results The result of analyzing documents (expert conclusion application) presented for attestation shows that teachers of schools, optional education and pre-school educational institutions, etc. participate in non- formal advanced training by doing the work stated. The teacher’s portfolio includes preparation and giving a report at a pedagogical council, seminar or conference, work and presentations at school methodological association, «open» classes or being a jury in contests, etc. If we compare the number of teachers finishing advanced training in formal and non-formal conditions (according to the survey of teachers of Moscow and Moscow Region) we can see that non-formal education prevails. It proves the necessity of posing the status of non-formal education as a constituent part of teachers’ professional development. Discussions Today it is necessary to create a network of organizations that will be able to carry out quality educational activities. It should be borne in mind that education is a licensed activity. Licensing, in turn, involves the presence of an educational organization of a certain regulatory material base that meets the requirements for educational activities, and human resources that can carry out this activity. This ability must be confirmed by the relevant documents recognized by the licensing authorities. It follows that in the system of continuing education there should be enough organizations to carry out educational activities on the required scale: - educational institutions should have a sufficient number of places for those wishing to take a course of study; - educational institutions should have a sufficient number of places for those wishing to take a course of study; - educational institutions should have a sufficient number of qualified teachers and other specialists to train everyone in accordance with their needs; - educational institutions should have a sufficient number of qualified teachers and other specialists to train everyone in accordance with their needs; ain everyone in accordance with their needs; - availability of variable programs, teaching materials, textbooks, teaching AIDS and other resources necessary for the implementation of educational activities. - availability of variable programs, teaching materials, textbooks, teaching AIDS and other resources necessary for the implementation of educational activities. Conclusion Thus, we came to the conclusion that it is necessary to integrate formal and informal professional and additional education in the process of continuous professional development of teachers. This should be done at the legislative level of the Ministry of Education and Science of the Russian Federation in order to take into account these forms of education in the certification of teachers, which does not always happen on the ground. Continuing education should be both formal and non-formal. The latter is a qualitatively new phenomenon in social and educational practice, the content of which is based on its own principles, performs certain functions, solving many old problems in a new way. Modern strategies and models considered in the article are under the significant influence of information and communication technologies and are based on informal mechanisms of knowledge transfer and competence formation. Continuing education of teachers will overcome difficulties and meet the needs for the formation and development of new professional competencies. References Burlakova, I. I. (2014). Use of interactive technologies in the system of professional training of students. Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 67 Scientific notes of Orlov State University, 5, 335-339. Chechel, I. D. (2014). Post-technocratic model of continuous professional development of heads of educational institutions. Standards and Monitoring in Education, 2, 47-56. Commission of the European Communities. (2000). A Memorandum on Lifelong Learning. Retrieved from https://www.hsu-hh.de/wb/wp-content/uploads/sites/647/2017/10/Anthology- Memorandum_on_Lifelong_Learning-EU-2000.pdf Commission of the European Communities. (2000). A Memorandum on Lifelong Learning. Retrieved from https://www.hsu-hh.de/wb/wp-content/uploads/sites/647/2017/10/Anthology- Dashkovskaya, O. (2001). Lessons of a successful reform. Journal of School Management, 12. Retrieved from http://upr.1september.ru/article.php?ID=200101201 Fullan, M. (2010). All systems go. Thousand Oaks, CA: Corwin Press. Fullan, M. (2010). All systems go. Thousand Oaks, CA: Corwin Press. Gracheva, E. Y. (2012). Peculiarities of mentorship of a new teacher in a German school. The Emissia. Offline Letters, 2. Retrieved from http://www.emissia.org/offline/2012/1747.htm International Labour Organization. (2009). International standard classification of occupations. Retrieved from http://www.ilo.org/public/english/bureau/stat/isco/index.htm Klimov, S. M. (1998) Informal education: problems of economics and management. St. Petersburg: Znanie. Kok, W. (2004). Facing the Challenge: The Lisbon Strategy for Growth & Employment. Retrieved from http://ec.europa.eu/research/evaluations/pdf/archive/fp6-evidence- base/evaluation_studies_and_reports/evaluation_studies_and_reports_2004/the_lisbon_strategy_f or_growth_and_employment__report_from_the_high_level_group.pdf Korshunov, I. A., & Gaponova, O. S. (2017). Continuous education of adults in the context of economical development and quality of state government. Educational Studies Moscow, 4, 36-55. McConnell, K. R., & Brue, S. L. (1992). Economics: principles, problems and policy. Moscow: Respublika. Ministry of Education and Science of Russian Federation. (2012). Federal Law on education in the Russian Federation. Retrieved from https://xn--80abucjiibhv9a.xn-- p1ai/%D0%B4%D0%BE%D0%BA%D1%83%D0%BC%D0%B5%D0%BD%D1%82%D1%8B/ 2974 Ministry of Education and Science of Russian Federation. (2015). Concept of development of continuous education of adults in the Russian Federation in the period until 2025. Retrieved from http://ipk.bspu.ru/sites/default/files/koncepciya_razvitiya_nepreryvnogo_obrazovaniya_vzroslyh.d ocx.pdf Ministry of Education and Science of Russian Federation. (2015). On the federal targeted programme of developing education during 2016-2020. Retrieved from https://xn--80abucjiibhv9a.xn- p1ai/%D0%B4%D0%BE%D0%BA%D1%83%D0%BC%D0%B5%D0%BD%D1%82%D1%8B/ 5930/%D1%84%D0%B0%D0%B9%D0%BB/4787/FCPRO_na_2016-2020_gody.pdf Romashina, S. Y., & Mayer, A. A. (2011). Facilitative pedagogy. Barnaul: Azbuka. The World Bank. (2003). Lifelong Learning in the Global Knowledge Economy: Challenges for Developing Countries. A World Bank Report. Retrieved from http://documents.worldbank.org/curated/en/528131468749957131/pdf/multi0page.pdf Tyunnikov, Y. S., & Maznichenko, M. A. (2014). Development of the system of optional professional education: modern challenges, theory and practice. Moscow: Flinta. Nauka.
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https://www.frontiersin.org/articles/10.3389/fimmu.2023.1139980/pdf
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Neutralizing activity against Omicron BA.5 after tixagevimab/cilgavimab administration comparable to those after Omicron BA.1/BA.2 breakthrough infections
Frontiers in immunology
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TYPE Original Research PUBLISHED 02 March 2023 DOI 10.3389/fimmu.2023.1139980 OPEN ACCESS OPEN ACCESS EDITED BY Greta Forlani, University of Insubria, Italy REVIEWED BY Luuk Hilbrands, Radboud University, Netherlands Daniela Dalla Gasperina, University of Insubria, Italy *CORRESPONDENCE Yoo-kyung Lee leeykyoung@korea.kr Jae-Hoon Ko jaehoon.ko@gmail.com †These authors have contributed equally to this work and share first authorship ‡These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology RECEIVED 10 January 2023 ACCEPTED 20 February 2023 PUBLISHED 02 March 2023 CITATION Yang J, Won G, Baek JY, Lee YH, Kim H, Huh K, Cho SY, Kang C-I, Chung DR, Peck KR, Lee KW, Park JB, Yoon SE, Kim SJ, Kim WS, Yim MS, Kim K, Hyeon S, Kim BC, Lee Y-k and Ko J-H (2023) Neutralizing activity against Omicron BA.5 after tixagevimab/cilgavimab administration comparable to those after Omicron BA.1/ BA.2 breakthrough infections. Front. Immunol. 14:1139980. OPEN ACCESS doi: 10.3389/fimmu.2023.1139980 COPYRIGHT OPEN ACCESS EDITED BY Greta Forlani, University of Insubria, Italy REVIEWED BY Luuk Hilbrands, Radboud University, Netherlands Daniela Dalla Gasperina, University of Insubria, Italy *CORRESPONDENCE Yoo-kyung Lee leeykyoung@korea.kr Jae-Hoon Ko jaehoon.ko@gmail.com †These authors have contributed equally to this work and share first authorship ‡These authors have contributed equally to this work Jinyoung Yang 1†, Gunho Won 2†, Jin Yang Baek 1,3†, Young Ho Lee 1, Haein Kim 1, Kyungmin Huh 1, Sun Young Cho 1, Cheol-In Kang 1, Doo Ryeon Chung 1, Kyong Ran Peck 1, Kyo Won Lee 4, Jae Berm Park 4, Sang Eun Yoon 5, Seok Jin Kim 5, Won Seog Kim 5, Min Su Yim 2, Kwangwook Kim 2, Seokhwan Hyeon 2, Byung Chul Kim 2, Yoo-kyung Lee 2*‡ and Jae-Hoon Ko 1*‡ 1Division of Infectious Diseases, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea, 2Division of Vaccine Development Coordination, Center for Vaccine Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju, Republic of Korea, 3Asia Pacific Foundation for Infectious Diseases (APFID), Seoul, Republic of Korea, 4Division of Transplantation Surgery, Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea, 5Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea CITATION Yang J, Won G, Baek JY, Lee YH, Kim H, Huh K, Cho SY, Kang C-I, Chung DR, Peck KR, Lee KW, Park JB, Yoon SE, Kim SJ, Kim WS, Yim MS, Kim K, Hyeon S, Kim BC, Lee Y-k and Ko J-H (2023) Neutralizing activity against Omicron BA.5 after tixagevimab/cilgavimab administration comparable to those after Omicron BA.1/ BA.2 breakthrough infections. Front. Immunol. 14:1139980. doi: 10.3389/fimmu.2023.1139980 Introduction: The effect of tixagevimab/cilgavimab (Evusheld™; AstraZeneca, UK) should be evaluated in the context of concurrent outbreak situations. Methods: For serologic investigation of tixagevimab/cilgavimab during the BA.5 outbreak period, sera of immunocompromised (IC) hosts sampled before and one month after tixagevimab/cilgavimab administration and those of healthcare workers (HCWs) sampled one month after a 3rd shot of COVID-19 vaccines, five months after BA.1/BA.2 breakthrough infection (BI), and one month after BA.5 BI were investigated. Semi-quantitative anti-spike protein antibody (Sab) test and plaque reduction neutralizing test (PRNT) against BA.5 were performed. Domestic outbreak situations and healthy adult sera for immunity level in population Passive immunization using convalescent plasma or monoclonal antibodies has been widely studied since the coronavirus disease 2019 (COVID-19) pandemic began, but the narrow administration time window (effective only at the early course of illness), short half-life, and rapidly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants limited its clinical use (1–3). Tixagevimab/cilgavimab (Evusheld™; AstraZeneca, Cambridge, England, UK) was designed as a combination of two monoclonal antibodies that simultaneously bind to distinct non-overlapping epitopes of the viral spike protein and therefore exhibited broad coverage to variants (4–7). In addition, by reengineering Fc variant amino acids, the half-life of tixagevimab/cilgavimab was extended to approximately 90 days and the effectiveness in both pre-exposure prophylaxis and early outpatient treatment was demonstrated (5–7). Although the Omicron (B.1.1.529) variant and its subvariants, which contain numerous mutations at the spike protein regions, dominated the COVID-19 global outbreak in 2022, in vitro studies have demonstrated that tixagevimab/cilgavimab retains decreased, but still active neutralizing activity against the Omicron subvariants including BA.1, BA.2, BA.4, and BA.5 (4, 8–10). In South Korea, tixagevimab/cilgavimab has been prescribed for immunocompromised (IC) hosts who may not obtain sufficient vaccine-induced immunity from August 2022, when BA.5 became a dominant strain after a large outbreak wave of BA.1/BA.2. To estimate the clinical significance of tixagevimab/cilgavimab administration in the context of concurrent outbreak situations, we investigated serologic responses induced by tixagevimab/ cilgavimab administration in IC hosts in comparison with those obtained from three-dose vaccinations, past infections from BA.1/ BA.2, and recent infections from BA.5 among healthy adults. Information about domestic outbreak situation including the proportion of circulating SARS-CoV-2 variants and number of confirmed cases was collected from the weekly report of the Korea Disease Control and Prevention Agency (11). When a variant occupied more than half of the domestic strains, the variant was defined as a dominant strain. Accordingly, the BA.1/BA.2 outbreak period was corresponding to week 3 (January 2022) to week 29 (July 2022) (Figure 1). The transition from BA.1 to BA.2 occurred at week 12 (March 2022), but it was considered as a single BA.1/BA.2 outbreak period as the outbreak continued as one large wave (the 5th domestic outbreak wave). BA.5 dominance was observed from week 30 (July 2022) and continued with two distinct outbreak waves (the 6th and 7th waves). During the 7th wave, increase of the BA.2.75 sublineage (mainly BN.1) was observed from week 44 (November 2022). COPYRIGHT © 2023 Y The ND50 of one month after BA.5 BI (ND50 1,272.5) was highest among tested groups, but statistical difference was not noticed with tixagevimab/ cilgavimab 600 mg. Conclusion: Tixagevimab/cilgavimab provided a comparable neutralizing activity against the BA.5 with a healthy adult population who were vaccinated with a 3rd shot and experienced BA.1/BA.2 BI. KEYWORDS COVID-19, SARS-CoV-2, Evusheld, neutralizing antibody, BA.5 variant COVID-19, SARS-CoV-2, Evusheld, neutralizing antibody, BA.5 variant COPYRIGHT © 2023 Y COPYRIGHT © 2023 Yang, Won, Baek, Lee, Kim, Huh, Cho, Kang, Chung, Peck, Lee, Park, Yoon, Kim, Kim, Yim, Kim, Hyeon, Kim, Lee and Ko. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: A total of 19 IC hosts (five received tixagevimab/cilgavimab 300 mg and 14 received 600 mg) and 41 HCWs (21 experienced BA.1/BA.2 BI and 20 experienced BA.5 BI) were evaluated. Baseline characteristics did not differ significantly between IC hosts and HCWs except for age and hypertension. Sab significantly increased after tixagevimab/cilgavimab administration (median 130.2 BAU/mL before tixagevimab/cilgavimab, 5,665.8 BAU/mL after 300 mg, and 10,217 BAU/mL after 600 mg; both P < 0.001). Sab of one month after the 3rd shot (12,144.2 BAU/mL) or five months after BA.1/BA.2 BI (10,455.8 BAU/mL) were comparable with that of tixagevimab/cilgavimab 600 mg, while Sab of one Frontiers in Immunology 01 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 month after BA.5 BI were significantly higher (22,216.0 BAU/mL; P < 0.001). BA.5 PRNT ND50 significantly increased after tixagevimab/cilgavimab administration (median ND50 29.6 before tixagevimab/cilgavimab, 170.8 after 300 mg, and 298.5 after 600 mg; both P < 0.001). The ND50 after tixagevimab/cilgavimab 600 mg was comparable to those of five months after BA.1 BI (ND50 200.9) while ND50 of one month after the 3rd shot was significantly lower (ND50 107.6; P = 0.019). The ND50 of one month after BA.5 BI (ND50 1,272.5) was highest among tested groups, but statistical difference was not noticed with tixagevimab/ cilgavimab 600 mg. month after BA.5 BI were significantly higher (22,216.0 BAU/mL; P < 0.001). BA.5 PRNT ND50 significantly increased after tixagevimab/cilgavimab administration (median ND50 29.6 before tixagevimab/cilgavimab, 170.8 after 300 mg, and 298.5 after 600 mg; both P < 0.001). The ND50 after tixagevimab/cilgavimab 600 mg was comparable to those of five months after BA.1 BI (ND50 200.9) while ND50 of one month after the 3rd shot was significantly lower (ND50 107.6; P = 0.019). Domestic outbreak situations and healthy adult sera for immunity level in population BA.5 sublineages such as BQ.1, BQ.1.1, and BF.7 were detected from week 41 (November 2022), but did not exceed 5% by November 2022. For the evaluation of immunity level in healthy adult population when tixagevimab/cilgavimab was introduced in Korea, sera obtained from a heterologous booster shot cohort of healthcare workers (HCWs) were utilized (12). HCWs in this booster shot cohort received a 3rd dose of mRNA COVID-19 vaccine (BNT162b2; Comirnaty®, Pfizer, NY, USA) after two standard doses of adenovirus vector (Adv) vaccines (ChAdOx1; Vaxzevria®, AstraZeneca, Oxford, UK). In the booster shot cohort, 21 HCWs who experienced breakthrough infections (BI) during the BA.1/BA.2 outbreak period were included in the present analysis. Sera sampled one month after the 3rd shot (mid-December Frontiers in Immunology 02 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 FIGURE 1 Schematic flow of sampling points presented with variant proportions and the number of domestic cases of COVID-19 per week in 2022, South Korea *Most of the BA.2.75 sublineages are reported to be BN.1. †Twenty-one HCWs from a pre-existing booster shot cohort and 20 newly- recruited HCWs who experienced BA.5 BI. HCW, healthcare worker; IC, immunocompromised; BI, breakthrough infection; COVID-19, coronavirus disease 2019. Schematic flow of sampling points presented with variant proportions and the number of domestic cases of COVID-19 per week in 2022, South Korea *Most of the BA.2.75 sublineages are reported to be BN.1. †Twenty-one HCWs from a pre-existing booster shot cohort and 20 newly- recruited HCWs who experienced BA.5 BI. HCW, healthcare worker; IC, immunocompromised; BI, breakthrough infection; COVID-19, coronavirus disease 2019. cell transplant or chimeric antigen receptor (CAR)-T cell therapy within six months, 3) those who have received a solid organ transplant within one year, 4) those who have human immunodeficiency virus infection and are not expected to have CD4 T cell ≥200/mm2 with sufficient treatment, and 5) those who have primary immunodeficiency. IC hosts who met the above criteria for tixagevimab/cilgavimab administration and exhibited poor antibody response either to three doses of COVID-19 vaccinations or to SARS-CoV-2 infection were prospectively recruited. Poor antibody response was defined as anti-spike protein antibody (Sab) response < 3,719.0 BAU/mL, which is the lowest level of the booster shot HCW cohort measured one month after the 3rd shot (n = 43, median 12,918.0 BAU/mL, IQR 8,989.0–17,304.0 BAU/mL, and range 3719.0–38,544.0 BAU/mL). The determination to receive 300 mg (one vial) or 600 mg (two vials) of tixagevimab/cilgavimab was solely the patient’s discretion. Domestic outbreak situations and healthy adult sera for immunity level in population Blood sampling was conducted before and one month after tixagevimab/cilgavimab administration. Informed consent was obtained from all the participants and this study was approved by our local institutional review board (SMC 2020-03-113, 2021-01-165, and 2021-11-0506). 2021) and five months after BA.1/BA.2 BI (late July 2022) were used. Follow-up sera from HCWs of the booster shot cohort who did not experience BI were not included in the present comparison, because the antibody titers and neutralizing activities would have waned and would be significantly lower than those of the sera collected one month after the 3rd shot (12). Since a limited number of HCWs in the booster shot cohort experienced BI during the BA.5 outbreak period, 20 HCWs who received three doses of vaccines (adv/adv/mRNA) and experienced BA.5 BI (after week 32 when BA.5 accounted for more than 90% of domestic cases) were additionally recruited. Sampling was performed one month after BA.5 BI (early October 2022). Baseline characteristics of the study population Data on the baseline characteristics of age, sex, height, weight, body mass index (BMI), body surface area (BSA), underlying disease, date and type of COVID-19 vaccination, and history of SARS-CoV-2 infection were collected. The diagnosis of SARS-CoV-2 infection was based on the positive result of either reverse transcription-polymerase chain reaction or antigen testing of respiratory specimens. For the semi- quantitative measurement of Sab, an electrochemiluminescence immunoassay kit (Elecsys Anti-SARS-CoV-2 S, Roche Diagnostics, Basel, Switzerland) was utilized. Ant-SARS-CoV-2 S antibody concentration ≥0.8 U/mL was considered positive, and the linear range was 0.4–250 U/mL (13). Automated dilution was performed for up to a 1:50 dilution in the cobas e analyzer, and an additional manual dilution of up to 1:200 was applied for the saturated specimens. A linear titer-correlation of the kit and plaque reduction neutralization test (PRNT) was presented in previous publications (14). To standardize binding assay results to the binding antibody units (BAU) recommended by the World Health Organization, a correction factor of 0.972 provided by the manufacturer was multiplied to the result (15, 16). To the evaluate neutralizing activity against BA.5, PRNT was conducted for the selected specimens (12, 17, 18). Considering the capacity of biosafety level-3 laboratory and time required for PRNT, 45 specimen were planned for PRNT. As binding assays were tested earlier, specimens with sufficient remaining volume were selected preferentially. SARS-CoV-2 dilutions to 40–50 PFU/well (BA.5 sublineage, Incheon, GRA clade, NCCP No. 43426) were prepared. Vero E6 cells were inoculated with serum and virus mixtures on a 12-well plate and incubated at 37°C, 5% CO2 for one hour. After the inoculums were removed, the cells were overlaid with 1 ml of modified Eagle’s medium (Gibco, Gaithersburg, MD, USA) containing 0.75% agarose and 2% fetal bovine serum (Gibco). The plates were incubated at 37°C with 5% CO2 for two or three days. Stain solution (0.07% crystal violet, 10% formaldehyde, and 5% ethanol) was then added to the cells, and the visualized plaques were counted. The 50% neutralizing dose (ND50) titer was calculated using the Karber formula (19). For the serologic investigation of tixagevimab/cilgavimab administration during the BA.5 outbreak period, a total of 19 IC hosts (five received tixagevimab/cilgavimab 300 mg and 14 received 600 mg) and 41 HCWs (21 who had experienced BA.1/BA.2 BI and 20 who had experienced BA.5 BI) were evaluated (Figure 1). Table 1 shows the baseline characteristics of the patients. IC cohort with poor antibody response The indications for tixagevimab/cilgavimab administration in South Korea were those who have not been confirmed to be SARS-CoV-2 positive within seven days, who are ≥12 years of age, weigh ≥40 kg, and meet one of the following conditions: 1) those receiving immunosuppressive therapy or high-intensity chemotherapy, 2) those who have received hematopoietic stem Frontiers in Immunology frontiersin.org 03 Yang et al. 10.3389/fimmu.2023.1139980 Baseline characteristics of the study population The average age of the IC hosts (53.9 ± 13.8 years) was older than the HCWs with BA.1/BA.2 BI (43.3 ± 9.1 years) or HCWs with BA.5 BI (43.2 ± 10.0 years). The sex distribution, BMI, and BSA were not significantly different between the groups. More patients in the IC hosts exhibited hypertension (36.8%) compared to the HCWs with BA.1/BA.2 BI (4.8%), while other underlying diseases except for the main causes of IC did not differ between the groups. All the HCWs received three doses of COVID-19 vaccine with Adv/Adv/ mRNA vaccine schedules. Among the IC hosts, 13 patients (68.4%) received two or more doses of COVID-19 vaccines (two patients received four doses of vaccines, eight patients received three doses of vaccines, and three patients received two doses of vaccines), while six patients did not receive any vaccinations (31.6%). All non- vaccinated and two-dose-vaccinated IC hosts experienced SARS- CoV-2 infections. Poor antibody responses were determined by the samples taken either one month after three doses of vaccines or at the convalescent status of SARS-CoV-2 infection (< 3,719.0 BAU/mL). Serologic response after tixagevimab/ cilgavimab, three-dose vaccination, and BI Table 2 and Figure 2 present the serologic response after tixagevimab/cilgavimab, in comparison with three-dose vaccination and BI. Sab was measured in all the collected specimens. All the tixagevimab/cilgavimab groups exhibited a statistically significant increase in Sab titers compared to before administration (median 130.2 BAU/mL before tixagevimab/ cilgavimab, 5,665.8 BAU/mL after tixagevimab/cilgavimab 300 mg, 10,217 BAU/mL after tixagevimab/cilgavimab 600 mg, both P < 0.001; Figure 2A), and the titers significantly increased more when 600 mg was administered versus 300 mg (P = 0.002). Compared with tixagevimab/cilgavimab 600 mg administration, there was no statistically significant difference in the Sab titers one month after the 3rd shot (12,144.2 BAU/mL, P = 0.175) or five months after BA.1 BI (10,455.8 BAU/mL, P = 0.778). In contrast, the titers measured one month after BA.5 BI (22,216.0 BAU/mL) were significantly higher than those after tixagevimab/cilgavimab 600 mg administration (P < 0.001). When compared with tixagevimab/cilgavimab 300 mg administration, Sab titers were significantly higher one month after the 3rd shot, five months after BA.1 BI, and one month after BA.5 BI (P = 0.008, P = 0.019, and P < 0.001, respectively). Statistical analysis For comparative analysis of baseline characteristics, analysis of variance (ANOVA) and Tukey’s multiple comparisons test were used for continuous variables, and chi-square or Fisher’s exact tests were used for categorical variables. For the comparison of serologic tests, the Mann-Whitney U test was utilized. In the investigation of correlation between physical measurement values and antibody titers, a linear regression model was used. All P values were two- sided tests, and those < 0.050 were considered statistically significant. GraphPad Prism version 9.4.1 (GraphPad Software, San Diego, CA, USA) were used for all statistical analyses. Frontiers in Immunology 04 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 A total of 45 specimens underwent PRNT against BA.5. Both i i b/ il i b d 6 i d d i i ll cilgavimab 600 mg, both P < 0.001; Figure 2B). A statistically i ifi diff i d b i i b/ TABLE 1 Baseline characteristics of the study population. Frontiers in Immunology Statistical analysis IC hosts with poor antibody response (n = 19) HCWs with BA.1/BA.2 BI (n = 21) HCWs with BA.5 BI (n = 20) P value Demographics Age, year 53.9 ± 13.8 43.3 ± 9.1* 43.2 ± 10.0* 0.003 Male sex 13 (68.4) 9 (42.9) 8 (40.0) 0.149 BMI, kg/m2 23.2 ± 2.9 21.8 ± 2.4 23.6 ± 3.1 0.098 BSA‡, m2 1.5 ± 0.3 1.4 ± 0.3 1.6 ± 0.4 0.361 Underlying disease Hypertension 7 (36.8) 1 (4.8)* 3 (15.0) 0.029 Diabetes mellitus 1 (5.3) 0 (0.0) 0 (0.0) 0.334 Dyslipidemia 1 (5.3) 0 (0.0) 1 (5.0) 0.572 Chronic heart disease 3 (15.8) 0 (0.0) 1 (5.0) 0.127 Thyroid cancer 1 (5.3) 0 (0.0) 1 (5.0) 0.572 Diseases causing IC condition B-cell malignancy 9 (47.4) none none NA Lymphoma 8 (42.1) none none NA ALL 1 (5.3) none none NA Organ transplantation 9 (47.4) none none NA Kidney 8 (42.1) none none NA Heart 1 (5.3) none none NA Good’s syndrome 1 (5.3) none none NA Vaccination status Four doses Adv/Adv/mRNA/mRNA 2 (10.5) 0 (0.0) 0 (0.0) 0.107 Three doses 8 (42.1) 21 (100.0)* 20 (100.0)* 0.020 Adv/Adv/mRNA 1 (5.3) 21 (100.0)* 20 (100.0)* <0.001 Adv/mRNA/mRNA 2 (10.5) 0 (0.0) 0 (0.0) 0.107 mRNA/mRNA/mRNA 5 (26.3) 0 (0.0)* 0 (0.0)* 0.003 Two doses mRNA/mRNA 3 (15.8) 0 (0.0) 0 (0.0) 0.033 None† 6 (31.6) 0 (0.0)* 0 (0.0)* 0.001 SARS-CoV-2 infection 10 (52.6) 21 (100.0)* 20 (100.0)* <0.001 BA.1/BA.2 period 10 (52.6) 21 (100.0)* 0 (0.0)* <0.001 BA.5 period 0 (0.0) 0 (0.0) 20 (100.0)* <0.001 Data are expressed as the number (%) of patients or mean ± standard deviation. *Significantly different in a two-group comparison with IC hosts. †All non-vaccinated hosts experienced SARS- CoV-2 infection and exhibited poor antibody responses after convalescence. ‡BSA was calculated based on the Mosteller method. IC, immunocompromised; HCW, healthcare worker; BI, breakthrough infection; BMI, body mass index; ALL, acute lymphoblastic leukemia; NA, not applicable; Adv, adenovirus vector vaccines; mRNA, messenger ribonucleic acid vaccines; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. TABLE 1 Baseline characteristics of the study population. Data are expressed as the number (%) of patients or mean ± standard deviation. *Significantly different in a two-group comparison with IC hosts. †All non-vaccinated hosts experienced SARS- CoV-2 infection and exhibited poor antibody responses after convalescence. ‡BSA was calculated based on the Mosteller method. Statistical analysis BI, breakthrough infection; Sab, anti-spike protein antibody; BAU, binding antibody unit; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; IC, immunocompromised; tixa./cilga., tixagevimab/cilgavimab; HCW, healthcare worker; ab, antibody; mo, month; IQR, interquartile range. BA.1 BI (ND50 200.9, P = 0.514), while ND50 of one month after the 3rd shot was significantly lower (ND50 107.6, P = 0.019). The ND50 of one month after BA.5 BI (ND50 1,272.5) was highest among tested groups, but statistical difference was not noticed with tixagevimab/cilgavimab 600 mg (P = 0.151). When compared with tixagevimab/cilgavimab 300 mg administration, BA.5 PRNT ND50 titers were not significantly different with those measured one month after the 3rd shot, five months after BA.1 BI, and one month after BA.5 BI (P = 0.222, P = 0.691, P = 0.151, respectively). A A B FIGURE 2 (A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing activity against BA.5, presented as PRNT ND50. Binding and neutralizing antibody responses after tixagevimab/cilgavimab in comparison with those to vaccination and BI *Statistically significant difference was noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./ cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; ns, not significant. A To investigate the effect of physical characteristics on the effect of tixagevimab/cilgavimab, we calculated the correlation of Sab titers measured after the administration of tixagevimab/cilgavimab 600 mg with BMI, BSA, height, and weight (Figure 3). All physical characteristics demonstrated statistically significant reverse correlations with Sab (all P < 0.050), and the highest R2 value was noticed with BSA (R2 = 0.595). The BA.5 PRNT ND50 values also exhibited a reverse trend with physical measurement values, but statistical significances were not observed. B FIGURE 2 (A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing activity against BA.5, presented as PRNT ND50. Binding and neutralizing antibody responses after tixagevimab/cilgavimab in comparison with those to vaccination and BI *Statistically significant difference was noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./ cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; ns, not significant. B FIGURE 2 B Statistical analysis IC, immunocompromised; HCW, healthcare worker; BI, breakthrough infection; BMI, body mass index; ALL, acute lymphoblastic leukemia; NA, not applicable; Adv, adenovirus vector vaccines; mRNA, messenger ribonucleic acid vaccines; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. A total of 45 specimens underwent PRNT against BA.5. Both tixagevimab/cilgavimab 300 mg and 600 mg induced a statistically significant increase in BA.5 PRNT ND50 than before administration (median ND50 29.6 before tixagevimab/cilgavimab, 170.8 after tixagevimab/cilgavimab 300 mg, and 298.5 after tixagevimab/ cilgavimab 600 mg, both P < 0.001; Figure 2B). A statistically significant difference was not noticed between tixagevimab/ cilgavimab 300 mg and 600 mg (P = 0.310). Compared with tixagevimab/cilgavimab 600 mg administration, there was no statistically significant difference in the values five months after Frontiers in Immunology 05 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 ologic responses to tixagevimab/cilgavimab in comparison with those to vaccination and BI. Groups Sampling point Sab, BAU/mL BA.5 PRNT, ND50 IC hosts with poor antibody response (n = 19) Before tixa./cilga. Binding ab test, n = 19 PRNT, n = 15 130.2 (0.4–1,165.4) 29.6 (17.0–41.2) After tixa./cilga. 300 mg Binding ab test, n = 5 PRNT, n = 5 5,665.8 (3,162.9–8,105.0) 170.8 (124.2–379.0) After tixa./cilga. 600 mg Binding ab test, n = 14 PRNT, n = 10 10,216.7 (8,602.2–11,274.2) 298.5 (166.9–540.8) HCWs with BA.1/BA.2 BI (n = 21) 1 mo after 3rd shot Binding ab test, n = 21 PRNT, n = 5 12,144.2 (8,386.9–16,562.9) 107.6 (83.3–202.6) 5 mo after BA.1/BA.2 BI Binding ab test, n = 21 PRNT, n = 5 10,455.8 (7,679.8–19,418.6) 200.9 (118.9–1,144.4) HCWs with BA.5 BI (n = 20) 1 mo after BA.5 BI Binding ab test, n = 20 PRNT, n = 5 22,216.0 (15,272.1–33,148.1) 1,272.5 (415.3–2,448.7) Data are expressed as median (IQR). BI, breakthrough infection; Sab, anti-spike protein antibody; BAU, binding antibody unit; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; IC, immunocompromised; tixa./cilga., tixagevimab/cilgavimab; HCW, healthcare worker; ab, antibody; mo, month; IQR, interquartile range. Data are expressed as median (IQR). BI, breakthrough infection; Sab, anti-spike protein antibody; BAU, binding antibody unit; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; IC, immunocompromised; tixa./cilga., tixagevimab/cilgavimab; HCW, healthcare worker; ab, antibody; mo, month; IQR, interquartile range. Data are expressed as median (IQR). Discussion This result is in line with other study which suggested tixagevimab/cilgavimab maintained the neutralization activity against BA.5 in spite of a 30.7 fold reduction compared to against the ancestral strain (10). To date, several real-world studies suggested that pre-exposure tixagevimab/cilgavimab administration would be effective in IC hosts during the early Omicron period, but these studies did not reflect the emergence and outbreak of the BA.5 subvariant (21, 22). Our serologic investigation supports the continued use of tixagevimab/ cilgavimab during BA.5 predominance, and additional cohort study would be required to evaluate the clinical effects at this period. In another study by Benotmane et al., evaluating serologic effect of tixagevimab/cilgavimab administration among kidney transplant recipients, only 9.5% obtained neutralizing activity against the BA.1 subvariant after the administration of tixagevimab/cilgavimab 300 mg, and there was no neutralizing activity when Sab titers < 2,500 BAU/ml after tixagevimab/cilgavimab administration (23). In the present study, only one patient exhibited low Sab titer of 2145.2 BAU/mL and showed the lowest PRNT titer of ND50 99.4. and higher than those vaccinated with the 3rd dose. Those who received tixagevimab/cilgavimab 300 mg also exhibited comparable neutralizing activity with these comparators. PRNT ND50 of 118.25 has been suggested as the 50% protective neutralizing titer in our previous study (12). Since most patients exhibited BA.5 PRNT ND50 over 118.25 after tixagevimab/cilgavimab administration except for one (ND50 99.4), the protective role of tixagevimab/ cilgavimab is anticipated during the BA.5 dominant outbreak period. This result is in line with other study which suggested tixagevimab/cilgavimab maintained the neutralization activity against BA.5 in spite of a 30.7 fold reduction compared to against the ancestral strain (10). To date, several real-world studies suggested that pre-exposure tixagevimab/cilgavimab administration would be effective in IC hosts during the early Omicron period, but these studies did not reflect the emergence and outbreak of the BA.5 subvariant (21, 22). Our serologic investigation supports the continued use of tixagevimab/ cilgavimab during BA.5 predominance, and additional cohort study would be required to evaluate the clinical effects at this period. Benotmane et al. suggested that low tixagevimab/cilgavimab- induced antibody titers would be associated with high BMI. In the present study, we also investigated the correlation of tixagevimab/cilgavimab-induced antibody titers with physical characteristics and noticed that BSA exhibited the best reverse correlation with antibody titers. Discussion After the emergence of the Omicron variant with high transmissibility, approximately a half of the South Korean population experienced SARS-CoV-2 infection and IC hosts continue to be at risk of exposure to the virus (11, 20). As subvariants of Omicron succeeded the following outbreak waves, it became necessary to investigate the serologic status of the community together when estimating the serologic impact of monoclonal antibody agents to a circulating variant. FIGURE 2 (A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing activity against BA.5, presented as PRNT ND50. Binding and neutralizing antibody responses after tixagevimab/cilgavimab in comparison with those to vaccination and BI *Statistically significant difference was noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./ cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; ns, not significant. GU (A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing activity against BA.5, presented as PRNT ND50. Binding and neutralizing antibody responses after tixagevimab/cilgavimab in comparison with those to vaccination and BI *Statistically significant difference was noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./ cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; ns, not significant. Of note, the administration of tixagevimab/cilgavimab 600 mg exhibited neutralizing activity against the BA.5 subvariant comparable with healthy adults who experienced BA.1/BA.2 BI, Frontiers in Immunology frontiersin.org 06 Yang et al. 10.3389/fimmu.2023.1139980 A B D C FIGURE 3 Correlation of Sab titers with physical measurement values after the administration of tixagevimab/cilgavimab 600 mg. Correlation with (A) BMI, (B) BSA, (C) height, and (D) weight is presented, respectively. Sab, anti-spike protein antibody; BMI, body mass index; BSA, body surface area; BAU, binding antibody unit. B A B C D D C FIGURE 3 Correlation of Sab titers with physical measurement values after the administration of tixagevimab/cilgavimab 600 mg. Correlation with (A) BMI, (B) BSA, (C) height, and (D) weight is presented, respectively. Sab, anti-spike protein antibody; BMI, body mass index; BSA, body surface area; BAU, binding antibody unit. and higher than those vaccinated with the 3rd dose. Those who received tixagevimab/cilgavimab 300 mg also exhibited comparable neutralizing activity with these comparators. PRNT ND50 of 118.25 has been suggested as the 50% protective neutralizing titer in our previous study (12). Since most patients exhibited BA.5 PRNT ND50 over 118.25 after tixagevimab/cilgavimab administration except for one (ND50 99.4), the protective role of tixagevimab/ cilgavimab is anticipated during the BA.5 dominant outbreak period. Ethics statement All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The studies involving human participants were reviewed and approved by the Institutional Review Board of Samsung Medical Center. The patients/participants provided their written informed consent to participate in this study. Discussion This finding may explain the finding of the present analysis that BA.5 PRNT ND50 titers obtained by tixagevimab/cilgavimab 300 mg administration were not significantly different from those after tixagevimab/cilgavimab 600 mg, as patients who received 300 mg had lower average BSA (1.4 ± 0.2 m2) than those who received 600 mg (1.6 ± 0.3 m2). Additional studies about the optimal dosage of monoclonal antibody agents according to body measurements should be conducted. This study has several limitations. First, the present study was conducted with a relatively small cohort, which likely affected statistical significance. However, we tried to enroll a typical population that may represent the immune status of the community at the time period of tixagevimab/cilgavimab administration, and the present study may provide important insight into the serologic implications of tixagevimab/cilgavimab administration during the BA.5 outbreak period. Second, the study focused on serologic analysis and actual preventive effect of tixagevimab/cilgavimab was not evaluated. Since the dominant SARS-CoV-2 variant of each outbreak wave changes rapidly and the evaluation of preventive effect for the changing variant requires a considerable time, it would be important to investigate In another study by Benotmane et al., evaluating serologic effect of tixagevimab/cilgavimab administration among kidney transplant recipients, only 9.5% obtained neutralizing activity against the BA.1 subvariant after the administration of tixagevimab/cilgavimab 300 mg, and there was no neutralizing activity when Sab titers < 2,500 BAU/ml after tixagevimab/cilgavimab administration (23). In the present study, only one patient exhibited low Sab titer of 2145.2 BAU/mL and showed the lowest PRNT titer of ND50 99.4. Frontiers in Immunology frontiersin.org 07 Yang et al. 10.3389/fimmu.2023.1139980 Funding This study was supported by a research program fund (#2021- ER2303-00) from the Korea Disease Control and Prevention Agency and a Samsung Medical Center Grant (#SMO1230121). Author contributions neutralizing activity of a monoclonal antibody agent against the dominant variant in comparison with the immune status of the community. In this context, our serologic study findings supported the administration of tixagevimab/cilgavimab during the BA.5 outbreak period in South Korea. Third, the present study does not provide information about how tixagevimab/cilgavimab would provide preventive effect for severe disease. Unlike active vaccination or natural infection that induce T cell immunity and B cell memory (24, 25), the role of passive immunization in preventing severe disease is still uncertain. To answer this question, clinical cohort study need to be conducted for IC hosts who were infected after tixagevimab/cilgavimab administration in comparison with those infected without tixagevimab/cilgavimab. Finally, since the measurement of antibody titers was only performed one month after tixagevimab/cilgavimab administration and was limited to neutralization of the BA.5 variant, additional studies evaluating waning time points and newly emerging variants such as BA.5 subvariants (e.g. BQ.1, BQ.1.1, or BF.7) and BA.2.75 subvariants (e.g. BN.1) or recombinants (e.g. XBB) are necessary. JY, BK, Y-kL, and J-HK were involved in the design of this study. JY, YL, HK, KH, SC, C-IK, DC, KP, KL, JP, SY, SK, WK, and J-HK enrolled the participants and collected specimens. GW, JB, MY, KK, and SH performed the experiments. JY, GW, JB, and J-HK assembled the data. JY, GW, KP, Y-kL, and J-HK were involved in the writing. All authors contributed to the article and approved the submitted version. Data availability statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors. 8. Dejnirattisai W, Huo J, Zhou D, Zahradnı́k J, Supasa P, Liu C, et al. SARS-CoV-2 omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses. Cell (2022) 185:467–484.e415. doi: 10.1016/j.cell.2021.12.046 Acknowledgments In conclusion, tixagevimab/cilgavimab provided a comparable neutralizing activity against the BA.5 subvariant with those in a healthy adult population who had previous experiences with BA.1/ BA.2 BI after three-dose vaccinations. The authors would like to express their gratitude to all the participants who participated voluntarily in this study. 5. Levin MJ, Ustianowski A, De Wit S, Launay O, Avila M, Templeton A, et al. Intramuscular AZD7442 (Tixagevimab–cilgavimab) for prevention of covid-19. New Engl J Med (2022) 386:2188–200. doi: 10.1056/NEJMoa2116620 7. 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Sci Transl Med (2022) 14:eabl8124. doi: 10.1126/scitranslmed.abl8124 5. Levin MJ, Ustianowski A, De Wit S, Launay O, Avila M, Templeton A, et al. Intramuscular AZD7442 (Tixagevimab–cilgavimab) for prevention of covid-19. New Engl J Med (2022) 386:2188–200. doi: 10.1056/NEJMoa2116620 6. Loo YM, Mctamney PM, Arends RH, Abram ME, Aksyuk AA, Diallo S, et al. The SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman primates and has an extended half-life in humans. Sci Transl Med (2022) 14:eabl8124. doi: 10.1126/scitranslmed.abl8124 7. Montgomery H, Hobbs F, Padilla F, Arbetter D, Templeton A, Seegobin S, et al. Efficacy and safety of intramuscular administration of tixagevimab-cilgavimab for early outpatient treatment of COVID-19 (TACKLE): A phase 3, randomised, double-blind, placebo-controlled trial. Lancet Respir Med (2022) 10:985–96. doi: 10.1016/S2213-2600(22) 00180-1 8. Dejnirattisai W, Huo J, Zhou D, Zahradnı́k J, Supasa P, Liu C, et al. 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J Clin Virol (2021) 139:104820. doi: 10.1016/ j.jcv.2021.104820 25. Yang J, Lee KW, Baek JY, Bae S, Lee YH, Kim H, et al. Augmented humoral and cellular immunity against severe acute respiratory syndrome coronavirus 2 after breakthrough infection in kidney transplant recipients who received 3 doses of coronavirus disease 2019 vaccine. Am J Transplant (2023) S1600-6135(22)30227-7. doi: 10.1016/j.ajt.2022.12.022 09 09 Frontiers in Immunology frontiersin.org
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Individualizing Follow-Up Strategies in High-Grade Soft Tissue Sarcoma with Flexible Parametric Competing Risk Regression Models
Cancers
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Individualizing follow-up strategies in high-grade soft tissue sarcoma with flexible parametric competing risk regression models Smolle, M.A.; Sande, M. van de; Callegaro, D.; Wunder, J.; Hayes, A.; Leitner, L.; ... ; Szkandera, J. Citation Smolle, M. A., Sande, M. van de, Callegaro, D., Wunder, J., Hayes, A., Leitner, L., … Szkandera, J. (2020). Individualizing follow-up strategies in high-grade soft tissue sarcoma with flexible parametric competing risk regression models. Cancers, 12(1). doi:10.3390/cancers12010047 Citation Version: Publisher's Version License: Creative Commons CC BY 4.0 license Downloaded from: https://hdl.handle.net/1887/3182044 License: Note: To cite this publication please use the final published version (if applicable). Note: To cite this publication please use the final published version (if applica cancers cancers Individualizing Follow-Up Strategies in High Soft Tissue Sarcoma with Flexible Parametric Competing Risk Regression Models Haas 16,17,*,‡ and Joanna Szkandera 12,*,‡ 1 Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria 1 Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria 2 Department of Orthopaedic Surgery, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 3 Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy 1 Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria 2 Department of Orthopaedic Surgery, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 3 Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy 4 University Musculoskeletal Oncology Unit, Mount Sinai Hospital, University of Toronto, Toronto, ON M5G IX5, Canada 5 1 Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria 2 Department of Orthopaedic Surgery, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 3 Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy 4 University Musculoskeletal Oncology Unit, Mount Sinai Hospital, University of Toronto, Toronto, ON M5G IX5, Canada 5 f l d l d d 5 Department of Surgery, Royal Marsden Hospital NHS Foundation Trust, London SW3 6JJ, UK 6 6 Sarcoma Centre, HELIOS-Klinikum Berlin-Buch, 13125 Berlin, Germany 7 Institute of Mathematics, Leiden University Medical Centre, 2333 ZA Leiden, T 7 Institute of Mathematics, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 8 M di l St ti ti D t t f Bi di l D t S i L id U i it M di l C t 7 Institute of Mathematics, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 8 Medical Statistics, Department of Biomedical Data Science, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 9 Princess Máxima Center for Pediatric Oncology, Trial and Data Center, 3584 CT Utrecht, The Nethe 9 Princess Máxima Center for Pediatric Oncology, Trial and Data Center, 3584 CT Utrecht, The Netherlands 10 Department of Orthopaedics and Traumatology, Medical University of Vienna, 1090 Vienna, Austria 11 Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands 12 Division of Clinical Oncology, Department of Medicine, Medical University of Graz, 8036 Graz, Austria 13 Institute of Pathology, Medical University of Graz, 8010 Graz, Austria Princess Máxima Center for Pediatric Oncology, Trial and Data Center, 3584 CT Utrecht, The Netherla 10 Department of Orthopaedics and Traumatology, Medical University of Vienna, 1090 Vienna, Austria p p gy y 11 Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands 12 Division of Clinical Oncology Department of Medicine Medical University of Graz 8036 Graz Austria 11 Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands 11 Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands 12 Division of Clinical Oncology, Department of Medicine, Medical University of Graz, 8036 Graz, Austria 13 Institute of Pathology, Medical University of Graz, 8010 Graz, Austria Division of Clinical Oncology, Department of Medicine, Medical University of Graz, 8036 Graz, Austria 13 Institute of Pathology, Medical University of Graz, 8010 Graz, Austria 13 Institute of Pathology, Medical University of Graz, 8010 Graz, Austria 14 Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz, 8036 Graz, Austria 15 Sarcoma Centre, HELIOS Klinikum Bad Saarow, 15526 Bad Saarow, Germany 16 Department of Radiotherapy, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 17 Department of Radiotherapy, The Netherlands Cancer Institute Amsterdam, 1 CX A d Th N h l d 17 Department of Radiotherapy, The Netherlands Cancer Institute Amsterdam, 1066 CX Amsterdam, The Netherlands * Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkandera@medunigraz.at (J.S.); Tel.: +31-20-5122135 (R.L.H.); +43-316-385-13115 (J.S.) * Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkandera@medunigraz.at (J.S.); T l 31 20 5122135 (R L H ) 43 316 385 13115 (J S ) * Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkand Tel.: +31-20-5122135 (R.L.H.); +43-316-385-13115 (J.S.) * Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkandera@medunigraz.at (J.S.); † These authors share the first authorship. Individualizing Follow-Up Strategies in High Soft Tissue Sarcoma with Flexible Parametric Competing Risk Regression Models Maria Anna Smolle 1,† , Michiel van de Sande 2,† , Dario Callegaro 3, Jay Wunder 4, Andrew Hayes 5, Lukas Leitner 1 , Marko Bergovec 1, Per-Ulf Tunn 6, Veroniek van Praag 2, Marta Fiocco 7,8,9, Joannis Panotopoulos 10 , Madeleine Willegger 10, Reinhard Windhager 10, Sander P. D. Dijkstra 2, Winan J. van Houdt 11, Jakob M. Riedl 12 , Michael Stotz 12, Armin Gerger 12, Martin Pichler 12, Herbert Stöger 12, Bernadette Liegl-Atzwanger 13, Josef Smolle 14, Dimosthenis Andreou 15, Andreas Leithner 1, Alessandro Gronchi 3, Rick L. Individualizing Follow-Up Strategies in High Soft Tissue Sarcoma with Flexible Parametric Competing Risk Regression Models ‡ These authors share the last authorship. Received: 1 December 2019; Accepted: 19 December 2019; Published: 21 December 2019 Received: 1 December 2019; Accepted: 19 December 2019; Published: 21 December 2019 Abstract: Currently, patients with extremity soft tissue sarcoma (eSTS) who have undergone curative resection are followed up by a heuristic approach, not covering individual patient risks. The aim of this study was to develop two flexible parametric competing risk regression models (FPCRRMs) for local recurrence (LR) and distant metastasis (DM), aiming at providing guidance on how to individually follow-up patients. Three thousand sixteen patients (1931 test, 1085 validation cohort) with high-grade eSTS were included in this retrospective, multicenter study. Histology (9 categories), grading (time-varying covariate), gender, age, tumor size, margins, (neo)adjuvant radiotherapy (RTX), and neoadjuvant chemotherapy (CTX) were used in the FPCRRMs and performance tested with Harrell-C-index. Median follow-up was 50 months (interquartile range: 23.3–95 months). Two hundred forty-two (12.5%) and 603 (31.2%) of test cohort patients developed LR and DM. Factors Cancers 2020, 12, 47; doi:10.3390/cancers12010047 www.mdpi.com/journal/cancers www.mdpi.com/journal/cancers 2 of 13 Cancers 2020, 12, 47 significantly associated with LR were gender, size, histology, neo- and adjuvant RTX, and margins. Parameters associated with DM were margins, grading, gender, size, histology, and neoadjuvant RTX. C-statistics was computed for internal (C-index for LR: 0.705, for DM: 0.723) and external cohort (C-index for LR: 0.683, for DM: 0.772). Depending on clinical, pathological, and patient-related parameters, LR- and DM-risks vary. With the present model, implemented in the updated Personalised Sarcoma Care (PERSARC)-app, more individualized prediction of LR/DM-risks is made possible. Keywords: soft tissue sarcoma; follow-up; flexible parametric competing risk regression model; local recurrence; distant metastasis Keywords: soft tissue sarcoma; follow-up; flexible parametric competing risk regression model; local recurrence; distant metastasis 1. Introduction Patients with high-grade extremity soft tissue sarcoma (eSTS) are at risk of developing local recurrences (LR) and even more so of developing distant metastases (DM) after having undergone surgical resection of the primary tumor [1–5]. These rates differ substantially per size, grade, and subtype [6]. Close follow-up regimens are currently used in order to detect LR and DM at stages where they are still potentially treatable by re-resection or metastasectomy, respectively [7]. There is no clear consensus when, by what means, and how often to perform follow-up in eSTS patients, with many centers and guidelines having introduced a heuristic approach: for the first 3 years after surgery, patients would be checked three or four times a year, then biannually for the following two years and annually thereafter [8,9]. Imposing all eSTS patients on these strict follow-up regimens has raised public, scientific, and health economic concerns over the last years. Numerous factors interact with the risk of developing LR or DM, such as histological STS-subtypes, surgical margins, tumor size, grade, administration of neo(adjuvant) radiotherapy (RTX) or chemotherapy (CTX), and patient-derived factors [1–4,10–12]. Consequently, the current approach of “one-size-fits-all” may not account for the unequal risk of recurrence in the heterogeneous eSTS population, involving an excessive number of surveillance imaging, possibly leading to unnecessary delivery of imaging-induced radiation exposure, and the inherent burden for radiology departments, as well as inappropriately refraining from it, a high number of outpatient visits and financial costs and emotional stress for each individual patient [13]. However, an evaluation of prognostic factors for LR and DM taking into consideration the time-varying rate for the occurrence of events in a multicenter cohort, including important patient-(i.e., age, gender), tumor-(e.g., size, grade, histological subtype), and treatment-related features (e.g., margins, (neo)adjuvant CTX/RTX), is currently missing. Therefore, the aim of the present study was to estimate and validate two models predicting risks of LR and DM over the first 5 years of follow-up by applying flexible parametric competing risk regression modeling in a large, multicenter cohort of patients with primary localized high-grade eSTS. The results have been implemented into the Personalised Sarcoma Care (PERSARC)-app [14] for Individualized Sarcoma Care and follow-up. 2. Results Patients had undergone surgery with curative intent between January 1994 and October 2014 for the test cohort and between January 2000 and December 2013 for the validation cohort, respectively. There was a slight male predominance (n = 1038; 53.8%) and the median patient age was 59 years (interquartile range (IQR): 44.7–70 years). With 55.8%, 17.9%, and 13.9%, most tumors in the test cohort were located in the thigh (n = 1078), upper arm (n = 346), and lower leg (n = 268), while the lower arm (n = 142), the foot or toes (n = 65), and the hand or fingers (n = 32) were affected in 7.3%, 3.4%, and 1.7%, respectively. Further clinicopathological features for both the test and validation cohort are listed in Table 1. 3 of 13 Cancers 2020, 12, 47 Table 1. Patient characteristics and clinical, pathological, and treatment-related parameters. Table 1. Patient characteristics and clinical, pathological, and treatment-related parameters. Variables Test Cohort (n = 1931) Validation Cohort (n = 1085) N (%) Missing (%) N (%) Missing (%) p-Value * Age (continuous; years; median + IQR) 59 (44.7–70) 45 (2.3) 61 (47–74) 0 (0.0) <0.0001 Gender Male 1038 (53.8) 0 (0.0) 615 (56.7) 0 (0.0) 0.121 Female 893 (46.2) 470 (43.3) Tumor Location Upper Extremity 520 (26.9) 1 (0.05) 312 (28.8) 0 (0.0) 0.285 Lower Extremity 1410 (73.1) 773 (71.2) Depth Epifascial 518 (26.9) 2 (0.1) 291 (26.8) 0 (0.0) 0.984 Subfascial 1411 (73.1) 794 (73.2) Tumor Size (continuous; cm; median + IQR) 7 (4–11) 30 (1.6) 7.5 (5–12) 5 (0.5) 0.026 Histology Myxoid liposarcoma 222 (11.6) 16 (0.8) 111 (10.2) 0 (0.0) <0.0001 MPNST 83 (4.3) 43 (4.0) Myxofibrosarcoma 451(23.6) 104 (9.6) Synovial Sarcoma 174 (9.1) 79 (7.3) UPS 325 (17.0) 375 (34.6) Angiosarcoma/Vascular Sarcoma 22 (1.1) 20 (1.8) Dedifferentiated/Pleomorphic Liposarcoma 141 (7.4) 85 (7.8) Leiomyosarcoma 221 (11.5) 118 (10.9) Others 276 (14.4) 150 (13.8) Grading G2 719 (37.8) 30 (1.6) 382 (35.8) 19 (1.8) 0.282 G3 1182 (62.2) 684 (64.2) Margins R0 1494 (77.4) 0 (0.0) 768 (70.8) 0 (0.0) <0.0001 R1/2 437 (22.6) 317 (29.2) CTX No 1408 (73.0) 1 (0. 2. Results 05) 1039 (95.8) 0 (0.0) <0.0001 Neoadjuvant 262 (13.6) 40 (3.7) Adjuvant 209 (10.8) 6 (0.6) Neoadjuvant + Adjuvant 51 (2.6) 0 (0.0) RTX No 619 (32.9) 50 (2.6) 335 (30.9) 0 (0.0) <0.0001 Neoadjuvant 303 (16.1) 460 (42.4) Adjuvant 956 (50.8) 275 (25.4) Neoadjuvant + Adjuvant 3 (0.2) 15 (1.4) Follow-up (continuous; months; median + IQR) 50 (23.3–95) 11 (0.6) 56 (21–91) 0 (0.0) 0.254 * p-values calculated with Wilcoxon rank sum test for continuous variables, with chi2-test for binary and categorical variables. p-values in bold are considered statistically significant. Abbreviations: CTX = chemotherapy. IQR = interquartile range. MPNST = Malignant Peripheral Nerve Sheat Tumor. RTX = radiotherapy. UPS = Undifferentiated pleiomorphic sarcoma. * p-values calculated with Wilcoxon rank sum test for continuous variables, with chi2-test for binary and categorical variables. p-values in bold are considered statistically significant. Abbreviations: CTX = chemotherapy. IQR = interquartile range. MPNST = Malignant Peripheral Nerve Sheat Tumor. RTX = radiotherapy. UPS = Undifferentiated pleiomorphic sarcoma. Five- and 10-year overall survival (OS) was 73.6% (95%CI: 71.3–75.7) and 62.7% (95%CI: 59.8–65.5) in the test cohort. In the validation cohort, 5- and 10-year OS were 64.9% (95%CI: 61.8–67.8) and 52.9% (95%CI: 48.9–56.8), respectively. Gender, tumor size, histological subtype (except for angiosarcoma/vascular sarcoma (p = 0.127) and dedifferentiated/pleomorphic liposarcoma (p = 0.254), margins, neoadjuvant and adjuvant RTX, as well as adjuvant CTX (all p < 0.05) had a significant influence on risk of LR in the stepwise backward selection of the Fine and Gray model. Grading as a time-dependent effect was kept in the model (p = 0.108), while age (p = 0.082) and neoadjuvant CTX (p = 0.214) were excluded. Consequently, gender, grading, tumor size, neoadjuvant and adjuvant RTX, histological subtype, and adjuvant CTX were included in the flexible parametric competing risk regression model (Table 2). The subdistribution hazard and cumulative incidence functions for LR using ten clinical examples are shown in Figure 1A,B (definition of these examples found in Table S1, together with estimated conditional risks of LR). As an example, a male patient with a G2 myxofibrosarcoma sized 10 cm, with contaminated resection margins (R1/2), no neoadjuvant or adjuvant RTX, and no adjuvant CTX, has a significantly increased risk of developing LR, especially within the first 15 months of follow-up (=clinical example IX). 2. Results On the other hand, a male patient with a 6 cm large, G3 synovial sarcoma, resected with clear margins (R0), without adjuvant CTX or (neo-)adjuvant RTX, has a moderate LR risk during the first 15 months, and an estimated low risk thereafter (=clinical example VIII). Five- and 10-year overall survival (OS) was 73.6% (95%CI: 71.3–75.7) and 62.7% (95%CI: 59.8–65.5) in the test cohort. In the validation cohort, 5- and 10-year OS were 64.9% (95%CI: 61.8–67.8) and 52.9% (95%CI: 48.9–56.8), respectively. Gender, tumor size, histological subtype (except for angiosarcoma/vascular sarcoma (p = 0.127) and dedifferentiated/pleomorphic liposarcoma (p = 0.254), margins, neoadjuvant and adjuvant RTX, as well as adjuvant CTX (all p < 0.05) had a significant influence on risk of LR in the stepwise backward selection of the Fine and Gray model. Grading as a time-dependent effect was kept in the model (p = 0.108), while age (p = 0.082) and neoadjuvant CTX (p = 0.214) were excluded. Consequently, gender, grading, tumor size, neoadjuvant and adjuvant RTX, histological subtype, and adjuvant CTX were included in the flexible parametric competing risk regression model (Table 2). The subdistribution hazard and cumulative incidence functions for LR using ten clinical examples are shown in Figure 1A,B (definition of these examples found in Table S1, together with estimated conditional risks of LR). As an example, a male patient with a G2 myxofibrosarcoma sized 10 cm, with contaminated resection margins (R1/2), no neoadjuvant or adjuvant RTX, and no adjuvant CTX, has a significantly increased risk of developing LR, especially within the first 15 months of follow-up (=clinical example IX). On the other hand, a male patient with a 6 cm large, G3 synovial sarcoma, resected with clear margins (R0), without adjuvant CTX or (neo-)adjuvant RTX, has a moderate LR risk during the first 15 months, and an estimated low risk thereafter (=clinical example VIII). 4 of 13 Cancers 2020, 12, 47 Table 2. Estimated coefficients along with their 95% confidence intervals for local recurrence. Table 2. Estimated coefficients along with their 95% confidence intervals for local recurrence. 2. Results Variables Coefficient 95%-CI p-Value Lower Upper Local Recurrence Gender Male 1 0.011 Female 0.698 0.529 0.921 Grading G2 1 0.199 G3 0.816 0.598 1.113 Tumor size 1.026 1.004 1.049 0.019 Margins R0 1 <0.001 R1/R2 2.761 2.021 3.774 Histology Myxoid Liposarcoma 1 MPNST 4.227 1.837 9.729 0.001 Myxofibrosarcoma 4.156 2.056 8.400 <0.001 Synovial Sarcoma 3.116 1.429 7.014 0.005 UPS 3.373 1.620 7.025 0.001 Angiosarcoma/Vascular Sarcoma 3.316 0.981 12.341 0.074 Dedifferentiated/Pleomorphic Liposarcoma 1.727 0.719 4.143 0.221 Leiomyosarcoma 2.779 1.294 5.966 0.009 Others 2.385 1.123 5.065 0.024 Neoadjuvant RTX No 1 <0.001 Yes 0.298 0.178 0.494 Adjuvant RTX No 1 0.001 Yes 0.603 0.447 0.814 Adjuvant CTX No 1 0.008 Yes 1.711 1.154 2.538 Restricted cubic spline 1 2.104 1.851 2.392 <0.001 Restricted cubic spline 2 1.332 1.230 1.442 <0.001 Restricted cubic spline 3 0.980 0.937 1.026 0.391 Restricted cubic spline for time-dependent effect of grading 0.944 0.813 1.096 0.449 Constant 0.048 0.024 0.097 <0.001 Death Gender Male 1 0.005 Female 0.736 0.595 0.910 Grading G2 1 <0.001 G3 2.215 1.655 2.964 Tumor size 1.065 1.048 1.081 <0.001 Margins R0 1 0.296 R1/R2 1.153 0.883 1.057 Histology Myxoid Liposarcoma 1 MPNST 1.205 0.664 2.187 0.540 Myxofibrosarcoma 1.208 0.795 1.836 0.375 Synovial Sarcoma 1.461 0.888 2.404 0.136 UPS 1.150 0.753 1.758 0.517 Angiosarcoma/Vascular Sarcoma 4.729 2.335 9.580 <0.001 Dedifferentiated/Pleomorphic Liposarcoma 1.420 0.863 2.338 0.167 Leiomyosarcoma 2.154 1.402 3.309 <0.001 Others 1.516 0.975 2.356 0.065 Neoadjuvant RTX No 1 0.007 Yes 1.543 1.127 2.111 Adjuvant RTX No 1 0.296 Yes 1.145 0.888 1.476 Adjuvant CTX No 1 0.022 Yes 0.679 0.488 0.946 Restricted cubic spline 1 4.220 3.393 5.250 <0.001 Restricted cubic spline 2 1.487 1.329 1.663 <0.001 Restricted cubic spline 3 0.965 0.921 1.011 0.139 Restricted cubic spline for time-dependent effect of grading 0.714 0.580 0.889 0.002 Constant 0.050 0.032 0.078 <0.001 p-values in bold are considered statistically significant. CI = Confidence interval. RTX = radiotherapy. CTX = chemotherapy. UPS = undifferentiated pleomorphic sarcoma. MPNST = Malignant Peripheral Nerve Sheat Tumour. 5 of 13 RTX, inical 5 of 13 RTX, inical Cancers 2020, 12, 47 synovial sarcom has a moderate Figure 1. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for local recurrence using ten clinical examples (constellation of parameters shown in Tables S1 and S2). A B Figure 1. 2. Results Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for local recurrence using ten clinical examples (constellation of parameters shown in Tables S1 and S2). A B B Figure 1. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for local recurrence using ten clinical examples (constellation of parameters shown in Tables S1 and S2). Figure 1. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for local recurrence using ten clinical examples (constellation of parameters shown in Tables S1 and S2). In the stepwise backward selection of the Fine and Gray model for distant metastasis (DM) histological subtype (except for myxofibrosarcoma (p = 0.641), angiosarcoma/vascular sarcoma (p = 0.067) and dedifferentiated/pleomorphic liposarcoma (p = 0.592), grading, tumor size, gender, margins, and neoadjuvant RTX (all p < 0.05) were significantly associated with development of metastases. Age (p = 0.852), adjuvant RTX (p = 0.116), neoadjuvant CTX (p = 0.095), and adjuvant CTX (p = 0.536) were excluded via stepwise backward selection. Thus, histological subtype, grading, tumor size, margins, gender, and neoadjuvant RTX were included in the flexible parametric competing risk regression model (Table 3). In the stepwise backward selection of the Fine and Gray model for distant metastasis (DM) histological subtype (except for myxofibrosarcoma (p = 0.641), angiosarcoma/vascular sarcoma (p = 0.067) and dedifferentiated/pleomorphic liposarcoma (p = 0.592), grading, tumor size, gender, margins, and neoadjuvant RTX (all p < 0.05) were significantly associated with development of metastases. Age (p = 0.852), adjuvant RTX (p = 0.116), neoadjuvant CTX (p = 0.095), and adjuvant CTX (p = 0.536) were excluded via stepwise backward selection. Thus, histological subtype, grading, tumor size, margins, gender, and neoadjuvant RTX were included in the flexible parametric competing risk regression model (Table 3). In Figure 2A,B, subdistribution hazard and cumulative incidence functions for DM (using the same ten clinical examples as in Figure 1A,B) are shown. Once again referring to clinical examples IX (male, myxofibrosarcoma, G2, 10 cm, R1/2-margins, no neoadjuvant RTX) and VIII (male, synovial sarcoma, G3, 6cm, R0-margins, no neoadjuvant RTX), risk of DM is lower in clinical example IX in comparison to clinical example VI, while LR-risks are just the opposite, highlighting the importance of an individualized follow-up strategy. 2. Results The conditional risks of these ten clinical examples changing over time estimated based on the models presented above are provided in Table S1 for LR and Table S2 for DM. Conditional risks for all possible combinations of prognostic factors may be estimated and have been implemented in the updated version of the PERSARC app. The Harrell C index for LR was equal to 0.705 and 0.683 for the internal and external cohort, respectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for the external cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that the LR model tended to underestimate the actual patient risk, especially in the validation cohort. On the other hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very good model calibration. 6 of 13 Cancers 2020, 12, 47 Table 3. Estimated coefficients along with their 95% confidence intervals for distant metastasis. Table 3. Estimated coefficients along with their 95% confidence intervals for distant metastasis. 2. Results Coefficient 95%-CI p-Value Lower Upper Distant Metastasis Gender Male 1 <0.001 Female 0.720 0.605 0.857 Grading G2 1 <0.001 G3 1.737 1.412 2.136 Tumor size 1.069 1.056 1.083 <0.001 Margins R0 1 0.006 R1/R2 1.347 1.087 1.669 Histology Myxoid Liposarcoma 1 MPNST 1.825 1.158 2.875 0.009 Myxofibrosarcoma 1.064 0.750 1.508 0.729 Synovial Sarcoma 1.986 1.343 3.976 0.001 UPS 1.445 1.033 2.022 0.032 Angiosarcoma/Vascular Sarcoma 2.016 1.022 3.797 0.043 Dedifferentiated/Pleomorphic Liposarcoma 1.209 0.786 1.861 0.387 Leiomyosarcoma 2.689 1.900 3.797 <0.001 Other 1.835 1.293 2.604 0.001 Neoadjuvant RTX No 1 0.005 Yes 1.351 1.097 1.663 Restricted cubic spline 1 2.928 2.591 3.308 <0.001 Restricted cubic spline 2 1.458 1.374 1.547 <0.001 Restricted cubic spline 3 0.965 0.926 1.006 0.096 Restricted cubic spline 4 1.040 1.020 1.062 <0.001 Restricted cubic spline 5 0.995 0.982 1.008 0.427 Restricted cubic spline for time-dependent effect of grading 0.723 0.640 0.817 <0.001 Constant 0.108 0.078 0.149 <0.001 Death Gender Male 1 0.864 Female 0.968 0.666 1.407 Grading G2 1 0.018 G3 1.873 1.116 3.145 Tumor size 1.023 0.997 1.050 0.087 Margins R0 1 0.198 R1/R2 1.378 0.846 2.244 Histology Myxoid Liposarcoma 1 MPNST 2.506 0.844 7.442 0.098 Myxofibrosarcoma 3.136 1.325 7.418 0.009 Synovial Sarcoma 0.600 0.150 2.416 0.472 UPS 1.781 0.714 4.443 0.216 Angiosarcoma/Vascular Sarcoma 11.165 * 3.507 * 35.542 * <0.001 * Dedifferentiated/Pleomorphic Liposarcoma 3.331 1.259 8.812 0.015 Leiomyosarcoma 1.798 0.675 4.782 0.241 Other 2.408 0.963 4.782 0.060 Neoadjuvant RTX No 1 0.048 Yes 0.541 0.295 0.993 Restricted cubic spline 1 3.604 2.494 5.211 <0.001 Restricted cubic spline 2 1.270 1.060 1.523 0.010 Restricted cubic spline 3 0.952 0.863 1.049 0.316 Restricted cubic spline 4 0.953 0.908 1.001 0.057 Restricted cubic spline 5 0.974 0.569 1.199 0.097 Restricted cubic spline for time-dependent effect of grading 0.826 0.569 1.199 0.314 Constant 0.010 0.004 0.025 <0.001 p-values in bold are considered statistically significant; CI = Confidence interval. RTX = radiotherapy. CTX = chemotherapy UPS = undifferentiated pleomorphic sarcoma * too few events 7 of 13 in the 7 of 13 in the Cancers 2020, 12, 47 all possible com d d Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for distant metastasis using the same ten clinical examples as in Figures 1A and B (constellation of parameters shown in Tables S1 and S2). A B Figure 2. 2. Results Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for distant metastasis using the same ten clinical examples as in Figure 1A,B (constellation of parameters shown in Tables S1 and S2). Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for distant metastasis using the same ten clinical examples as in Figures 1A and B (constellation of parameters shown in Tables S1 and S2). The Harrell C index for LR was equal to 0.705 and 0.683 for the internal and external cohort, respectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for the external cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that the LR model tended to underestimate the actual patient risk, especially in the validation cohort. On the other hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very good A Figure 2. Subdistribution hazard function (A) an parametric competing risk regression model fo examples as in Figures 1A and B (constellation of The Harrell C index for LR was equal to 0.7 ti l F DM H ll C t ti ti B d cumulative incidence function (B) of the flexible or distant metastasis using the same ten clinical parameters shown in Tables S1 and S2). 705 and 0.683 for the internal and external cohort, ual to 0 723 fo the i te al oho t a d 0 772 fo the B Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for distant metastasis using the same ten clinical examples as in Figures 1A and B (constellation of parameters shown in Tables S1 and S2). Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible parametric competing risk regression model for distant metastasis using the same ten clinical examples as in Figure 1A,B (constellation of parameters shown in Tables S1 and S2). pectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for ernal cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that model tended to underestimate the actual patient risk, especially in the validation cohort. 2. Results On her hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very go The Harrell C index for LR was equal to 0.705 and 0.683 for the internal and external cohort, respectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for the external cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that the LR model tended to underestimate the actual patient risk, especially in the validation cohort. On the other hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very good model calibration. Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). Di i The Harrell C index for LR was equal to 0.70 respectively. For DM, Harrell C statistics was equa external cohort. Calibration plots for LR (Figure 3A LR model tended to underestimate the actual patie other hand, calibration plots for DM with test and model calibration. 05 and 0.683 for the internal and external cohort al to 0.723 for the internal cohort and 0.772 for the ) using test and validation cohort showed that the nt risk, especially in the validation cohort. On the validation cohort (Figure 3B) showed very good espe l to Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). 3. Discussion 3. Discussion Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). 3. Discussion In the present retrospective multicenter cohort study, flexible parametric competing risk regression modeling was applied in order to estimate individual three-to-six-month risks for local In the present retrospective multicenter cohort study, flexible parametric competing risk regression modeling was applied in order to estimate individual three-to-six-month risks for local In the present retrospective multicenter cohort study, flexible parametric competing risk regression modeling was applied in order to estimate individual three-to-six-month risks for local recurrence and distant metastasis during the first 5 years of follow-up in patients undergoing curative resection for high-grade extremity soft tissue sarcoma. It offers an evidence-based opportunity to individually schedule follow-up visits instead of adhering to calendar-based guidelines [8,9]. The number of radiological investigations for assessing disease status, especially after R0 resections and taking histological subtype into account, could be significantly restricted, reducing patient- and healthcare burden. The advantage of using flexible parametric competing risk regression models to estimate LR- and DM-risks in eSTS-patients is based on the fact that these rates strongly vary upon time (i.e., they do not constantly increase or decrease). To overcome this issue, flexible parametric competing risk regression models represent the baseline distribution function as a restricted cubic spline function of log time instead of a linear function of log time [15]. Moreover, it allows smooth estimation of both the cause-specific hazard rates and cumulative incidence functions. Both models performed well at internal and external calibration, with c-indexes comparable to previously published studies [14,16]. One of the limitations of the present study is its retrospective design, resulting in possible selection biases regarding diagnosis, treatment, and follow-up of patients included, due to slightly differing policies at the respective centers. By incorporating these factors in the statistical models, we aimed at reducing this bias. Moreover, during the study period, several histological STS-subtypes were Cancers 2020, 12, 47 8 of 13 reclassified (i.e., malignant fibrous histiocytoma to undifferentiated pleomorphic sarcoma). At some, but not all, participating centers, all histological diagnoses had been reevaluated by pathologists and, if applicable, reclassified according to the current classification systems. In order to limit the impact of this limitation, we only included patients treated in tertiary reference sarcoma centers with experienced and dedicated sarcoma pathologists. 3. Discussion 3. Discussion p g Another limitation of the present study is that the models were developed based on patient cohorts from experienced, tertiary tumor centers. This implies that generalizability of the predicted risks to patients not treated at such centers has to be questioned. Moreover, considering that we did only include patients with high-grade (G2/3), primary eSTS who had undergone surgery with curative intent, the risks estimated are not applicable to patients with low-grade disease or metastases at initial presentation. Furthermore, estimated risks of the current models should be applied with caution after patients have already developed an event (i.e., LR or DM) during follow-up. Due to the retrospective design of the study, not all variables could be ascertained in every patient, thus potentially reducing the statistical power. However, it can be assumed that in this large patient collective, missing data may have little to no bias to the conclusions made, wherefore cases with missing information on clinical and/or pathological variables were not excluded [17]. As outlined in the introduction, current follow-up strategies follow a heuristic approach, with 3- to 4-months intervals for the first three years, followed by biannual check-ups until the end of the 5th year and annual appointments thereafter [8,9]. In clinical practice, it is not only of interest to estimate a patient’s cumulative risk after a specific period of time but also to know about the conditional risks from one follow-up appointment to the next, in case no event had occurred. We addressed this question by calculating conditional risks for LR and DM depending on different, clinically relevant, examples. Notably, the present model allows risk prediction at any constellation of variables, which are at the moment included in the updated PERSARC app. This app allows the patient’s individualized risk of LR and DM to be estimated by entering relevant prognostic parameters, such as histological subtype, tumor size, and resection margins. With the estimated event-risk in time, physicians and patients may decide together when the next follow-up examination should be scheduled. In light of the heterogeneity of eSTS with part significantly differing outcomes, estimated event risks would facilitate planning of an individualized follow-up protocol for each patient. 3. Discussion 3. Discussion Arbitrary thresholds of 4% for LR and 2% for DM were chosen in the present study to be of clinical “relevance”, considering that LR is usually detected during clinical examination or even noticed by patients themselves, while DM (most commonly to the lungs) require visualization by chest x-ray or thoraci computed tomography (CT) scan [18,19]. However, thresholds should be changed on patients’ preference and clinical significance. Previously published studies have well-investigated risks of LR, DM, and overall survival (OS) in large, retrospective cohorts of patients with eSTS [14,16,20,21]. The nomogram for OS by Kattan et al. [21] in 2002 and the two more recent nomograms for DM and OS by Callegaro et al. [16] published in 2016 added significant value to predict individual patient risks. Both studies used Cox-regression models as the basis for their nomograms. In comparison to Cox-regression models, flexible parametric competing risk regression models have a major advantage; while the Cox-regression models only estimate the relative effects (i.e., hazard rates), flexible parametric competing risk regression models estimate the baseline hazard using restricted cubic splines [22]. The cumulative incidence functions of LR and DM predicted from flexible parametric competing risk regression models demonstrate the clear variance in event rates. By applying a flexible parametric competing risk regression model, we aimed at incorporating non-constant hazards, time-varying covariates, and death as the competing event in order to obtain a robust, comprehensible, and accurate prediction of individual patient risks. Moreover, with the clinical examples provided, the risk peaks during the first year of follow-up is clearly visible. Although appointments may be safely skipped in some patients due to very low risks of LR and/or DM, others would benefit from closer follow-up intervals. Cancers 2020, 12, 47 9 of 13 Cancers 2020, 12, 47 Potentially due to the application of the present statistical model, interesting results emerged: Female gender was independently associated with a significantly lower risk of LR and DM. An association between gender and overall survival (OS) has been observed by Maretty-Nielsen et al. and Wu et al. [2,23]. However, an association between gender and LR-free as well as DM-free survival has not been described thus far [24]. Moreover, tumor grading, which is a well-known prognostic factor of LR, was not significantly associated with an altered risk in the current flexible parametric competing risk regression model. 3. Discussion 3. Discussion This may be explained by the fact that patients with usually fast-growing, highly-aggressive G3 tumors will present with LR at early time points, while in those with relatively slower-growing G2 tumors, LR is most probably detected at a later date. This hypothesis is corroborated by the fact that grading did not meet the proportional hazards assumption, wherefore it was treated as a time-varying covariate. On the other hand, another recently published multicenter study for grade III eSTS did not incorporate grade II in the multivariate model for OS [14]. The current model has broader applicability as it also incorporates patients with grade II eSTS. Additionally, margins as classified in the current study only divide “clear” from “contaminated” margins, not taking into consideration that histological subtypes with infiltrative growth pattern as undifferentiated pleiomorphic sarcoma (UPS) and myxofibrosarcoma potentially require broader margins to markedly reduce LR-risk [25]. Thus, unsurprisingly, also in the current flexible parametric competing risk regression model for LR, these histological subtypes showed significantly higher LR-risks in comparison to other histologies. Statistical Analysis We focused on the first five years of follow-up to predict the conditional risk at the usually scheduled follow-up times (every 3 months from 1st to 3rd year; every 6 months in 4th and 5th year), i.e., the risk of experiencing an event at X + Y, given that the patient has not developed an event before X months. The variables age and tumor size were centered at their mean value in order to allow prediction at the average in case variables were not specified upon calculation. We used the Royston and Parmar approach to fit a flexible parametric competing risk regression model in order to estimate the risk of LR and DM, with death as the competing event [30]. In this model, the baseline distribution is modeled as a restricted cubic spline function of log time [15,22]. Splines constitute flexible mathematical functions defined by piecewise polynomials together with distinct constraints, ensuring that the overall curve is smooth [22]. A feature of restricted cubic splines as used in the present model is that the fitted function is forced to be linear before the first and after the last knot [31]. As automatic stepwise backward selection of variables is currently not available for the flexible parametric competing risk regression model, variable selection for the LR and DM models was based on a stepwise backward procedure using a multivariable Fine and Gray model [32]. Variables with a p-value < 0.05 were excluded from the model, except for histology, where all subtypes were kept in the analyses. The LR and DM models were fit on the log cumulative subdistribution hazard scale, directly modeling the cause-specific cumulative incidence function. Grading was incorporated in the model as a time-dependent effect for LR and DM, as it did not meet the proportional hazards assumption. The number of knots of the flexible parametric competing risk regression model for LR and DM was chosen based on the lowest AIC (=Akaike Information Criterion) after fitting several models with knots from 0 to 5. For the flexible parametric competing risk regression model estimating the risk of LR, two knots turned out as most accurate, while for the model predicting the risk of DM, four knots were used (with no internal knots for grading as a time-dependent covariate). Cumulative incidence functions were estimated based on the defined models. 4. Materials and Methods In this retrospective multicenter study, 1931 consecutive patients with primary nonmetastatic high-grade (G2/3) eSTS managed with surgery at a curative intent were included in the test cohort, with patient information deriving from prospectively maintained STS databases at 5 participating tertiary sarcoma referral centers. Patients with missing information on oncological follow-up (i.e., development of LR/DM) had to be excluded (n = 42). Extremity STS were defined as tumors from the shoulder to the fingers (=upper limb) and from the pelvic girdle, excluding intrapelvic STS, to the foot (=lower limb). The validation cohort consisted of 1085 patients with identical inclusion criteria as for the test cohort from two independent tertiary sarcoma referral centers. As described above, patient monitoring after surgery usually followed a standardized approach with clinical examination, radiography using chest X-ray (CXR) or chest CT-scan (chest-CT) for control of DM and sonography or magnetic resonance imaging (MRI) for control of LR. Demographic variables (patient age at diagnosis, gender), tumor-related parameters (tumor size, depth, location, grading, histological subtype), treatment (histological margins, (neo)adjuvant CTX/RTX), and outcome variables (date of LR or DM, date of death/last follow-up) were reported. Histological resection margins were divided into “clear” margins (=R0) and “contaminated” margins (=R1/2), as classification and definition of margin status have changed over time [26–28]. Histological subtypes were classified into 9 categories, with myxoid liposarcoma as the reference, compliant with previous studies and the current World Health Organisation (WHO) classification (Table 1) [6,16]. The FNCLCC grading system (Fédération Française des Centres de Lutte Contre le Cancer/French Federation of Centres for the Fight against Cancer) was used to categorize tumors into either intermediate (=G2) or high-grade (G3) [29]. (Neo-)adjuvant RTX and CTX had been administered in case a high risk of LR or DM had been anticipated by the multidisciplinary tumor board, according to locally preferred guidelines, LR was defined as a radiologically and/or histologically confirmed tumor recurrence. DM must have been confirmed by radiography (sonography, MRI, CXR, chest-CT) and/or histologically. In the case of pulmonary nodules without subsequent surgical exploration, an increase in size of the suspected metastasis must have been present. Patient, tumor, and treatment-related factors were ascertained using medical records, histological reports, and prospectively maintained databases at the respective centers. 10 of 13 Cancers 2020, 12, 47 Ethics approval was obtained in each participating center. 4. Materials and Methods The study was performed according to the Declaration of Helsinki and approved by the Ethics Committee of the Medical University of Graz, Austria (IRB-approval-number: 31-046 ex 18/19; date of approval: 24 May 2019). Statistical Analysis Conditional risks at the 3–6-months intervals were calculated based on the cumulative incidence functions of the flexible parametric competing risk regression model. Threshold was set to 4% for LR, considering that they are often palpable and diagnosed during the clinical examination or by patients themselves [18]. On the other hand, a 2% threshold for DM was chosen, as DM (and most commonly lung metastases) can only safely be diagnosed by chest X-ray or CT-scans of the thorax [19]. Model discrimination was tested using the Harrell C index, estimating the probability of concordance between observed and predicted outcomes. A value of 0.5 indicates no predictive discrimination, while a value of 1.0 indicates a perfect separation of patients with different outcomes [33]. Furthermore, calibration plots were compiled to assess model calibration in the test and validation cohort. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/1/47/s1, Table S1: Conditional Risks for Local Recurrence–Threshold 4%; Table S2: Conditional Risks for Distant Metastasis– Threshold 2%. References 1. Italiano, A.; Le Cesne, A.; Mendiboure, J.; Blay, J.Y.; Piperno-Neumann, S.; Chevreau, C.; Delcambre, C.; Penel, N.; Terrier, P.; Ranchere-Vince, D.; et al. Prognostic factors and impact of adjuvant treatments on local and metastatic relapse of soft-tissue sarcoma patients in the competing risks setting. Cancer 2014, 120, 3361–3369. [CrossRef] [PubMed] 2. Maretty-Nielsen, K.; Aggerholm-Pedersen, N.; Safwat, A.; Jorgensen, P.H.; Hansen, B.H.; Baerentzen, S.; Pedersen, A.B.; Keller, J. Prognostic factors for local recurrence and mortality in adult soft tissue sarcoma of the extremities and trunk wall: A cohort study of 922 consecutive patients. Acta Orthop. 2014, 85, 323–332. [CrossRef] [PubMed] 2. Maretty-Nielsen, K.; Aggerholm-Pedersen, N.; Safwat, A.; Jorgensen, P.H.; Hansen, B.H.; Baerentzen, S.; Pedersen, A.B.; Keller, J. Prognostic factors for local recurrence and mortality in adult soft tissue sarcoma of the extremities and trunk wall: A cohort study of 922 consecutive patients. Acta Orthop. 2014, 85, 323–332. [CrossRef] [PubMed] 3. Novais, E.N.; Demiralp, B.; Alderete, J.; Larson, M.C.; Rose, P.S.; Sim, F.H. Do surgical margin and local recurrence influence survival in soft tissue sarcomas? Clin. Orthop. Relat. Res. 2010, 468, 3003–3011. [CrossRef] [PubMed] 3. Novais, E.N.; Demiralp, B.; Alderete, J.; Larson, M.C.; Rose, P.S.; Sim, F.H. Do surgical margin and local recurrence influence survival in soft tissue sarcomas? Clin. Orthop. Relat. Res. 2010, 468, 3003–3011. [CrossRef] [PubMed] 4. Willeumier, J.; Fiocco, M.; Nout, R.; Dijkstra, S.; Aston, W.; Pollock, R.; Hartgrink, H.; Bovee, J.; van de Sande, M. High-grade soft tissue sarcomas of the extremities: Surgical margins influence only local recurrence not overall survival. Int. Orthop. 2015, 39, 935–941. [CrossRef] [PubMed] 4. Willeumier, J.; Fiocco, M.; Nout, R.; Dijkstra, S.; Aston, W.; Pollock, R.; Hartgrink, H.; Bovee, J.; van de Sande, M. High-grade soft tissue sarcomas of the extremities: Surgical margins influence only local recurrence not overall survival. Int. Orthop. 2015, 39, 935–941. [CrossRef] [PubMed] p 5. Singer, S.; Demetri, G.D.; Baldini, E.H.; Fletcher, C.D. Management of soft-tissue sarcomas: An overview and update. Lancet Oncol. 2000, 1, 75–85. [CrossRef] 6. Fletcher, C.D.; Bridge, J.A.; Hodgendoorn, P.C.W.; Mertens, F. WHO Classification of Tumours of Soft Tissue and Bone; IARC: Lyon, France, 2013. 7. Smolle, M.A.; van Praag, V.M.; Posch, F.; Bergovec, M.; Leitner, L.; Friesenbichler, J.; Heregger, R.; Riedl, J.M.; Pichler, M.; Gerger, A.; et al. Surgery for metachronous metastasis of soft tissue sarcoma—A magnitude of benefit analysis using propensity score methods. Eur. J. Surg. Oncol. 2018, 45, 242–248. [CrossRef] 8. 5. Conclusions In conclusion, the present study provides a model to individually predict patient’s LR and DM risks during follow-up, applying a flexible parametric competing risk regression approach. These models are at the moment being included in the updated version of the PERSARC app for Individualized Sarcoma Care and follow-up. Although a risk-threshold of 4% for LR and 2% for DM was chosen in the present study, the “optimal” threshold upon which an individual patient should undergo imaging with MRI, chest-CT, or CXR, is still subjected to experts’ opinion and should be further discussed with patients concerned. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/1/47/s1, Table S1: Conditional Risks for Local Recurrence–Threshold 4%; Table S2: Conditional Risks for Distant Metastasis– Threshold 2%. 11 of 13 11 of 13 Cancers 2020, 12, 47 Author Contributions: Conceptualization, M.A.S., A.L., R.L.H., and J.S. (Joanna Szkandera); data curation, M.A.S., D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., M.F., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger), M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi); formal analysis, J.S. (Josef Smolle); investigation, D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.D., W.J.v.H., J.M.R., M.S., A.G. (Alessandro Gronchi), M.P., H.S., B.L.-A., and A.G. (Armin Gerger); methodology, M.v.d.S., M.F., S.D., J.S. (Josef Smolle), R.L.H., and J.S. (Joanna Szkandera); project administration, M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); supervision, M.v.d.S., D.A., and R.L.H.; validation, V.v.P., M.F., D.A. and J.S. (Joanna Szkandera); visualization, M.A.S. and J.S. (Josef Smolle); writing—Original draft, M.A.S., M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); writing—Review and editing, D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger), M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi). All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.A.S., A.L., R.L.H., and J.S. (Joanna Szkandera); data curation, M.A.S., D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., M.F., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger), M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi); formal analysis, J.S. (Josef Smolle); investigation, D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.D., W.J.v.H., J.M.R., M.S., A.G. (Alessandro Gronchi), M.P., H.S., B.L.-A., and A.G. (Armin Gerger); methodology, M.v.d.S., M.F., S.D., J.S. (Josef Smolle), R.L.H., and J.S. (Joanna Szkandera); project administration, M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); supervision, M.v.d.S., D.A., and R.L.H.; validation, V.v.P., M.F., D.A. and J.S. (Joanna Szkandera); visualization, M.A.S. and J.S. 5. Conclusions (Josef Smolle); writing—Original draft, M.A.S., M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); writing—Review and editing, D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger), M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi). All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Dutch Cancer Society (DCS)—KWF Kankerbestrijding [UL2015-8028]. The funding source had no role in the design of this study; execution, analyses, and interpretation of the data; report writing; or decision to submit the article for publication. Conflicts of Interest: Author van de Sande reports grants from Daiichi Sankyo, outside the submitted work. The remaining authors (Maria A Smolle, Dario Callegaro, Jay Wunder, Andrew J. Hayes, Lukas Leitner, Marko Bergovec, Per-Ulf Tunn, Veroniek van Praag, Marta Fiocco, Joannis Panotopoulos, Madeleine Willegger, Reinhard Windhager, Sander Djikstra, Winan J van Houdt, Jakob M Riedl, Michael Stotz, Armin Gerger, Martin Pichler, Herbert Stöger, Bernadette Liegl-Atzwanger, Josef Smolle, Dimosthenis Andreou, Andreas Leithner, Alessandro Gronchi, Rick L. Haas, and Joanna Szkandera) have no conflicts of interest to declare. References Casali, P.G.; Abecassis, N.; Bauer, S.; Biagini, R.; Bielack, S.; Bonvalot, S.; Boukovinas, I.; Bovee, J.; Brodowicz, T.; Broto, J.M.; et al. Soft tissue and visceral sarcomas: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 2018, 29, iv51–iv67. [CrossRef] 9. von Mehren, M.; Randall, R.L.; Benjamin, R.S.; Boles, S.; Bui, M.M.; Ganjoo, K.N.; George, S.; Gonzalez, R.J.; Heslin, M.J.; Kane, J.M., 3rd; et al. Soft Tissue Sarcoma, Version 2.2018, NCCN Clinical Practice Guidelines in Oncology. J. Natl. Compr. Cancer Netw. 2018, 16, 536–563. [CrossRef] 10. Gingrich, A.A.; Bateni, S.B.; Monjazeb, A.M.; Darrow, M.A.; Thorpe, S.W.; Kirane, A.R.; Bold, R.J.; Canter, R.J. Neoadjuvant Radiotherapy is Associated with R0 Resection and Improved Survival for Patients with Extremity Soft Tissue Sarcoma Undergoing Surgery: A National Cancer Database Analysis. Ann. Surg. Oncol. 2017, 24, 3252–3263. [CrossRef] 12 of 13 Cancers 2020, 12, 47 11. Gronchi, A.; Ferrari, S.; Quagliuolo, V.; Broto, J.M.; Pousa, A.L.; Grignani, G.; Basso, U.; Blay, J.Y.; Tendero, O.; Beveridge, R.D.; et al. Histotype-tailored neoadjuvant chemotherapy versus standard chemotherapy in patients with high-risk soft-tissue sarcomas (ISG-STS 1001): An international, open-label, randomised, controlled, phase 3, multicentre trial. Lancet Oncol. 2017, 18, 812–822. [CrossRef] 12. Posch, F.; Partl, R.; Doller, C.; Riedl, J.M.; Smolle, M.; Leitner, L.; Bergovec, M.; Liegl-Atzwanger, B.; Stotz, M.; Bezan, A.; et al. Benefit of Adjuvant Radiotherapy for Local Control, Distant Metastasis, and Survival Outcomes in Patients with Localized Soft Tissue Sarcoma: Comparative Effectiveness Analysis of an Observational Cohort Study. Ann. Surg. Oncol. 2018, 25, 776–783. [CrossRef] [PubMed] 13. Royce, T.J.; Punglia, R.S.; Chen, A.B.; Patel, S.A.; Thornton, K.A.; Raut, C.P.; Baldini, E.H. Cost-Effectiveness of Surveillance for Distant Recurrence in Extremity Soft Tissue Sarcoma. Ann. Surg. Oncol. 2017, 24, 3264–3270. [CrossRef] [PubMed] 14. van Praag, V.M.; Rueten-Budde, A.J.; Jeys, L.M.; Laitinen, M.K.; Pollock, R.; Aston, W.; van der Hage, J.A.; Dijkstra, P.D.S.; Ferguson, P.C.; Griffin, A.M.; et al. A prediction model for treatment decisions in high-grade extremity soft-tissue sarcomas: Personalised sarcoma care (PERSARC). Eur. J. Cancer 2017, 83, 313–323. [CrossRef] [PubMed] 15. Royston, P.; Lambert, P.C. Flexible Parametric Survival Analysis Using Stata: Beyond the Cox Model; Stata Press: College Statoin, TX, USA, 2011. 16. Callegaro, D.; Miceli, R.; Bonvalot, S.; Ferguson, P.; Strauss, D.C.; Levy, A.; Griffin, A.; Hayes, A.J.; Stacchiotti, S.; Pechoux, C.L.; et al. References Development and external validation of two nomograms to predict overall survival and occurrence of distant metastases in adults after surgical resection of localised soft-tissue sarcomas of the extremities: A retrospective analysis. Lancet Oncol. 2016, 17, 671–680. [CrossRef] 17. Kang, H. The prevention and handling of the missing data. Korean J. Anesthesiol. 2013, 64, 402–406. [CrossRef] [PubMed] 18. Puri, A.; Ranganathan, P.; Gulia, A.; Crasto, S.; Hawaldar, R.; Badwe, R.A. Does a less intensive surveillance protocol affect the survival of patients after treatment of a sarcoma of the limb? updated results of the randomized TOSS study. Bone Jt. J. 2018, 100-B, 262–268. [CrossRef] 19. Hovgaard, T.B.; Nymark, T.; Skov, O.; Petersen, M.M. Follow-up after initial surgical treatment of soft tissue sarcomas in the extremities and trunk wall. Acta Oncol. 2017, 56, 1004–1012. [CrossRef] 20. Rueten-Budde, A.J.; van Praag, V.M.; PERSARC studygroup; van de Sande, M.A.J.; Fiocco, M. Dynamic prediction of overall survival for patients with high-grade extremity soft tissue sarcoma. Surg. Oncol. 2018, 27, 695–701. [CrossRef] 21. Kattan, M.W.; Leung, D.H.; Brennan, M.F. Postoperative nomogram for 12-year sarcoma-specific death. J. Clin. Oncol. 2002, 20, 791–796. [CrossRef] 22. Lambert, P.C.; Royston, P. Further development of flexible parametric models for survival analysis. Stata J. 2009, 9, 265–290. [CrossRef] 23. Wu, J.; Qian, S.; Jin, L. Prognostic factors of patients with extremity myxoid liposarcomas after surgery. J. Orthop. Surg. Res. 2019, 14, 90. [CrossRef] [PubMed] 24. Trovik, C.S.; Bauer, H.C.; Alvegard, T.A.; Anderson, H.; Blomqvist, C.; Berlin, O.; Gustafson, P.; Saeter, G.; Walloe, A. Surgical margins, local recurrence and metastasis in soft tissue sarcomas: 559 surgically-treated patients from the Scandinavian Sarcoma Group Register. Eur. J. Cancer 2000, 36, 710–716. [CrossRef] 5. Fujiwara, T.; Stevenson, J.; Parry, M.; Tsuda, Y.; Tsoi, K.; Jeys, L. What is an adequate margin for infiltra soft-tissue sarcomas? Eur. J. Surg. Oncol. 2019. [CrossRef] 26. Kainhofer, V.; Smolle, M.A.; Szkandera, J.; Liegl-Atzwanger, B.; Maurer-Ertl, W.; Gerger, A.; Riedl, J.; Leithner, A. The width of resection margins influences local recurrence in soft tissue sarcoma patients. Eur. J. Surg. Oncol. 2016, 42, 899–906. [CrossRef] [PubMed] 27. Tunn, P.U.; Kettelhack, C.; Durr, H.R. Standardized approach to the treatment of adult soft tissue sarcoma of the extremities. Recent Results Cancer Res. 2009, 179, 211–228. 28. Wittekind, C.; Compton, C.C.; Greene, F.L.; Sobin, L.H. TNM residual tumor classification revisited. Cancer 2002, 94, 2511–2516. [CrossRef] 29. © 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/). References Trojani, M.; Contesso, G.; Coindre, J.M.; Rouesse, J.; Bui, N.B.; de Mascarel, A.; Goussot, J.F.; David, M.; Bonichon, F.; Lagarde, C. Soft-tissue sarcomas of adults; study of pathological prognostic variables and definition of a histopathological grading system. Int. J. Cancer 1984, 33, 37–42. [CrossRef] 13 of 13 13 of 13 Cancers 2020, 12, 47 30. Mozumder, S.I.; Rutherford, M.J.; Lambert, P.C. stpm2cr: A flexible parametric competing risks model using a direct likelihood approach for the cause-specific cumulative incidence function. Stata J. 2017, 17, 462–489. [CrossRef] 31. Durrleman, S.; Simon, R. Flexible regression models with cubic splines. Stat. Med. 1989, 8, 551–561. [CrossRef] 32. Fine, J.P.; Gray, R.J. A proportional hazards model for the subdistribution of a competing risk. J. Am. Stat. Assoc. 1999, 94, 496–509. [CrossRef] 32. Fine, J.P.; Gray, R.J. A proportional hazards model for the subdistribution of a competing risk. J. Am. Stat. Assoc. 1999, 94, 496–509. [CrossRef] 33. Harrell, F.E., Jr.; Lee, K.L.; Mark, D.B. Multivariable prognostic models: Issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors. Stat. Med. 1996, 15, 361–387. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4361810727
https://aacr.figshare.com/articles/journal_contribution/Data_Supplement_from_High-Throughput_Time-Resolved_FRET_Reveals_Akt_PKB_Activation_as_a_Poor_Prognostic_Marker_in_Breast_Cancer/22402640/1/files/39848441.pdf
English
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Data Supplement from High-Throughput Time-Resolved FRET Reveals Akt/PKB Activation as a Poor Prognostic Marker in Breast Cancer
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pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer Supplementary Figure 1: Specificity of human anti-Akt (panAkt, pT308) antibodies assessed by quantitative Western blot analysis in SKBR3 cells. Supplementary Figure 1: Specificity of human anti-Akt (panAkt, pT308) antibodies assessed by quantitative Western blot analysis in SKBR3 cells. SKBR3 cells starved for 3h followed by pretreatment with 50µM LY294002 for 30 min prior to 100ng/ml EGF stimulation for 5min as indicated. Western blot analysis of (A) phospho pAkt (pT308) and (B) total Akt (panAkt) was performed using the LI- COR/Odyssey system, with (C) merged. (D) Quantification of Akt phosphorylation (pT308) normalized to the amount of total Akt (panAkt) from three independent experiments (***, p<0.0001). Supplementary Figure 2: Localization of endogenous Akt and pAkt (pT308) in SKBR3 cells and FFPE human breast tumor tissues using TSA amplified immunofluorescence. (A) SKBR3 cells were pretreated with LY294002 (50µM) for 30 min prior to EGF stimulation (100ng/ml) for 5 min as indicated. Confocal images show the localization of endogenous panAkt and pT308 using specific primary antibodies (as described in Supporting Information Fig 1). Labeled Fab fragments were used as secondary antibodies: anti-mouse Fab-ORG488 (green) and anti-rabbit Fab-ALX594 (red). (B) SKBR3 cells were treated as in (A). However the signals were detected using a Fab fragment-HRP based TSA immunofluorescence assay as described in Materials and Methods. Confocal microscopy images show the localization of endogenous panAkt (green) using an anti-mouse Fab-ORG488, and pT308 (red) using an anti-rabbit Fab- 1 1 pAkt is a marker for poor prognosis in breast cancer HRP and TSA-ALX594. (C) FFPE human breast tumor tissue was labeled with panAkt and pT308 for TSA amplified immunofluorescence, as in (B) pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer HRP and TSA-ALX594. (C) FFPE human breast tumor tissue was labeled with panAkt and pT308 for TSA amplified immunofluorescence, as in (B) Supplementary Figure 3: Optimization of TSA amplification in MCF7 cells using TSA amplified immunofluorescence assay. Supplementary Figure 3: Optimization of TSA amplification in MCF7 cells using TSA amplified immunofluorescence assay. MCF7 cells were stimulated with EGF (100ng/ml) for 5 min. Confocal images show the localization of endogenous panAkt (green, white arrow) and pT308 (red, white arrow). Labeled Fab fragments were used as secondary antibodies: anti-mouse Fab- ORG488 and anti-rabbit Fab-HRP/TSA-ALX594. The TSA amplification reaction was allowed to develop for 5, 15, 20 or 30 min as indicated. The bottom panel shows the control, with no primary antibody included. Cells were counter stained with DAPI (blue) for DNA. Supplementary Figure 4: Quantification of endogenous pAkt (pT308) using whole IgG secondary antibodies in fixed FFPE human breast tumor by amplified FRET assay. (A) Intensity images and lifetime maps (scales are relative and in pseudo-color) of donor (panAkt) and donor+acceptor (panAkt+pT308) for FFPE human breast tumor tissues. panAkt was detected using an anti-mouse IgG(whole)-ORG488 and pT308 using a secondary anti-rabbit IgG(whole)-HRP and TSA-ALX594. Box and whiskers plot shows quantification of FRET efficiency of 3 independent experiments, each experiment consisting of measurements from at least 10 different regions of the same 2 2 pAkt is a marker for poor prognosis in breast cancer tissue section. (B) The same tissue sections prepared for (A) were imaged by confocal microscopy. Supplementary Figure 5: Molecular heterogeneity of Akt activation in human colon TMA revealed by amplified FRET. Supplementary Figure 5: Molecular heterogeneity of Akt activation in human colon TMA revealed by amplified FRET. (A) H&E staining of TMAs prepared from colon tumor biopsies obtained from 7 patients. For each patient, 4 cores (circles 1 to 4) were selected from different regions within each biopsy. In total 28 tumor cores (4 x 7 patients) were spotted to make the TMA. The lower image shows an expanded view of one core divided into 4 sectors for further analysis. The panels on the right show 3 different magnifications of sector 4. Intensity images and lifetime maps (scales are relative and in pseudo-color) of the TMAs stained with (B) donor alone (panAkt) or (C) donor+acceptor (panAkt+pT308), followed by TSA amplification. TMAs were mapped and images automatically acquired using our FRET/FLIM platform. (D) For each patient, the maximum FRET efficiency of each core is shown; (inset) for each patient the mean FRET efficiency for the 4 cores is shown. Bars represent mean. Supplementary Figure 6: Distribution of Akt activation revealed by amplified FRET for all breast carcinoma patients (ER-/ER+). Supplementary Figure 6: Distribution of Akt activation revealed by amplified FRET for all breast carcinoma patients (ER-/ER+). (A) Mean FRET efficiency for the patients grouped by ER status (***, p<0.0001). (B) Mean intensity ratio for the patients grouped by ER status (ns, not significant). (C) Population distribution of patients according to FRET quartiles, grouped by ER status 3 pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer (black, high FRET quartile; light gray, low FRET quartile). (D) FRET efficiencies are shown as box and whiskers plots representing the age distribution of patients according to FRET tertiles (ns, not significant). Supplementary Figure 7: Quantification of endogenous pAkt (pT308) in fixed FFPE human normal and tumor breast tissue using amplified FRET. Supplementary Figure 7: Quantification of endogenous pAkt (pT308) in fixed FFPE human normal and tumor breast tissue using amplified FRET. (A) Intensity images and lifetime maps (scales are relative and in pseudo-color) of FFPE human breast tissues from two different normal (normal 1, normal 2) tissues and one tumor tissue labeled with donor alone (panAkt) or donor+acceptor (panAkt+pT308). FRET efficiencies are shown as box and whiskers plots representing the mean ±SEM for at least 10 different regions from the same tissue section (****, p<0.0001). The increased FRET efficiency represents the phosphorylation status of endogenous Akt and shows the differential activation of pT308 in normal and tumor tissues. 4 4
https://openalex.org/W4320031564
http://pdf.blucher.com.br/designproceedings/ped2022/6427782.pdf
Portuguese
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Design para Acessibilidade e Inclusão: Design Positivo e DOP no desenvolvimento de projetos em saúde.
Blucher Design Proceedings
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Keywords: Positive Design, Health, Accessibility, Inclusion. RESUMO Design e Psicologia sempre caminharam juntos, ambos com mesmo objetivo de proporcionar bem-estar ao indivíduo; no caso do design, por meio de soluções às necessidades e desejos do homem e no caso da psicologia por meio de tratamento de ‘doenças’ ou situações relacionados a mente. Nesse sentido buscamos firmar a compreensão de bem-estar relacionado à saúde, como já o fez a Psicologia Positiva ao adotá-la como último elemento do modelo PERMAH que é utilizado como estratégia para mensurar o índice de bem-estar de uma pessoa. Dessa forma, o presente artigo propõe investigar a Psicologia Positiva e o Design Positivo, aplicado a projetos na área da saúde pela perspectiva do DOP – Design Orientado para Possibilidades – com atenção para as premissas de acessibilidade e inclusão, evidenciadas como particularidades a serem adotadas no desenvolvimento de qualquer projeto de design. Palavras-chave: Design Positivo, Saúde, Acessibilidade, Inclusão. Design para Acessibilidade e Inclusão: Design Positivo e DOP no desenvolvimento de projetos em saúde. Design for Accessibility and Inclusion: Positive Design and DOP in the development of health projects. 1. Introdução A relação entre Design e Psicologia aponta na direção das primeiras instituições para formação de desenhistas industriais, a exemplo das escolas Bauhaus e Ulm, que sofreram grande influência da Psicologia por meio da Teoria da Gestalt. Nesse contexto, há evidências da influência da psicologia no campo do Design e do Design através de elementos da psicologia em projetos na área da saúde, o que podemos constatar ao analisarmos alguns projetos destacados na presente pesquisa. Projetos na área da saúde, por sua própria natureza, buscam atender necessidades e requisitos de acessibilidade e inclusão, contribuindo diretamente para o desenvolvimento de políticas públicas. De frente a essa posição o designer pode ver-se provocado a promover e difundir propostas metodológicas e possíveis soluções que atendam demandas na área da saúde se valendo de sua capacidade de interpretação e criatividade. Desse modo, o designer se posiciona como um possível agente de transformação social, por estender sua atuação a relações com outras áreas propondo projetos com implicações tão especificas e importantes, no que tange ao bem-estar individual com consequências sociais significativas. Na virada dos anos 2000 surge a Psicologia Positiva com a proposta de expandir o foco das pesquisas e práticas clínicas da área da psicologia para além do diagnóstico e tratamento de psicopatologias, olhando também para a construção de qualidades positivas. (MANDELLI et al, 2019, p.41). A atividade do design, segundo Desmet e Hassenzahl (2012, p.1.2) é entendida como uma atividade centrada na remoção de problemas (ou seja, para tornar algo mais fácil, mais limpo, mais barato, mais seguro ou mais pequeno), muitas vezes motivada por discrepâncias muito concretas entre a forma atual e uma forma aparentemente ideal de fazer algo. A aspiração é tornar o mundo um lugar melhor através da resolução dos seus problemas. [...] A concepção orientada para problemas centra-se na "cura de doenças", ou seja, na eliminação de problemas predominantes, em vez de se concentrar diretamente naquilo que nos faz felizes. 1. Introdução Para a Psicologia Positiva e o Design Positivo abordagens tradicionais, a exemplo do design como solução de problemas são limitantes e não geram experiências, diferente do que propõe o Design Positivo pela abordagem do Design Orientado para Possibilidade (DOP) que busca contribuir para geração de experiências positivas e melhor qualidade de vida das pessoas Portanto, este artigo propõe uma reflexão entre Psicologia e Design, com foco no Design Positivo pela abordagem DOP e as possibilidades de aplicações dessa abordagem em projetos na área da saúde preconizando os princípios da acessibilidade e de inclusão, com o objetivo de contribuir para geração de experiências e o bem-estar dos envolvidos. Abstract Design and Psychology have always gone together, both with the same goal of providing well-being to the individual, in the case of design, through solutions to the needs and desires of man, and in the case of psychology through the treatment of 'diseases' or situations related to the mind. In this sense, we seek to establish the understanding of well-being related to health, as Positive Psychology has already done by adopting it as the last element of the PERMAH model, which is used as a strategy to measure a person's well-being index. Thus, this article proposes to investigate Positive Psychology and Positive Design, applied to projects in the health area from the perspective of PDO - Possibility-Oriented Design - with attention to the premises of accessibility and inclusion, evidenced as particularities to be adopted in the development of any design project. Keywords: Positive Design, Health, Accessibility, Inclusion. 3. Psicologia Positiva Em 1998 a psicologia, na pessoa do Dr Martin Seligman (2012), à época presidente da Associação Americana de Psicologia, sofria a intervenção de um modelo não convencional de atenção à saúde dos pacientes, a saber, a Psicologia Positiva, defendida e justificada por ele como ‘um movimento científico e profissional’, em seu livro FLORESCER: Uma nova compreensão sobre a natureza da felicidade e do bem-estar. (SELIGMAN, 2012, p.9) Seligman é considerado, precursor desse movimento, que em um primeiro momento, contrariando os métodos tradicionais da Psicologia, abordava a temática da felicidade como objetivo principal, o que se tornaria a primeira teoria da Psicologia Positiva: a Felicidade Autêntica. Seligman (2012, p.9) afirma que o objetivo da Psicologia, em seus moldes tradicionais era aliviar o sofrimento e eliminar as condições debilitantes da vida, de acordo com o autor, um dos objetivos é romper com o Monismo1 Aristotélico de que: [...] todo conhecimento e todo trabalho visa a algum bem, quais afirmamos ser os objetivos da ciência política e qual é o mais alto de todos os bens que se podem alcançar pela ação. Verbalmente, quase todos estão de acordo, pois tanto o vulgo como os homens de cultura superior dizem ser esse fim a felicidade e identificam o bem viver e o bem agir como o ser feliz. Diferem, porém, quanto ao que seja a felicidade, [...]. (ARISTOTELES, 322 a.C.) Para Seligman, (2012, p.13), o objetivo dessa ciência é explorar aquilo que faz a vida valer a pena e produzir condições para isso, afirmando que, o intuito de compreender o bem-estar e produzir condições capacitadoras de vida de modo algum se iguala ao de compreender o sofrimento e desfazer as condições debilitantes da vida, proposta da psicologia tradicional. No ano de 2002, Seligman estabelece a Teoria da Felicidade Autêntica e em 2005, durante disciplina ministrada por ele na Universidade Huntsman Hall, Filadélfia, EUA, ao abordar a Teoria da Felicidade Autêntica, questionado por sua aluna Senia Maymin, o autor repensa sua primeira teoria e sugere a Teoria do Bem-estar, concluindo que a felicidade não poderia ser o objetivo da psicologia positiva. (SELIGMAN, 2012, p.14) A teoria do Bem-estar, proposta por Seligman (2012, p.16), é sustentada a partir da análise de cinco elementos que se afastam do monismo da felicidade - emoção positiva, engajamento, sentido, relacionamentos positivos e realização. 2. Referencial Teórico Para o desenvolvimento da pesquisa adotamos para discorrer sobre Psicologia Positiva, SELIGMAN (2012). Para abordamos Design, definições e finalidade, utilizaremos como literatura (CARDOSO (2008); MORAES, 2010; VERGANTI (2012), MANDELLI et al (2019). Design Positivo será discutido a partir de DESMET (2013); POLHMEYER (2013); CAETANO e VAN DER LINDEM (2018), PIMENTEL (2020); ROSA E TONETTO (2020); COTRIN (2021), e por fim, sobre Design e Saúde FREIRE (2016), BRITO (2018); ULBRICHT et al (2017), AMPHILÓQUIO E SOBRAL (2018). Design e Saúde FREIRE (2016), BRITO (2018); ULBRICHT et al (2017), AMPHILÓQUIO E SOBRAL (2018). 3. Psicologia Positiva E ainda por se tratar de uma teoria de livre escolha cujos elementos possuem três importantes propriedades: contribuir para a formação do bem-estar; a busca pelo elemento em si e não por satisfação pessoal; e, deve ser definido e mensurado independente dos outros elementos. Portanto, o objetivo da Psicologia Positiva é o Florescimento Humano, principal critério para a avaliação do Bem-Estar. Segundo Seligman: A teoria da felicidade autêntica é uma tentativa de explicar uma coisa real - a felicidade -, definida pela satisfação com a vida, (...). A teoria 1 [Filosofia] Sistema filosófico segundo o qual a realidade (tudo o que existe) se reduz somente em um único princípio, estando os seres condicionados a ele. do bem-estar nega que o tema da psicologia positiva seja uma coisa real; ele é, antes, um construto - o bem-estar -, que por sua vez tem diversos elementos mensuráveis, cada um deles uma coisa real e cada um deles contribuindo para formar o bem-estar, mas nenhum deles o definindo. (SELIGMAN, 2012, p.15) do bem-estar nega que o tema da psicologia positiva seja uma coisa real; ele é, antes, um construto - o bem-estar -, que por sua vez tem diversos elementos mensuráveis, cada um deles uma coisa real e cada um deles contribuindo para formar o bem-estar, mas nenhum deles o definindo. (SELIGMAN, 2012, p.15) A partir de então, a Psicologia Positiva passa a aplicar diversos elementos no desenvolvimento de sua própria abordagem no tratamento dos pacientes assistidos por essa alternativa, contrariando os métodos tradicionais com foco no tratamento da ‘doença’ ou, na linguagem do design, na solução de problemas. Portanto, a Psicologia Positiva, pelos moldes da Teoria do bem-estar se propõe não apenas a romper com o Monismo de Aristóteles, bem como contribuir para o bem-estar das pessoas e melhor qualidade de vida. 3 Missão DIOPD - https://diopd.org/about-us/mission/ 03 de março de 2022. – Interpretação livre 3.1 Design Positivo Segundo Caetano e Van Der Lindem (2018, p.56), o debate do papel do bem-estar no Design se inicia com Desmet (2011), ao propor que o bem-estar individual dos usuários poderia ser melhorado através do uso de produtos sugerindo que, os produtos poderiam dar suporte em atividades que contribuíssem para o bem-estar dos usuários através do desfrute de prazeres, favorecimento de conexões sociais e ajuda no seu crescimento pessoal. No entanto, constatou que os produtos não eram a fonte direta do bem-estar dos participantes de sua pesquisa, mas sim davam suporte em atividades que promovessem o seu bem-estar. Ainda de acordo com Pohlmeyer (2013, 540-541), em anos anteriores a Assembleia Geral das Nações Unidas, sobre temática da felicidade para políticas públicas, no ano de 2011, notou-se, um interesse crescente acerca da ciência da felicidade com ênfase na Psicologia Positiva; porém, pouco foi explorado, por parte do design, no tocante ao seu potencial, relevância e aplicabilidade, e das percepções e resultados da referida teoria para criar e melhorar produtos e serviços para o bem-estar subjetivo e florescimento humano Nesse contexto, Jimenez, et al (2015, p.607) afirma que: O designer ainda dá pouca importância a Psicologia Positiva como atributo fundamental para o desenvolvimento de sua atividade, ainda há alguns equívocos por parte dos designers, como exemplo, muitos consideram que o designer precisa ‘apenas’ de ferramentas e métodos, que é necessário considerar mudanças, tendências e diferenças sociais (ignorando as suas origens) e se limitam apenas a ‘conversar’ com potenciais usuários, não se aprofundando no que as pessoas sentem e porque sentem, nem em que características despertam tais sentimentos. (Idem) O Design Positivo parte da abordagem do Design Orientado para Possibilidades, contrariando a abordagem tradicional do Design concentrada na solução do problema, propondo a geração de experiências e o bem-estar. De acordo com Cotrin (2021, p.45) enquanto alternativa a essa abordagem tradicional, diversos pesquisadores, entre eles, Jordan (2000), Desmet (2002,2007), Norman (2004), McCarthy & Wright (2004), Hassenzahl (2010), Gaver (2002), Dunne (2006) e Zimmerman (2009), têm impulsionado essa corrente de pensamento: Para os autores tanto da abordagem pelo viés do problema, quanto pelo viés da possibilidade, buscam a modificação do estado atual para um estado futuro. Ambas reconhecem a existência de um problema/questão da pesquisa, porém a diferença está no tipo de reação ao problema. 3.1 Design Positivo A ONU – Organização das Nações Unidas – em 2011, convocada pelo Butão, país que adotou o Felicidade Interna Bruta (FIB) como índice de avaliação para medir o desenvolvimento de sua população (GOMES, 2013, p.77-73), realizou a Assembleia Geral das Nações Unidas, convocando os 193 países participantes a incluir a felicidade e o bem-estar na promoção de políticas públicas. No mesmo ano surge o Delf Institute of Positive Design (DIOPD) formado por pesquisadores, designers e psicólogos, que compreendem a responsabilidade de gerar conhecimento que permitam aos designers formular estratégias eficazes de forma a contribuir para a felicidade das pessoas. Esse conhecimento não deve apenas ajudá-los em suas tentativas de projetar deliberadamente relações significativas entre produto e usuário, mas, em última análise, projetar produtos que contribuam para uma sociedade saudável, para tornar o mundo um lugar melhor2. Em 2013, os pesquisadores Pieter Desmet e Anna Pohlmeyer propuseram o Design Positivo com objetivo principal de responder “Como o design pode contribuir para o bem-estar dos usuários? ”, sugerindo que futuros estudos no design deveriam fomentar atividades que apoiassem a busca de uma vida mais gratificante para os usuários de produtos e consequentemente favorecessem seu bem-estar. No mesmo ano, Pohlmeyer propôs o Manifesto do Design Positivo, com propósito de englobar estudos, pesquisas e intenções em Design Segundo os pesquisadores, Design Positivo é um termo para todas as formas de Design, pesquisa de Design e intenção de Design que prestam atenção explícita ao impacto do Design no bem-estar subjetivo de indivíduos e comunidades, que cria possibilidades, promove florescimento humano, permite atividades significativas, abraça a complexidade humana, aceita a responsabilidade e apoia todas as partes interessadas3. 3 Missão DIOPD - https://diopd.org/about-us/mission/ 03 de março de 2022. – Interpretação livre Segundo Caetano e Van Der Lindem (2018, p.56), o debate do papel do bem-estar no Design se inicia com Desmet (2011), ao propor que o bem-estar individual dos usuários poderia ser melhorado através do uso de produtos sugerindo que, os produtos poderiam dar suporte em atividades que contribuíssem para o bem-estar dos usuários através do desfrute de prazeres, favorecimento de conexões sociais e ajuda no seu crescimento pessoal. No entanto, constatou que os produtos não eram a fonte direta do bem-estar dos participantes de sua pesquisa, mas sim davam suporte em atividades que promovessem o seu bem-estar. 3.1 Design Positivo Enquanto o design orientado para solução de problemas foca no presente, o design orientado para possibilidades foca no futuro, nas potencialidades; o primeiro contém um caráter de busca de uma solução, sendo ela singular e excludente, enquanto o segundo lida com multiplicidade de natureza expandida e inclusiva. (COTRIN, 2021, p.45.46) É importante notarmos que, a fala de Cotrin (2021), motivada por importantes pesquisadores - psicólogos e designers - argumentam a favor do compromisso do design com o desenvolvimento de abordagens que rompam com os métodos tradicionais do design, que tem por fundamento a solução de problemas, semelhante ao que propõe a Psicologia Positiva em relação aos métodos da Psicologia Tradicional ao tratar o paciente simplesmente pela cura da doença. em relação aos métodos da Psicologia Tradicional ao tratar o paciente simplesmente pela cura da doença. em relação aos métodos da Psicologia Tradicional ao tratar o paciente simplesmente pela cura da doença. Portanto, o Design Positivo provoca pesquisadores e designers a pensarmos desenvolvimento de projetos na área da saúde pela perspectiva do Design Orientado para Possibilidades, de forma a contribuirmos para o maior bem-estar das pessoas, por meio da geração de experiências positivas e não apenas da solução de problemas. Anna Pohlmeyer (2018) observou que, segundo a Organização Mundial da Saúde, a saúde é mais do que a ausência de doença – é também uma questão de bem-estar mental e social e que a saúde física vem sendo abordada pelo design por décadas e mais recentemente o design para o bem-estar psicológico mereceu atenção no campo do design. Tal observação foi apresentada em palestra realizada em 1º de junho de 2018 na Conferência Saúde Apoiada pela Tecnologia, organizada pelo Centro de Pesquisa em Health & Wellbeing da Universidade de Twente, localizada em Enschede, nos Países Baixos, com o objetivo de discutir o progresso e desenvolvimento de intervenções em saúde e introduzir novos métodos e modelos para mudança de comportamento usando tecnologias de monitoramento. A pesquisadora e psicóloga também abordou as oportunidades para o design, a exemplo de tecnologias de intervenções comportamentais na forma de aplicativos pessoais que apoiam pessoas com treinamentos e serviços objetivando melhorar a saúde e bem-estar. 3.1 Design Positivo Porém, a pesquisadora afirma que, apesar de haver evidências cientificas sobre a eficácia dessas intervenções, estas ainda enfrentam grandes desafios, a exemplo do alcance limitado e que o Design pode ter impactos no bem-estar das pessoas através de rotinas já existentes no dia a dia4. 3.2 Design Orientado para Possibilidade Como ocorreu com a Psicologia Positiva surgida na década de 1990, com uma nova abordagem para assuntos tratados pelos métodos tradicionais, o Design Positivo também emerge, a partir do Design Orientado para Possibilidades, como alternativa aos métodos tradicionais do design. Enquanto a Psicologia Positiva propõe uma nova abordagem para o tratamento de pessoas, como alternativa ao método tradicional, o Design Positivo, por meio da abordagem do Design Orientado para Possibilidade propõe ao Design tradicional, uma proposta de intervenção em Design por meio da geração de soluções em projetos cujo foco não seja o problema em si, mas o usuário, de maneira a promover experiências positivas para este. O Design Positivo surge como, uma alternativa à abordagem do design orientado para problemas, e pode contribuir criando experiências positivas (a vida prazerosa/hedonismo), mas também estimulando a consciência das pessoas, das suas capacidades, desafiando ou inspirando o usuário a agir ou pensar de uma forma diferente (a boa vida/eudaimonia). DESMET E HASSENZAHL (2012, p.1- 11) Desmet (2010, p.2) descreve o design orientado a problemas como mera tentativa de “manter os demônios dormindo”, uma metáfora em alusão às preocupações, valores e necessidades das pessoas, despertadas apenas quando a situação representa uma ameaça à sua realização. O design precisa de um ponto de partida, uma ideia, uma semente para nutrir e crescer. Enquanto uma abordagem orientada para o 4 https://diopd.org/brave-new-tech-at-health-supported-by-technology/ 06 de março de 2022 problema toma um problema como um começo, uma abordagem orientada para a possibilidade procura uma possibilidade. É importante ressaltar que essa possibilidade deve estar enraizada em nosso conhecimento da felicidade, da prática humana e nas necessidades humanas. Muito facilmente, uma tecnologia por si só, pode ser entendida como uma possibilidade. (DESMET & HASSENZAHL, 2012, p.11) Segundo Jimenez et al, (2014, p.607), falamos de "um problema" quando alguém quer alcançar um objetivo e os meios para o fazer não são imediatamente óbvios. A resolução de problemas é o processo de pensamento, em que aqueles meios são procurados intencionalmente. Há numerosos exemplos que ilustram esta visão tradicional orientada para o problema do design. [...]. Esta forma tradicional de resolução de problemas é relevante uma vez que aumenta a qualidade e eficácia dos produtos e serviços que todos nós usamos, procura minimizar situações problemáticas que pode ameaçar o nosso bem-estar, e geralmente torna nosso viver 'mais fácil' e 'melhor’. 3.2 Design Orientado para Possibilidade No entanto, neutralizando o negativo ao resolver os nossos problemas cotidianos não significa necessariamente proporcionando uma experiência positiva e valiosa (Desmet e Hassenzahl, 2012). Focando o lado positivo da solução espectro com uma abordagem orientada para as possibilidades promete um uma nova perspectiva sobre o papel do design. A visão orientada para a possibilidade sobre o design foi recentemente proposta na comunidade de pesquisa em design. Nas palavras de Hekkert e van Dijk (2011, p. 120), o trabalho de um designer é procurar possibilidades e futuros possíveis, em vez de simplesmente resolver problemas atuais. O design, portanto, pode contribuir para o bem-estar ao criar e mediar experiências positivas ao estimular a consciência das pessoas sobre suas capacidades e, pela perspectiva do DOP, pode criar ou apoiar novas possibilidades, considerando que o processo da abordagem do design para possibilidade não é tão evidente quanto a abordagem tradicional, apresentando desafios interessantes à pesquisa, à atuação do designer e ao ensino em design. 4. Design e Saúde Os tópicos anteriores trouxeram propostas de conceitos e teorias desenvolvidas em torno do Design Positivo e da Psicologia Positiva. Tais referências interessam na medida em que, possibilitam abrir espaço de diálogo para a relação entre Design e Saúde, vislumbrado novos caminhos para que o designer possa expandir sua atuação em projetos voltados para a área da saúde, com foco em acessibilidade e inclusão, favorecendo e contribuindo para o desenvolvimento de políticas públicas. O Design é uma área de atuação profissional de grande complexidade, com características que possibilitam ao profissional dessa área, o designer, propor soluções para demandas das mais diversas áreas. A definição do design, segundo Verganti (2012, p.22), é algo fluido e escorregadio, por haver várias discussões em torno do seu significado e, por consequência, a falta de convergência. O design compreendido pela definição funcionalista do século XX de que ‘a forma segue a função’, conceito aplicado pelo arquiteto americano Louis Sullivan (18965) é considerado por Verganti como limitante. compreendido pela definição funcionalista do século XX de que ‘a forma segue a função’, conceito aplicado pelo arquiteto americano Louis Sullivan (18965) é considerado por Verganti como limitante. compreendido pela definição funcionalista do século XX de que ‘a forma segue a função’, conceito aplicado pelo arquiteto americano Louis Sullivan (18965) é considerado por Verganti como limitante. No entanto, o autor sugere como definição, a proposta por Thomas Maldonado (1963), adotada pela International Council of Societies of Industrial Design (Conselho Internacional das Sociedade de Desenho Industrial) em 1969 e que passou por alterações, sendo inserido o design de serviços, processos e sistemas: Design é a atividade criativa cujo objetivo é estabelecer as multifacetadas qualidades de objetos, processos, serviços e seus sistemas no ciclo de vida completo. O design é, portanto, fator central para humanizar as inovações tecnológicas e um fator crucial de intercâmbio cultural e econômico. (VERGANTI, 2012, p.24) Design é a atividade criativa cujo objetivo é estabelecer as multifacetadas qualidades de objetos, processos, serviços e seus sistemas no ciclo de vida completo. O design é, portanto, fator central para humanizar as inovações tecnológicas e um fator crucial de intercâmbio cultural e econômico. (VERGANTI, 2012, p.24) Segundo Verganti (2012, p. 5 A frase "a forma segue a função" foi cunhada pelo arquiteto Louis H. Sullivan em seu ensaio de 1896 "The Tall Office Building Artistically Considered". "The Tall Office Building Artistically Considered". 4. Design e Saúde Se todos adquirirem alguma consciência do tamanho e do intricado das relações que regem o mundo hoje, será possível caminhar coletivamente em direção a um objetivo, seja qual for. Do ponto de vista histórico o autor afirma que: O grande trabalho do design tem sido ajustar conexões entres coisas que antes eram desconexas, hoje entendido por projetar interfaces. Trata-se, contudo, de um processo bem maior e mais abrangente do que imagina o projetista sentado à sua estação de trabalho. A parte de cada um é entender a sua parte no todo. (Idem). Em relação a Cardoso (2013), podemos compreender que, de fato, o designer possui um papel significativo no tocante ao desenvolvimento de soluções que, atendam às necessidades do mundo, enquanto sistema complexo, e é nessa perspectiva que esse campo tende a colaborar cada vez mais com o desenvolvimento de outras áreas, a exemplo de projetos que possibilitem sua intervenção, inclusive propondo novas metodologias, abordagens e reflexões. Portanto, o design não apenas pode propor novas abordagens metodológicas, a exemplo do Design Orientado para Possibilidade, como sugere o Design Positivo, como também prever sua atuação mais abrangente, no que corresponde a busca por novas possibilidades de sua atuação. Já a relação do design com a área da saúde não é necessariamente direta, mas pode ser sentida em projetos que não foram pensados inicialmente para saúde, mas possuem implicações e resultados positivos ou negativos, gerando de fato algum tipo de experiência ao usuário, como veremos a seguir em alguns exemplos. Verganti (2012, p.63) cita como referência o projeto do vídeo game Wii da fabricante Nitendo, lançado em 2006, considerado um projeto de produto revolucionário, pelo fato de que, no contexto de sua abordagem, este propôs uma mudança radical em significado, quando comparado aos seus concorrentes, passando de uma imersão passiva virtual acessível somente para poucos para um entretenimento que inclui atividade física real para todos, posteriormente, vindo a ser utilizado na prática de atividades físicas, como terapia e até mesmo em processo de reabilitação de pessoas acometidas pelo Acidente Vascular Encefálico (AVE), tornando-se elemento de socialização. É importante considerarmos que o design, de forma direta ou indireta, possui forte influência em aspectos relacionados ao bem-estar e, consequentemente, à saúde do indivíduo, já que estes dois elementos estão intrinsicamente relacionados. 4. Design e Saúde 37), o design engloba todas as profissões criativas que modificam seu ambiente: engenharia, medicina, administração, arquitetura e pintura estão preocupados não apenas com o que é essencial, mas com todas as possibilidades – não como as coisas são, mas como podem vir a ser, resumindo, com o design. Em sintonia e para além do que aponta Verganti (2012) “o design pela sua capacidade multidisciplinar e transversal, fornece rápidas respostas por meio de produtos, imagens e novas possibilidades de interação”, de acordo com MORAES (2010, p. 10). Nesse âmbito, segundo esse autor, o design como uma disciplina transversal (e mesmo ‘atravessável’), ao aceitar e propor interações multidisciplinares, amplia ainda o seu diálogo com disciplinas tecnológicas, econômicas e humanas, bem como com as do âmbito da gestão, da semiótica e da comunicação. É nessa diversidade presente na atividade do designer que compreendemos a responsabilidade, enquanto projetistas, ao propormos soluções para demandas de projetos existentes nas mais diversas áreas, inclusive na área da saúde, que exige por parte deste uma percepção maior de consequências futuras que um projeto mal resolvido possa provocar ou os tipos de experiências que um projeto nessa área, geralmente para atender uma necessidade ou demanda especifica, possa causar. Ao trabalhar o Design sob a ótica transversal e considerá-lo um campo político, que possui responsabilidade social e desenvolve soluções para as necessidades dos usuários, observa-se a importância do diálogo entre várias áreas, a fim de suprir as carências dos impactados. (PIMENTEL et al, 2020, p.178) CARDOSO (2008, p.225), ao referir-se ao Design e a importância de examinar a paisagem material que nos cerca, afirma que nos deparamos com problemas de Design crônicos em áreas como transportes, saúde, equipamentos urbanos e uma infinidade de outras instancias do cotidiano. Para este autor, mesmo fora do âmbito dos serviços públicos, em um setor privado voltado por definição para questões de competitividade e dinamismo, percebe-se um desconcerto frequente entre as preocupações da profissão e aquelas da própria sociedade. privado voltado por definição para questões de competitividade e dinamismo, percebe-se um desconcerto frequente entre as preocupações da profissão e aquelas da própria sociedade. privado voltado por definição para questões de competitividade e dinamismo, percebe-se um desconcerto frequente entre as preocupações da profissão e aquelas da própria sociedade. CARDOSO (2013, p.22) considera que, reconhecer a complexidade do sistema já é um grande avanço. 4. Design e Saúde O designer pode não ter pensado nas implicações da solução ou resposta a que se chegou para determinada demanda, necessidade ou oportunidade, mas possivelmente a aplicação e posterior utilização dessa resposta dada pelo design terá consequências na vida de determinado usuário ou conjunto de usuários, sendo possível e cabível ao designer, nesse contexto, prever o futuro ao analisar o que o uso de determinado design pode acarretar a comunidade. Um exemplo do impacto negativo do design na área da saúde, é o que ocorreu em 2003 com o modelo de carro Fox, da Volkswagen. Considerado resultado de um projeto inovador, um modelo compacto, estiloso e mais espaçoso que seus concorrentes, porém em 2004 começou a apresentar um problema recorrente que teve por consequência, diversos casos registrad a apresentar um problema recorrente que teve por consequência, diversos casos registrados de pessoas que tiveram dedos decepados pelo sistema de rebatimento do banco traseiro6. a apresentar um problema recorrente que teve por consequência, diversos casos registrados de pessoas que tiveram dedos decepados pelo sistema de rebatimento do banco traseiro6. O Wii, de forma positiva, e o Modelo Fox da Volkswagen, de forma negativa, nos advertem a respeito do impacto do design na sociedade e a grande responsabilidade no desenvolvimento de projetos independentes de qual área estejamos atuando. Porém, é importante notarmos que, só recentemente, a própria Psicologia Positiva, teria inserido a Saúde como novo elemento no Modelo PERMA, proposto inicialmente por Seligman, sendo Emiliya Zhivotovskaya, portanto, considerada responsável por, expandir o modelo PERMA, passando a ser PERMAH (Health) ou PERMA – V (Vitality). O acrônimo7 PERMAH ou PERMA-V significa: P = Positive Emotions (Emoções Positivas); E = Engajement (Engajamento); R = Relashionships (Relacionamentos); M = Meaning (Significado); A = Accomplishment (Realização); H = health (Saúde) ou V = Vitality (Vitalidade). O Conceito de saúde mais aceito foi desenvolvido em 1947 e definido pela Organização Mundial da Saúde (OMS) como "um estado de completa saúde física, mental, e bem-estar social e não apenas a ausência de doença ou enfermidade". Este conceito da OMS está, em parte, alinhado com o modelo holístico, e reforça as conotações positivas de saúde. 4. Design e Saúde (PAHO, 2018, p.4) Freire (2016, p.5) quanto ao Design voltado para projetos em saúde, afirma ser a principal contribuição do Design reduzir a complexidade do conjunto de atividades que caracterizam tal processo, sejam criando plataformas capazes de suportar essas interações, seja auxiliando o usuário como ele pode estar utilizando determinado sistema. Segundo a autora: [...] as experiências dos usuários não podem ser projetadas. Por consequência, os designers podem apenas analisar como elas se formam e, partindo disso, criar alternativas para os elementos que as definem. [...] uma vez entendida a experiência, o papel do designer é conduzir o processo de projeto de maneira a desenvolver interfaces, processos ou interações que carreguem não apenas as experiências estéticas, mas também os significados esperados pelos usuários nessa interação. (Ibidem, p.8) De acordo com Amphilóquio e Sobral (2018, p.171) o designer, enquanto agente de inovação social pode também provocar rupturas significativas na cultura, inclusive criar novas necessidades e comportamentos. O Design, no decorrer da história, sofreu importantes mudanças que alteraram o seu modo de projetar e de entender a sociedade, a preocupação deixou de ser apenas industrial e passou a ganhar novas modelagens, como o design participativo, o design thinking, o design sustentável e o design centrado no humano, que eles consideram serem aberturas significativas para a promoção de uma sociedade inclusiva. Segundo Brito (2018, p.23) o Design se insere na perspectiva da saúde como uma área do saber capaz de contribuir na obtenção dos melhores resultados em prol da promoção da saúde a 6 https://jornaldocarro.estadao.com.br/fanaticos/defeitos-famosos-o-vw-fox-que-decepava-dedos/ 14 de fevereiro de 2022, 12h45 Tenho dúvida sobre essa fonte. 7 Diz-se de ou palavra formada pela inicial ou por mais de uma letra de cada um dos segmentos sucessivos de uma locução, ou pela maioria dessas partes; acrossílabo; 7 Diz-se de ou palavra formada pela inicial ou por mais de uma letra de cada um dos segmentos sucessivos de uma locução, ou pela maioria dessas partes; acrossílabo; partir de uma série de atuações, seja no campo do desenvolvimento de produtos, comunicação, elementos gráficos, ambientes ou serviços. partir de uma série de atuações, seja no campo do desenvolvimento de produtos, comunicação, elementos gráficos, ambientes ou serviços. partir de uma série de atuações, seja no campo do desenvolvimento de produtos, comunicação, elementos gráficos, ambientes ou serviços. 4. Design e Saúde Ao desenvolver sua pesquisa em Design de Moda com o objetivo de minimizar medo e estresse provocados pelo processo de tratamento de crianças com câncer, por meio de uma proposta lúdica, Pimentel (2020) aproxima o design da área da saúde partindo de princípios essenciais ao design e a saúde para compor estratégias apresentadas como resultados em sua pesquisa, a exemplo do Mapa de Empatia e da Neurociência. A autora afirma que: A realidade da criança com câncer é múltipla e complexa. O contexto que envolve o diagnóstico e o tratamento da doença ultrapassa os limites farmacológicos e medicinais, [...]. As dificuldades se estendem para além da doença do corpo: atingem as emoções, as relações de afeto e aprendizagem e a forma de se relacionar com o mundo e com o futuro. Esses obstáculos permeiam também a relação da criança com o vestuário, trazendo novas necessidades. O campo do Design de Moda, aliado à Neurociência e à Psicologia, abre possibilidades para o vestuário, que pode tornar-se ferramenta de auxílio à criança com câncer potencializando a minimização dos sofrimentos. (Idem, p.179) Cotrin (2021, p.46) ao avaliar as contribuições do design positivo no tratamento de crianças com Transtorno de Espectro Autista, afirma que, pensar um projeto do ponto de vista das possibilidades para o desenvolvimento do potencial humano, pode contribuir para uma perspectiva ampliada. Cotrin (2021, p.46) ao avaliar as contribuições do design positivo no tratamento de crianças com Transtorno de Espectro Autista, afirma que, pensar um projeto do ponto de vista das possibilidades para o desenvolvimento do potencial humano, pode contribuir para uma perspectiva ampliada. Desmet e Hassenzahl (2012), acerca da abordagem do Design Orientado para Possibilidade, afirma sobre o projeto da prótese de pernas cheetah-foot da empresa Össur: Tradicionalmente, as próteses são desenvolvidas dentro do "modelo de descontração" da tecnologia: ter duas pernas humanas é visto como a situação ideal, não as tendo como um "problema". Como consequência, as próteses visam muitas vezes imitar completamente a função e especialmente a aparência da situação ideal - pernas reais. Deixando de lado esta abordagem focada no problema, contudo, permitiu a Össur desenvolver membros revolucionários de fibra de carbono, Cheetah Flex-Foot, que não imitam pernas humanas e que se tornaram famosos por atletas internacionais como Oscar Pistorius. Fonte: Google imagens (2021). Nesse sentido, o Design aplicado a projetos na área da saúde, por meio da abordagem DOP propõe novas perspectivas para esse campo de atuação, com a possibilidade de revisão dessa literatura, aperfeiçoando e intensificando o uso dessa metodologia como alternativa a metodologia tradicional e, contribuindo com a sociedade e o desenvolvimento, também, de novos modelos em políticas públicas. 8 Também conhecido como design sem barreiras e relacionado ao design universal e ao design inclusivo (LIDWELL et al, 2010). 4. Design e Saúde (DESMET E HASSENZAHL, 2012, p.5) Os autores explicam que, em vez de entenderem a ausência de pernas como um problema a resolver, os desenhadores utilizaram uma situação aparentemente problemática como uma possibilidade de explorar material e tecnologia para criar um novo tipo de pernas. Figura 1 – Prótese de pernas cheetah- foot da empresa Össur de pernas cheetah- Össur de pernas cheetah- Össur de pernas cheetah- Össur Figura 1 – Prótese foot da empresa Figura 1 – Prótese foot da empresa Fonte: Google imagens (2021). 5. Acessibilidade e Inclusão Ao tratarmos de acessibilidade e inclusão não limitamos o tema à área da saúde, são muito mais amplas estas definições e é preciso que seja pensado dessa maneira ao nos referirmos a estes assuntos, a começar por não os distinguir, mas aproximá-los, com o objetivo de explorarmos ainda mais a aplicação do design à saúde, que não se trata apenas dos aspectos físicos. Lidwell et al (2010, p.16) afirmam que objetos e ambientes devem ser projetados para serem utilizados, sem modificação, pelo maior número de pessoas8, pelo princípio da acessibilidade. Segundo os autores, a história nos mostra a acessibilidade concentrada em acomodar usuários com deficiências, porém, à medida que o conhecimento e a experiência com o design acessível aumentaram, ficou cada vez mais claro que muitas “adaptações” obrigatórias poderiam ser projetadas para beneficiar todos os usuários, sendo necessário compreender o design acessível a partir de suas quatro características: Perceptibilidade – Obtida quando o design é percebido por todos, independentemente de suas capacidades sensoriais; Operabilidade – Obtida quando todos conseguem utilizar o design independentemente de suas capacidades físicas; Simplicidade – Obtida quando todos conseguem compreender e utilizar o design sem dificuldade, independentemente dos níveis de experiência, alfabetização e concentração; e, Condescendência – Obtida quando os designs diminuem a ocorrência e as consequências dos erros. (LIDWELL et al, 2010, p.16) Cohen et al (2012, p.17) ao abordarem a acessibilidade relacionado a museus, afirmam que há uma dimensão política da acessibilidade, que está estreitamente relacionada ao exercício da cidadania, a qual, para contemplar adequadamente a pluralidade e a diversidade dos modos de ser e de estar no mundo, que caracterizam o conjunto de cidadãos, implica a adoção de uma visão ampliada do conceito de acessibilidade. Na fala dos autores, fica claro que, acessibilidade deve incluir todos, não apenas pessoas com deficiências, porém devem ser pensadas à luz dessas pessoas, sendo possível a partir dessa compreensão adentrarmos ao conceito de inclusão. No que trata do desenvolvimento de projetos, tanto no aspecto de acessibilidade quanto de inclusão, talvez uma premissa básica sugerida por Caya e Neto (2016, p. 5. Acessibilidade e Inclusão 55-56), que deva ser considerada e difundida, é o entendimento de dois conceitos: 1 - Adaptabilidade, que consiste na propriedade de um produto/sistema mudar ou ser mudado a fim de se adequar ou trabalhar melhor em alguma situação para algum propósito, ou seja, que permite que o usuário altere explicitamente certas características do mesmo, para adequá-los às suas vontades e necessidades. 2- Adaptatividade, que consiste na propriedade que um produto/sistema tem de se adaptar ao usuário, ou seja, apresenta uma capacidade para adaptação. O sistema se ‘adapta’ de acordo com sua percepção do usuário, e o usuário não solicita alterações no sistema, diferente do que ocorre com a adaptabilidade. (Ulbricht et al, 2009, p.34) Por fim, projetos em Design na área da saúde devem ter como premissas acessibilidade e inclusão. Estes projetos, como afirmam, Mandelli e Tonetto (2019, p.5) podem resultar em artefatos, serviços ou até dinâmicas que despertem experiências afetivas especificas, emoções, estados de humor, bem-estar subjetivo ou até mesmo empatia. 6. Considerações Finais Como principal abordagem metodológica, partimos do Design Orientado pela Possibilidade, fundamentada em pesquisa bibliográfica e revisão literária acerca da relevância do Design Positivo como alternativa à metodologia do design tradicional. Nesse sentido, a utilização dessa metodologia busca possibilitar o desenvolvimento de projetos em Design que, atendam à área da saúde proporcionando aos usuários bem-estar, principalmente, por meio de experiências positivas com foco nas premissas de acessibilidade e inclusão. Ao discorrermos sobre a relação do design e sua aplicabilidade a projetos que visam atender às necessidades na área da saúde, compreendemos que há grande potencial nessa relação e grandes possibilidades de obtermos resultados cada vez mais mensuráveis e validadas que possam ser úteis à expansão dessa literatura. Ao investigarmos a Psicologia Positiva e consequentemente o Design Positivo pela abordagem DOP, concluímos que a relação entre Design e Psicologia é histórica e pode ser positiva para ambos os campos de pesquisa, já que eles têm por objetivo proporcionar o alcance do bem- estar humano e social. Por fim, outro importante aspecto nessa relação são os princípios de acessibilidade e inclusão aqui analisados, porém de forma mais objetiva, ainda havendo muito a se investigar. Nesse sentido, o design é pensado de forma mais ampla, além do conceito estabelecido como característica fundamental do design da primeira modernidade, de caráter funcionalista, proposto por Louis Sullivan, de que ‘a forma segue a função’, sugerindo a partir dessa colocação, uma revisão na maneira como pensamos design, psicologia, saúde, bem-estar, acessibilidade e inclusão. 7. Referências Bibliográficas AMPHILÓQUIO, Willian; SOBRAL, João Eduardo C. Design e sociedade: uma reflexão sobre acessibilidade, interação e inclusão. HFD, v.7, n.13. Fev/jul. 2018, p.171. ARISTÓTELES, Ética à Nicômaco. Texto integral. Brasília, Editora Martin Claret, 2003, p.5. BRITO, Talita. Contribuições para o cuidado da doença de Alzheimer e outras demências. AMPHILÓQUIO, Willian; SOBRAL, João Eduardo C. Design e sociedade: uma reflexão sobre acessibilidade, interação e inclusão. HFD, v.7, n.13. Fev/jul. 2018, p.171. AMPHILÓQUIO, Willian; SOBRAL, João Eduardo C. Design e sociedade: uma reflexão sobre acessibilidade, interação e inclusão. HFD, v.7, n.13. Fev/jul. 2018, p.171. ARISTÓTELES, Ética à Nicômaco. Texto integral. Brasília, Editora Martin Claret, 2003, p.5. BRITO, Talita. Contribuições para o cuidado da doença de Alzheimer e outras demências. 2018, 115 p. Dissertação (Mestrado em Design) - Universidade de Brasília, 2018. CAETANO, Ulisses.; VAN DER LINDEN, Júlio. Além do Design Positivo. 2018, Cap. 3, p. 55-76. CARDOSO, Rafael. Uma Introdução a história do Design. São Paulo: Editora Edgard Blücher, 2008, p.225. CAETANO, Ulisses.; VAN DER LINDEN, Júlio. Além do Design Positivo. 2018, Cap. 3, p. 55-76. CARDOSO, Rafael. Uma Introdução a história do Design. São Paulo: Editora Edgard Blücher, 2008, p.225. CARDOSO, Rafael. Uma Introdução a história do Design. São Paulo: Editora Edgard Blücher, 2008, p.225. CARDOSO, Rafael. Design para um mundo complexo. São Paulo: Editora Cosac Naify, 2013. CASAIS, M., MUGGE, R. & DESMET, PMA. Using symbolic meaning as a means to design for happiness: The development of a card set for designers. 2016, p.1. COHEN, Regina; DUARTE, Cristiane R. de S.; BRASILEIRO, Alice de Barros H. Cadernos Museológicos: Acessibilidades a museus. Instituto Brasileiro de Museus. Brasília, 2012, p. 17. COTRIN, Michelle de A. Design e bem-estar: Contribuições do Design Positivo para crianças dentro do Transtorno do Espectro Autista. 2021, 169 p. Tese (Doutorado em Design) - Escola de Design da Universidade de Minas Gerais, Belo Horizonte. Delft Institute of Positive Design. Nossa Missão. DIOPD. Disponível em: <https://diopd.org/about-us/mission/>. Acesso em: 22/06/2021. DESMET, Pieter & HEKKERT, Paul. Special Issue Editorial: Design & Emotion. International Journal of Design, 2009, p.1. DESMET, Pieter & HASSENZAHL, Marc. Towards happiness: Possibility-driven design. 2012, p. 2.8. DESMET, Pieter. Design for Mood: Twenty Activity-Based Opportunities to Design for Mood Regulation. International Journal of Design Vol. 9 Nº. 2, 2015, p. 0-19. FREIRE, Karine de M.; DAMAZIO, Vera M. Design para serviços: uma intervenção em uma Unidade Básica de Saúde do Sistema Único de Saúde Brasileiro. Estudos em Design. 7. Referências Bibliográficas Revista (online). Rio de Janeiro, v.24. n.2. 2016, p.5.8. GOMES, Paulo Sérgio F. O Butão na ordem mundial do reconhecimento da sua identidade nacional à influência nas relações externas. 2013, 313 p. Tese (Doutorado em Teoria Jurídico- Política e Relações Internacionais) – Universidade de Évora, Portugal, 2013. JIMENEZ, Simon; POHLMEYER, Anna; DESMET, Pieter; HUZEN, Gerjan. Learning from the positive: A Structured Approach to Possibility-Oriented Design. Em J. Salamanca, P. Desmet, A. Burbano, G. Ludden, J. Maya (Eds.). Proceedings of Colors of Care: The 9th International Conference on Design & Emotion, 2014, 607-615. LIDWELL, Willian.; HOLDEN, Kritina.; BUTLER, Jill.; Princípios Universais do Design. 2010, p.16 MANDELLI, Roberta R.; TONETTO, Leandro M. Design para empatia: brinquedos e brincadeiras como oportunidade para promover o desenvolvimento emocional. Porto Alegre. Design Culture Symposium, 2019, p.41. MANDELLI, Roberta R.; ROSA, Valentina M.; TONETTO, Leandro M. Da emoção à felicidade: uma introdução ao bem-estar com foco no positive design. Rio de Janeiro. 2019, p.5. MORAES, Dijon De. Metaprojeto: O design do design. São Paulo: Blucher, 2010. P.4. Pan American Health Organization. Health Indicators. Conceptual and operational considerations. Washington, D.C.: PAHO; 2018, p.4. PIMENTEL, Marina C. Design de moda e ludicidade: minimização do medo e estresse de crianças em tratamento de câncer. Dissertação (Mestrado em Design) – Universidade Anhembi Morumbi. São Paulo, 2020, p. 18. POHLMEYER, Anne. Positive Design: New Challenges, Opportunities, and Responsibilities for Design. In: A. Marcus (Ed.): Design, User Experience, and Usability, Part III, LNCS 8014. Berlin, Germany: Springer-Verlag, 2013, p. 540. ROSA, Valentina M.; TONETTO, Leandro M. Modelo Conceitual de Design para o Bem-estar para pacientes pediátricos. Colóquio Internacional de Design. Edição, 2020, p.2. SELIGMAN, Martin E. P. Florescer: uma nova compreensão sobre a natureza da felicidade e do bem-estar. Ed. Objetiva, 2012. O carro que decepava dedos. Disponivel em: <https://jornaldocarro.estadao.com.br/fanaticos/defeitos-famosos-o-vw-fox-que-decepava- dedos/>. Acesso em: 07/03/2022. Pan American Health Organization. Health Indicators. Conceptual and operational considerations. Washington, D.C.: PAHO; 2018, p.4. SELIGMAN, Martin E. P. Florescer: uma nova compreensão sobre a natureza da felicidade e do bem-estar. Ed. Objetiva, 2012. ULBRICHT, Vania R.; FADEL, Luciane M.; BATISTA, Claudia R. Design para acessibilidade e inclusão. Ed. Blucher. São Paulo, 2017, p.34. VERGANTI, Roberto. Driven Inovation. Mudando as regras da competição: A inovação do significado de produtos. São Paulo: Editora canal Certo, 2012, p. 22.37.63 Vitalidade – O bem estar físico da psicologia positiva. 2021. Disponivel em: <https://bemestarnaessencia.com.br/vitalidade-o-bem-estar-fisico-do-modelo-perma-v/> Acesso em 14/08/2022.
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Testicularis adrenalis rest tumor a congenitalis adrenalis hyperplasia hátterében
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ESETISMERTETÉS ESETISMERTETÉS Testicularis adrenalis rest tumor a congenitalis adrenalis hyperplasia hátterében Brzózka Ádám dr.1 ■ Kuthi Levente dr.2 ■ Hajdú Erzsébet dr.3 Magony Sándor dr.4 ■ Király István Előd dr.3 Kincses Zsigmond Tamás dr.1 ■ Szatmári Angelika5 Szalma Emese oh. ■ Palkó András dr.1 ■ Fejes Zsuzsanna dr.1, 6 1Szegedi Tudományegyetem, Általános Orvostudományi Kar, Radiológiai Klinika, Szeged 2Szegedi Tudományegyetem, Általános Orvostudományi Kar, Pathologiai Intézet, Szeged 3Szegedi Tudományegyetem, Általános Orvostudományi Kar, Urológiai Klinika, Szeged 4Szegedi Tudományegyetem, Általános Orvostudományi Kar, I. Belgyógyászati Klinika, Szeged 5Szegedi Tudományegyetem, Egészségtudományi és Szociális Képzési Kar, Szeged 6Affidea Diagnosztika, Szeged A congenitalis adrenalis hyperplasia különféle megjelenési formáiban a testicularis adrenalis rest tumorok prevalenci­ ája eltérő. A tapintható, általában kétoldali hereelváltozás az esetek 90–95%-ában a 21-hidroxiláz enzim hiánya okoz­ ta mellékvesekéreg-hyperplasia következményeként jelenik meg. Az adrenalis rest tumorok, bár jól ismert elváltozá­ sok, diagnózisuk és kezelésük multidiszciplináris megközelítést igényel. Az endokrinológiai, urológiai, patológiai és radiológiai szakmák együttműködése kiemelkedően fontos. Amennyiben korai stádiumban felismerésre kerül, adek­ vát szteroidhormon-emelés segíthet a térfoglalás méretének csökkentésében. Késői felismerés esetén azonban irreve­ zibilis károsodás jön létre, mely infertilitáshoz vezet. Mivel a kezelés az egyéb jó- és rosszindulatú hereelváltozások­ hoz képest jelentősen különbözik, fontos a korai és pontos diagnózis felállítása. Esetismertetésünk kapcsán a testicularis adrenalis rest tumorok differenciáldiagnoszikai és kezelési nehézségeit multidiszciplináris szemszögből mutatjuk be. j Orv Hetil. 2020; 161(16): 623–631. Kulcsszavak: testicularis adrenalis rest tumor, férfieredetű meddőség (Beérkezett: 2019. november 30.; elfogadva: 2020. január 18.) Brzózka Á, Kuthi L, Hajdú E, Magony S, Király IE, Kincses ZsT, Szatmári A, Szalma E, Palkó A, Fejes Zs. [Testi­cular adrenal rest tumor in the background of congenital adrenal hyperplasia]. Orv Hetil. 2020; 161(16): 623–631. Rövidítések 4Dion = androszténdion; ACTH = (adrenocorticotropic hor­ mone) adrenokortikotrop hormon; anti-TPO = thyreoidea- peroxidáz elleni antitest; AR = androgénreceptor; CAH = con­ genitalis adrenalis hyperplasia; CRH = (corticotropin-releasing hormone) kortikotropinfelszabadító hormon; FSH = folli­ culusstimuláló hormon; LH = luteinizáló hormon; LST = ­Leydig-sejtes tumor; MR = (magnetic resonance) mágneses rezonancia; TART = testicularis adrenalis rest tumor; UH = ultrahang A jelen dolgozatban, egy eseten keresztül, több szak­ ma szempontjából szeretnénk bemutatni a testicularis adrenalis rest tumor (TART) felismerésének és differen­ ciáldiagnosztizálásának a buktatóit, valamint az inter­ diszciplináris kivizsgálásának részleteit. Az Orvosi Heti­ lapban egy hasonló, de más módon felderített eset is­ mertetését találtuk [2]. A scrotum ultrahang (UH)-vizsgálata könnyen, gyorsan, költséghatékonyan hozzáférhető eljárás, melynek során a térfoglaló folyamatok jól karakterizálhatók, továbbá a lá­ tottak a meddőségi állapothoz is fontos információt szolgáltatnak [1]. Mivel a páciensek nagy része meddő­ ségi kivizsgálás keretében érkezik, az így felismert eltéré­ sek állítják a leginkább kihívás elé az andrológiában és UH-diagnosztikában dolgozó szakembereket. A heré­ ben lévő tumorok a fiatal, fertilis korosztályt érintik, de a Testicular adrenal rest tumor in the background of congenital adrenal hyperplasia The prevalence of testicular adrenal rest tumours varies in different forms of congenital adrenal hyperplasia. Patients with 21-hydroxilase deficiency usually have bilateral and palpable testicular nodules. Although adrenal rest tumours are well documented in the literature, the diagnosis and management require a multidisciplinary approach: the coop­ erative work of endocrinologists, urologists, pathologists and radiologists is essential. In the case of an early diagnosis, appropriately increased corticosteroid treatment may reduce the tumour mass. In advanced stages, tumours can lead to irreversible parenchymal damage causing infertility. The importance of an early and accurate diagnosis cannot be emphasized enough, since the therapy differs significantly from other benign or malignant testicular neoplasia. A case of a testicular adrenal rest tumour is presented along with the multidisciplinary perspectives of the diagnosis and management of these lesions. Keywords: testicular adrenal rest tumour, male infertility Brzózka Á, Kuthi L, Hajdú E, Magony S, Király IE, Kincses ZsT, Szatmári A, Szalma E, Palkó A, Fejes Zs. [Testi­cular adrenal rest tumor in the background of congenital adrenal hyperplasia]. Orv Hetil. 2020; 161(16): 623–631. (Beérkezett: 2019. november 30.; elfogadva: 2020. január 18.) 2020 ■ 161. évfolyam, 16. szám ■ 623–631. DOI: 10.1556/650.2020.31696 ■ © Szerző(k) 623 Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS meddőségi kivizsgálás során találhatunk olyan intratesti­ cularis térfoglalást is, mely az esetek egy kis részében be­ nignus. Ezen esetek nagy körültekintést igényelnek a megfelelő terápia megválasztása és a fertilitás biztosítása érdekében. Esetismertetés A 24 éves férfit újszülöttkora óta congenitalis adrenalis hyperplasiával (CAH) gondozzák. Három hónappal a születése után, hányások miatt kezdődő kivizsgálás során került sor a 21-hidroxiláz enzim hiányának kimutatására, melynek következtében klasszikus sóvesztő CAH alakult 1. táblázat A beteg laborértékei és gyógyszeres kezelése a bemutatott időszakban Gyógyszeres kezelés 17-OH- progesz­ teron, nmol/l (0,6–2,5) Andro­ szténdion, nmol/l (2,9–9,5) DHEA, nmol/l (8,5–36,0) Teszto­ szteron, nmol/l (9,90– 28,00) SHBG, nmol/l (14,5– 48,4) DHEA-S, μmol/l (5,73– 13,40) LH, IU/l (1,7–8,6) FSH, IU/l (1,5–12,4) ACTH, pmol/l (1,60– 13,90) Első észlelés Hidrokortizon: 15–10–10 mg Fludrokortizon: 0,05 mg 358 >35 4,7 18,71 37,8 1,60   4,0   8,5 91,56 Terápia­ módosítás Hidrokortizon: 10–5–0 mg Fludrokortizon: 2 × 0,025 mg Prednizolon: 5 mg 114 16,5 4,2 14,69 42,7 1,19   5,8 15,7 17,04 I. kontroll Hidrokortizon: 10–5–0 mg Fludrokortizon: 2 × 0,025 mg Prednizolon: 7,5 mg   <0,3 – 3,9 16,92 69,5   4,4 10,8 12,07 II. kontroll Hidrokortizon: 10–5–0 mg Fludrokortizon: 2 × 0,025 mg Prednizolon: 2 × 7,5 mg >75,0 >35,0 3,3 11,21 69,2 1,90   2,8   7,8 64,79 III. kontroll Műtét >75,0 >35,0 4,1   8,66 62,3 1,51   5,8 10,5 16,39 Műtét utáni kontroll Hidrokortizon: 10–5–0 mg Fludrokortizon: 2 × 0,025 mg Prednizolon: 2 × 7,5 mg   12,8 – 4,9   7,03     45 0,82 15,5 15,6   0,92 ACTH = adrenokortikotrop hormon; DHEA = dehidroepiandroszteron; DHEA-S = dehidroepiandroszteron-szulfát; FSH = folliculusstimuláló hormon; LH = luteinizáló hormon; SHBG = nemihormon-kötő globulin 1. táblázat A beteg laborértékei és gyógyszeres kezelése a bemutatott időszakban ACTH = adrenokortikotrop hormon; DHEA = dehidroepiandroszteron; DHEA-S = dehidroepiandroszteron-szulfát; FSH = folliculusstimuláló hormon; LH = luteinizáló hormon; SHBG = nemihormon-kötő globulin 2020 ■ 161. évfolyam, 16. szám ORVOSI HETILAP 624 Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UT ESETISMERTETÉS ki. A beteg a diagnózis felállítása óta folyamatos minera­ lo- és glükokortikoid-szteroid kezelésben részesült gyer­ mekgyógyászaton. Kórelőzményében még adenotomia és appendectomia szerepel. A beteg gyermek- és serdü­ lőkori kontrollvizsgálatokra rendszeresen eljárt, gyógy­ szerelését igény szerint módosították. ki. A beteg a diagnózis felállítása óta folyamatos minera­ lo- és glükokortikoid-szteroid kezelésben részesült gyer­ mekgyógyászaton. Kórelőzményében még adenotomia és appendectomia szerepel. A beteg gyermek- és serdü­ lőkori kontrollvizsgálatokra rendszeresen eljárt, gyógy­ szerelését igény szerint módosították. att további gyógyszermódosítás történt: este 7,5 mg prednizolon szedését javasoltuk, egyéb gyógyszereinek meghagyásával. A következő reguláris kontroll során eredményei jó terápiás effektust igazoltak, a vizsgálati laborértékek a normáltartományon belülre kerültek. Ezen eredmények alapján a beteg kezelését változatlanul folytattuk. A beteget 21 éves korában irányították a felnőttendo­ krinológiára. Belszervi panaszt első észlelésekor, illetve a további kontrollok során sem említett, rákérdezésre sze­ xuális diszfunkciót sem. Ekkor gyógyszeres kezelése a következő volt: reggel 15 mg, délben 10 mg, este 10 mg hidrokortizon per os, valamint reggel 0,05 mg fludrokor­ tizon volt; emellett tachycardia miatt 5 mg nebivololt is szedett. Vérvétel történt, melyből a terápia és a betegkö­ vetés szempontjából legfontosabb értékeket táblázatos formában rögzítettük (1. táblázat). Az alap-laborered­ ményekből kiemelendő a normáltartományban lévő nát­ rium- és káliumszint: nátrium 141 mmol/l (136–145), kálium 4,3 mmol/l (3,5–5,1). A kalcium- és parathor­ monszint nem mutatott eltérést, a pajzsmirigyfunkció euthyreosist igazolt, az anti-TPO a normáltartományon belül volt. A hasi, kismedencei UH-vizsgálat kórosat nem mutatott a mellékvesék területén. A magas ACTH-, 17-OH-progeszteron- és 4Dion-érték miatt gyógyszeres kezelésének módosítását javasoltuk az alábbiak szerint: 10–5–0 mg hidrokortizon per os, 2 × 0,025 mg fludro­ kortizon, este 5 mg prednizolon per os, kiegészítve napi 600 mg kálium-klorid per os adásával. Soron következő kontrolljakor a hormonmérések a korábbiakhoz képest rosszabbul kezelt állapotot jeleztek: mind a 17-OH-progeszteron, mind a férfi-, valamint az adrenokortikotrop hormon kikerült a normáltartomány­ ból. Ezek az aktuálisan nem a céltartományban lévő eredmények – összehasonlítva a korábban céltartomány­ ban álló eredményekkel – felvetették a compliance-prob­ léma lehetőségét is, ezért a beteg és családja ismételt részletes felvilágosítást kapott a betegségről és annak ke­ zeléséről; emellett a prednizolon dózisát 2 × 5 mg-ra emeltük, egyéb gyógyszereinek változatlan dózisban tör­ ténő szedése mellett. Az ismételt ellenőrzés során az emelt dózisú glüko­ kortikoidkezelés ellenére az eredmények továbbra sem voltak a céltartományban. Urológiai vizsgálatot javasoltunk, amelynek során jö­ vőbeli fertilitási igény kivizsgálásakor azoospermia igazo­ lódott, ezért here-UH-vizsgálat történt. Korábban here- UH-vizsgálat nem volt. Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UT Az UH-vizsgálatot GE LOGIQ S8 (General Electric, Boston, MA, Amerikai Egyesült Államok) ultrahangkészülékkel végeztük. A vizsgálat so­ rán normális mérettartományban lévő heréket észlel­ tünk, melyeken belül inhomogén echogenitású, helyen­ ként részben piszkos hangárnyékot adó összefüggő képlet volt látható mindkét oldalon. A képletek főként a mediastinum testist érintették (1. ábra). A jobb here fel­ ső pólusán egy, nagyjából 35 × 25 × 30 mm-es, durván Ennek hatására hormonértékei a következő vizsgálat során javulást mutattak. Mind a 17-OH-progeszteron, az androszténdion és az adrenokortikotrop hormon szintje csökkent ugyan, de nem kerültek a normáltarto­ mányba. Így a csökkenő tendenciát mutató, de céltarto­ mány feletti ACTH- és 17-OH-progeszteron-érték mi­ 1. ábra Az ultrahangvizsgálat lineáris (7–14 MHz) fejjel történt; jól látható a kétoldali intratesticularis térfoglalás, mely echószegényebb a környező állomány­ nál, a jobb oldalon a here felső pólusára lokalizálódik elsősorban, bal oldalon testicularis adrenalis rest tumorra (TART) jellegzetes képet mutat, a mediastinum testisre lokalizálódik Az ultrahangvizsgálat lineáris (7–14 MHz) fejjel történt; jól látható a kétoldali intratesticularis térfoglalás, mely echószegényebb a környező állomány­ nál, a jobb oldalon a here felső pólusára lokalizálódik elsősorban, bal oldalon testicularis adrenalis rest tumorra (TART) jellegzetes képet mutat, a mediastinum testisre lokalizálódik 20 ■ 161. évfolyam, 16. szám 625 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Az intraoperatív mikroszkó­ pos képen látható a vékony hereparenchyma alatt a porckemény tapintatú terime. Jelentősen tágult heretubulus nem volt megfigyelhető (b) a) b) ESETISMERTETÉS b) a) 4. ábra Intraoperatívan a heretok intaktnak bizonyult. Egy szürkésfehér, porckemény szövet tűnt elő (a). Az intraoperatív mikroszkó­ pos képen látható a vékony hereparenchyma alatt a porckemény tapintatú terime. Jelentősen tágult heretubulus nem volt megfigyelhető (b) citoplazmájuk eozinofil, melyben helyenként lipidcsep­ pek voltak megfigyelhetők. Reinke-kristályok nem ábrá­ zolódtak. A citológiai atípia alacsonynak bizonyult, sejt­ osztódás nem volt jelen (5. ábra). Immunhisztokémiailag a sejtek diffúz CD56- és MelanA-pozitivitást, valamint részleges szinaptofizin- és androgénreceptor-pozitivitást mutattak. A látottak alapján a patológus a laesiót TART- nak véleményezte. A környező hereállományban súlyos parenchymakárosodás állt fenn. Ép spermogenezis sem a tumor mellett, sem pedig attól távolabb nem volt jelen. A tubulusok jórészt hialinizálódtak, egy-egy tubulusban pedig csupán Sertoli-sejtek voltak jelen (Johnsen-score 4/10) (6. ábra). Ez a szöveti megjelenés lényegében összhangban állt az obstruktív azoospermia klinikai di­ agnózisával. A beavatkozás során ép hímivarsejtet nem sikerült izo­lálni. sebészeti herebiopsziát végeztünk, melynek kapcsán – a páciens kérését is figyelembe véve – a térfoglaló folyama­ tokból is történt mintavétel. A műtét során a heretok mindkét oldalon intakt volt. A jobb herének körülbelül a kétharmadát igen tömött ta­ pintatú terime töltötte ki, a kicsiny ép szövet alatt egy porckemény, sárga színű elváltozás tűnt elő. A bal oldalon egy hasonló morfológiájú képlet mutatkozott (4. ábra). Az intraoperatív szövettani vizsgálat malignitást nem igazolt. A paraffinba ágyazott anyag szövettani elemzésé­ ből kiderült, hogy a megjelenés a két mintában egyezett. A kötőszövetes állományban egy-, illetve kétsejtes kö­ tegeket, valamint szolid fészkeket formáló celluláris pro­ liferáció volt kivehető. A laesionalis sejtek kicsik voltak, A műtét során a heretok mindkét oldalon intakt volt. A jobb herének körülbelül a kétharmadát igen tömött ta­ pintatú terime töltötte ki, a kicsiny ép szövet alatt egy porckemény, sárga színű elváltozás tűnt elő. A bal oldalon egy hasonló morfológiájú képlet mutatkozott (4. ábra). Az intraoperatív szövettani vizsgálat malignitást nem igazolt. A paraffinba ágyazott anyag szövettani elemzésé­ ből kiderült, hogy a megjelenés a két mintában egyezett. A kötőszövetes állományban egy-, illetve kétsejtes kö­ tegeket, valamint szolid fészkeket formáló celluláris pro­ liferáció volt kivehető. A laesionalis sejtek kicsik voltak, 5. ábra Kötegekbe, illetve szolid fészkekbe rendeződő sejtszaporulat, melynek környezetében kifejezett fokú kötőszövet-képződés észlelhető (hematoxilin-eozin festés, 100×-os nagyítás) (a). A laesionalis sejtek kicsik, a citoplazma vakuolizált. Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS 2. ábra Az ultrahangvizsgálat lineáris (7–14 MHz) volt, a B-módú vizsgálatot a gyér keringés kimutatására is érzékeny ’power’ Doppler-technikával egészítet­ tük ki. Jól látható, hogy a terimében a keringés igen gyér Az ultrahangvizsgálat lineáris (7–14 MHz) volt, a B-módú vizsgálatot a gyér keringés kimutatására is érzékeny ’power’ Doppler-technikával egészítet­ tük ki. Jól látható, hogy a terimében a keringés igen gyér 2. ábra jelzett, az ellenoldalon pedig egy 19 × 39 × 18 mm-es, szintén a mediastinum testis mellett elhelyezkedő, ha­ sonló megjelenésű képlet ábrázolódott (3. ábra). Mesze­ sedésre, necrosisra utaló jelek egyik oldalon sem voltak megfigyelhetők. A hasi MR-vizsgálat során csupán a mel­ lékvesék fokozott halmozása került leírásra, kóros nyi­ rokcsomó vagy metastasisra típusos eltérés nem volt ész­ lelhető. Radiológiai és urológiai konzílium történt. inhomogén terület, a bal oldalon pedig hasonló megjele­ nésű, 40 × 15 × 20 mm-es képlet került leírásra. Dopp­ ler-vizsgálattal fokozott vascularisatio nem volt látható az elváltozásokon belül (2. ábra). Annak ellenére, hogy a herékben lévő két eltérés ultrahangos megjelenése nem volt teljesen egyforma, a radiológiai vélemény, endokri­ nológussal konzultálva, elsősorban a TART lehetőségét vetette fel. Testicularis és hasi-kismedencei MR- (GE Discovery MR750w 3T; General Electric) vizsgálat is történt, mely a jobb herében egy 22 × 28 × 31 mm-es, T1-, T2-jel-szegény, a mediastinum testis magasságában elhelyezkedő, kissé lobulált kontúrú, éles szélű képletet Urológiai szakvizsgálat alkalmával ép külső genitáliák mellett normál nagyságú, azonban mindkét oldalon porckemény tapintatú herék voltak észlelhetők. Azoo­ spermia miatt sebészi spermiumnyerés céljából mikro­ 3. ábra Jól látható a B-módú ultrahangvizsgálat képein a bal herében a TART-ra jellegzetes térfoglalás (a). Az MRI T2-súlyozott képein jól látható a medias­ tinum testisben lévő jellegzetes lobulált, a hereparenchymához képest jelszegény elváltozás (b) MRI mágnesesrezonancia képalkotás; TART testicularis adrenalis rest tumor a) b) b) a) ható a B-módú ultrahangvizsgálat képein a bal herében a TART-ra jellegzetes térfoglalás (a). Az MRI T2-súlyozott képein jól látható a medias­ testisben lévő jellegzetes lobulált, a hereparenchymához képest jelszegény elváltozás (b) 3. ábra MRI = mágnesesrezonancia-képalkotás; TART = testicularis adrenalis rest tumor ORVOSI HETILAP 626 2020 ■ 161. évfolyam, 16. szám Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS Intraoperatívan a heretok intaktnak bizonyult. Egy szürkésfehér, porckemény szövet tűnt elő (a). Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC A citológiai atí­ pia minimális, sejtosztódás vagy Reinke-kristály nem látható (he­ matoxilin-eozin festés, 400×-os nagyítás) (b) A beteg további endokrinológiai kezelése, valamint rendszeres képalkotó vizsgálattal történő követése folya­ matban van. 6. ábra A herecsatornák jó részében csupán spermatogoniumok és ­Leydig-sejtek látszanak. Elvétve korai spermiocyták még jelen vannak, de kiérő spermiogenezis nem észlelhető. Az intersztíci­ umban kötőszövet-felszaporodás és enyhe fokú Leydig-sejt- hyperplasia figyelhető meg (hematoxilin-eozin festés, 200×-os nagyítás) A herecsatornák jó részében csupán spermatogoniumok és ­Leydig-sejtek látszanak. Elvétve korai spermiocyták még jelen vannak, de kiérő spermiogenezis nem észlelhető. Az intersztíci­ umban kötőszövet-felszaporodás és enyhe fokú Leydig-sejt- hyperplasia figyelhető meg (hematoxilin-eozin festés, 200×-os nagyítás) Kötegekbe, illetve szolid fészkekbe rendeződő sejtszaporulat, melynek környezetében kifejezett fokú kötőszövet-képződés észlelhető (hematoxilin-eozin festés, 100×-os nagyítás) (a). A laesionalis sejtek kicsik, a citoplazma vakuolizált. A citológiai atí­ pia minimális, sejtosztódás vagy Reinke-kristály nem látható (he­ matoxilin-eozin festés, 400×-os nagyítás) (b) 5. ábra 2020 ■ 161. évfolyam, 16. szám 627 ORVOSI HETILAP ORVOSI HETILAP Patológiai vonatkozások A TART elkülönítése a here csírasejtes tumoraitól, a ­lymphomától vagy a lágyrész-daganatoktól nem okoz ­diagnosztikai nehézséget, a TART differenciáldiagnosz­ tikájában viszont a legfontosabb entitás a Leydig-sejtes tumor (LST). A TART-ot felépítő sejtek egyszerre hor­ dozzák a mellékvesekéreg és a Leydig-sejtek jellemvoná­ sait, ami arra utal, hogy ezek a sejtek totipotens embrio­ nális őssejt eredetűek lehetnek [7]. Az LST-knek csupán a 3%-a bilaterális, ezzel szemben TART esetén a kétol­ daliság 83%-os. Míg az előbbi tumorok 10%-a rosszin­ dulatú, ezzel szemben az utóbbiak mindig jóindulatúak. Itt fontos hangsúlyozni, hogy a TART a neve ellenére hiperplasztikus folyamatnak és inkább pszeudotumor­ nak tekintendő, az LST viszont valódi neoplasiának felel meg. A két daganatféleség makroszkóposan is különbö­ zik, a TART ugyanis jellemzően egygócú, fekete laesió­ ként látható a here állományában, míg az LST gyakran több­gócú, sárga tumorok képében jelentkezik. Jelentős fokú fibrosis, zsírszöveti metaplasia, lymphoid sejtes aggregátumok, emelkedett citológiai atípia és kifejezett lipo­krómpigment-felhalmozódás gyakran megfigyelhe­ tő jelenségek TART esetén [8]. Továbbá az ilyen tumo­ rokban sejtosztódások egyáltalán nem vagy csak hosszas keresgélés után fedezhetők fel, jóllehet ezek az LST-ese­ tek 69%-ában jelen vannak. Habár a Reinke-kristályok­ ról úgy tartják, hogy patognomikusak a Leydig-sejtekre, ez a jellegzetesség az LST-knek csupán a 40%-ában mu­ tatható ki, és a TART-okban sosincs jelen, tehát a Rein­ ke-kristályok hiánya önmagában nem segíti a helyes di­ agnózis felállítását [6, 8]. Immunhisztokémiailag a legfon­tosabb marker a szinaptofizin, mely a TART-ese­ tek 88%-ában erős pozitív reakciót ad, ezzel szemben az LST-knek csupán a 8%-ában figyelhető meg hasonló in­ tenzitású pozitivitás. További fontos marker még a CD56, mely jellemzően pozitív a neuroendokrin tumo­ rokban, továbbá az androgénreceptor (AR), mely az előbbi daganat kapcsán általában negatív, az utóbbiban viszont pozitív reakciót ad. Nem segíti viszont a két en­ titás elkülönítését az inhibin-α, ez ugyanis mindkét tu­ morban expresszálódik. Az evidenciák alapján a klinikai adatok, a hormonszintek és a szövettani jellegzetességek mellett a szinaptofizint, CD56-ot és AR-t tartalmazó immunhiszto­kémiai panel segíti a helyes diagnózis felál­ lítását [9]. A környező hereállomány a folyamat kiter­ jedtségétől függően károsodott, a spermiogenezis kez­ detben kismértékben, majd pedig fokozatosan csökken. Előrehaladott esetekben csupán hialinizálódott vagy csak Sertoli-sejteket tartalmazó tubulusok vehetők ki a peritumoralis hereparenchymában. A spermiogramban a folyamat súlyosságától függően oligospermia vagy azoo­ spermia észlelhető. A TART és az LST közötti hasonló­ ságokat és különbségeket Wang és mtsai foglalták össze [10] Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS Patológiai vonatkozások Posztoperatív hormonális eredményei változatlan gyógyszeres kezelés mellett jó kezelési eredményt mutat­ tak, így gyógyszeres kezelését továbbra is változatlanul javasoltuk folytatni. A TART elkülönítése a here csírasejtes tumoraitól, a ­lymphomától vagy a lágyrész-daganatoktól nem okoz ­diagnosztikai nehézséget, a TART differenciáldiagnosz­ tikájában viszont a legfontosabb entitás a Leydig-sejtes tumor (LST). A TART-ot felépítő sejtek egyszerre hor­ dozzák a mellékvesekéreg és a Leydig-sejtek jellemvoná­ sait, ami arra utal, hogy ezek a sejtek totipotens embrio­ nális őssejt eredetűek lehetnek [7]. Az LST-knek csupán a 3%-a bilaterális, ezzel szemben TART esetén a kétol­ daliság 83%-os. Míg az előbbi tumorok 10%-a rosszin­ dulatú, ezzel szemben az utóbbiak mindig jóindulatúak. Itt fontos hangsúlyozni, hogy a TART a neve ellenére hiperplasztikus folyamatnak és inkább pszeudotumor­ nak tekintendő, az LST viszont valódi neoplasiának felel meg. A két daganatféleség makroszkóposan is különbö­ zik, a TART ugyanis jellemzően egygócú, fekete laesió­ ként látható a here állományában, míg az LST gyakran több­gócú, sárga tumorok képében jelentkezik. Jelentős fokú fibrosis, zsírszöveti metaplasia, lymphoid sejtes aggregátumok, emelkedett citológiai atípia és kifejezett lipo­krómpigment-felhalmozódás gyakran megfigyelhe­ tő jelenségek TART esetén [8]. Továbbá az ilyen tumo­ rokban sejtosztódások egyáltalán nem vagy csak hosszas keresgélés után fedezhetők fel, jóllehet ezek az LST-ese­ tek 69%-ában jelen vannak. Habár a Reinke-kristályok­ ról úgy tartják, hogy patognomikusak a Leydig-sejtekre, ez a jellegzetesség az LST-knek csupán a 40%-ában mu­ tatható ki, és a TART-okban sosincs jelen, tehát a Rein­ ke-kristályok hiánya önmagában nem segíti a helyes di­ agnózis felállítását [6, 8]. Immunhisztokémiailag a legfon­tosabb marker a szinaptofizin, mely a TART-ese­ tek 88%-ában erős pozitív reakciót ad, ezzel szemben az LST-knek csupán a 8%-ában figyelhető meg hasonló in­ tenzitású pozitivitás. További fontos marker még a CD56, mely jellemzően pozitív a neuroendokrin tumo­ rokban, továbbá az androgénreceptor (AR), mely az előbbi daganat kapcsán általában negatív, az utóbbiban viszont pozitív reakciót ad. Nem segíti viszont a két en­ titás elkülönítését az inhibin-α, ez ugyanis mindkét tu­ morban expresszálódik. Az evidenciák alapján a klinikai adatok, a hormonszintek és a szövettani jellegzetességek mellett a szinaptofizint, CD56-ot és AR-t tartalmazó immunhiszto­kémiai panel segíti a helyes diagnózis felál­ lítását [9]. A környező hereállomány a folyamat kiter­ jedtségétől függően károsodott, a spermiogenezis kez­ detben kismértékben, majd pedig fokozatosan csökken. Előrehaladott esetekben csupán hialinizálódott vagy csak Sertoli-sejteket tartalmazó tubulusok vehetők ki a peritumoralis hereparenchymában. A spermiogramban a folyamat súlyosságától függően oligospermia vagy azoo­ spermia észlelhető. A TART és az LST közötti hasonló­ ságokat és különbségeket Wang és mtsai foglalták össze [10]. Képalkotás – differenciáldiagnosztika bevérzést vagy meszesedést, továbbá a mediastinum tes­ tis mentén helyezkedik el [18]. Erezettsége a környeze­ ténél kissé csökkentebb. A lymphomától való elkülöní­ tésben nagyon fontos a color-Doppler-módban végzett vizsgálat, mivel a lymphoma vascularisatiója a környe­ zeténél fokozottabb, valamint a lymphoma ezen meg­ jelenése elsősorban nem a fiatal korosztályra jellemző megbetegedés. A primer testicularis lymphoma a non- Hodgkin-lymphomának egy viszonylag ritka formája, amelynél az átlagéletkor 66–68 év; a here malignus meg­ betegedéseinek kevesebb mint 5%-át teszi ki. A TART- nál ritkábban, csupán 6–10%-a kétoldali [13]. A herék térfoglaló folyamatai között a kétoldaliság gya­ korisága igen változatos megoszlást mutat. Gyermekkor­ ban az akut lymphoblastos leukaemia az esetek 5%-ában okozhat kétoldali eltérést [11]. Az elsősorban a fiatal populációt érintő, csírasejtes tumorok ritkán (1,8%-ban) válnak kétoldalivá [12]. Az idősebb populációban a csí­ rasejtes tumoroknál ritkábban előforduló lymphoma vagy az áttétek lehetnek kétoldaliak. A testicularis lym­ phoma a csírasejtes tumorokhoz képest sokkal ritkábban fordul elő [13]. Hereáttétek ismert tumoros betegekben nagyon ritkán, csupán 0,04%-ban fordulnak elő a heré­ ben, leggyakrabban a prosztatarák (35%), amelyet a tüdő-, a vese- és a vastagbélrák, valamint a melanoma malignum előrehaladott stádiumban adott áttétei követ­ nek. A metastasisok megjelenési gyakorisága 55 éves életkorra tehető. Az áttétek terjedése a lymphovascularis rendszeren keresztül történik, de a vese-, illetve a prosz­ tatarák áttéteinek kialakulásában fontos szerepe van a plexus pampiniformisban lévő vénatágulatnak és a vénás elégtelenség miatt kialakult ’reverse flow’-nak is. A ritkán előforduló intratesticularis metastasis fő feltételezett oka a vér–here-gát védőhatása, más régebbi elképzelések sze­ rint a herében lévő alacsonyabb hőmérséklet sem kedvez a daganatszövet növekedésének [14]. A korábbi térfog­ laló folyamatokkal szemben a TART jellemzően kétolda­ li elváltozást okoz. A differenciáldiagnózis megkönnyítésére Nuffer és ­mtsai döntési folyamatot dolgoztak ki. Ha az anemnesz­ tikus adatok alapján felmerül a TART, az UH-képen ­típusos lokalizációban echószegény, lobulált eltérés ­azonosítható akkor egyoldaliság esetén elsősorban a me­ szesedés jelenlétét kell kizárni, valamint a kontúrok meg­ jelenését kell jól megítélni. Amennyiben a kontúrok el­ mosódottak, vagy a vizsgált eltérésben meszesedés észlelhető, felmerül a tumoros eredet. Kétoldali eltérés esetén különösen figyelni kell a fokozott vascularisatióra, mely lymphomát jelezhet. A TART ultrahangos megjele­ nése a környező hereparenchymánál gyérebb vascularisa­ tiót mutat [18]. A TART-betegek MR-vizsgálatáról már a 2000-es évek előtt is jelentek meg cikkek, melyek leírták, hogy a TART a T1-szekvenciákon a normális hereállományhoz képest izo/hiperintenzív, a T2-szekvenciákon azonban hipointenzív megjelenésű [19, 20]. Az elváltozás mind­ két alapszekvencián homogén, tokkal nem határolt. Képalkotás – differenciáldiagnosztika Kontrasztanyag adása után az esetek 85%-ában volt meg­ figyelhető halmozás. Egy másik tanulmány szerint az összes TART homogén kontrasztanyag-halmozást mutat [21]. A cikkek szerzői egyetértenek abban, hogy az MR- vizsgálat (az alapszekvenciákat figyelembe véve) és az UH-vizsgálat diagnosztikus értéke megegyezik. Amen�­ nyiben egy beteg esetében szóba jön a részleges orchiec­ tomia, úgy az MR-vizsgálat már indokolttá válhat, mivel ez a műtéti tervezést segíti. Az eddig megjelent cikkek leginkább az alap-MR-szekvenciákat tanulmányozták TART esetében. Az MR-spektroszkópia hasznos lehet a hereállomány funkciójának megítélésében, mivel károso­ dott here esetében a colin csúcs csökkenést mutat, már a morfológiai elváltozások megjelenése előtt [22, 23]. A TART diagnózisának felállításában az UH és az MR képalkotó vizsgálatok nyújthatnak segítséget. Az elsődle­ ges szerep a jó elérhetőség és a költséghatékonyság miatt az UH-vizsgálatoké. A herék UH-vizsgálatának diagnosztikus hasznát már az 1970-es évektől kezdődően kutatták. 1977-ben Miskin az akkoriban csúcstechnológiás, 5 MHz-es transz­ducerrel végzett here-UH-vizsgálatok klinikai hasznát emelte ki [15]. A technika rohamosan fejlődött, és néhány éven belül megjelent az első közlemény, mely az UH-vizsgálatoknak a TART diagnózisában és köve­ tésében betöltött szerepéről szólt [16]. A szerzők szerint a CAH- és a primer mellékvese-elégtelenségben szen­ vedő betegek körében a herékben bilaterálisan, excentri­ kusan elhelyezkedő nodulusok – melyekben esetlegesen akusztikus árnyékolás is megfigyelhető – elsősorban TART-nak véleményezhetők. A TART ultrahangos megjelenése viszonylag típusos: a kezdeti stádiumban kis, multifokális, a környező here­ parenchymához viszonyítva echószegény képletek jelen­ nek meg a mediastinum testis területén; később ezek, összeolvadva, nagyobb echószegény területet képeznek, melyben hiperreflektív területek is lehetnek [17]. Az echódús megjelenés ritka. Éles szegélyű, lobulált, a here kontúrjait viszonylag kevéssé torzítja, rajta előboltosulást nem okoz. Differenciáldiagnosztikai kihívást jelent elkü­ löníteni a nem csírasejtes tumoroktól, úgymint a Leydig- sejtes vagy a Sertoli-sejtes tumortól. Fontos támpont le­ het, hogy a TART általában nem tartalmaz necrosist, Endokrinológiai háttér A 21-hidroxiláz-deficientia az egyik leggyakoribb mono­ génes anyagcsere-betegség, melynek magyarországi gya­ korisága 1 : 14 000 [3]. A CAH autoszomális recesszív módon öröklődő be­ tegségek összefogaló neve, melyeket a mellékvese sztero­ idhormon-szintézisének zavara jellemez. A különböző megjelenési formákban a mineralo-, glükokortikoid-, valamint a szexuálszteroidok bioszintézise károsodott. A 21-hidroxiláz enzim a mellékvesekéreg sejtjeinek sima felszínű endoplazmatikus reticulumában katalizálja a progeszteron és a 17-α-hidroxi-progeszteron hidroxilá­ lását. A 21-hidroxiláz enzim defektusának következté­ ben nem termelődik elég kortizol, ami miatt a hypotha­ lamusban, valamint a hypophysisben fokozódik a kortikotropinfelszabadító hormon (CRH) és az adreno­ kortikotrop hormon (ACTH) termelése. Ennek hatására egyrészt CAH alakul ki, másrészt pedig a szteroidszinté­ zis az androgének és ösztrogének felé tolódik. A megvál­ tozott hormontermelés következménye a lánygyerme­ kek virilisatiója, valamint a mindkét nemben megfigyel­ hető felgyorsult növekedés és csontosodás (klasszikus virilisatiós forma). Az enzimdefektus miatt az aldoszte­ ron szintézise is csökken, ami a betegek mintegy 75%- ában sóvesztéshez vezet (klasszikus sóvesztő forma), és korai, súlyos hyponatraemiás hypovolaemia kialakulását okozhatja. A 21-hidroxiláz enzim defektusának enyhébb formájában (nem klasszikus forma) a kortizol és az al­ doszteron szintézise normális tartományban van, az and­ rogének szintje azonban kissé emelkedett. Ezek a bete­ gek enyhébb tünetekkel jelentkezhetnek, mint például pubertas praecox, akné vagy fertilitási problémák [4].i A férfi CAH-betegek körében infertilitás alakulhat ki, melynek oka kettős: egyrészt a hypogonadismus, más­ részt a TART megjelenése, mely gyakori szövődménynek számít [3]. A TART kialakulásának patomechanizmusa nem teljesen ismert, feltehetőleg a magzati fejlődés során a here állományába került adrenocorticalis sejtekből ­alakul ki. Mivel a tumorok térfogata nagy dózisú kor­ tikoszteroidkezelésre általában csökken, valamint megje­ lenésének idején az ACTH-szint emelkedett, az valószí­ nűsíthető, hogy a magas szérum-ACTH-szint és luteini­ zálóhormon-hatás felelős a kialakulásukért. Egyes tanul­ mányok szerint a tumorsejteken ACTH-receptorok is megfigyelhetők, ami megerősíti, hogy embrionális kor­ ban mellékvese-eredetű sejtcsoportok szálltak le a herék­ kel együtt [5, 6]. A TART jellemzően a mediastinum testis területén jelenik meg, eredendően nem malignus, a mediastinum területén azonban fibrosist indukál, majd pedig obstruktív azoospermiához és meddőséghez vezet. 2020 ■ 161. évfolyam, 16. szám 628 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS Urológiai-andrológiai szempontok A CAH az infertilitás gyakori okozója. A testicularis ad­ renalis rest tumorok megjelenése jól ismert jelenség, és fontos komplikációnak számít, amely a betegek kb. 30– 94%-ában alakul ki. Tipikus megjelenési helye a medias­ tinum testis, ami miatt a heretubulusok elzáródhatnak, emellett az elváltozás a környező szövetek tartós károso­ dását okozza, ami következményes azoospermiához, így infertilitáshoz vezet [24]. A mechanikus obstrukció/ destrukció mellett a mellékvesekéreg-szteroidhormonok 2020 ■ 161. évfolyam, 16. szám 629 Megbeszélés és azok metabolitjainak lokális toxikus hatása is hozzájá­ rul a Sertoli- és/vagy a csírasejtek károsodásához [24, 25]. A nagy mennyiségben termelődő androgének pedig az LH és az FSH szuppresszióján keresztül tovább ront­ hatják a fertilitást [2]. A here térfoglaló folyamatainak hátterében döntően urológiai megbetegedések állnak, így a kivizsgálás és a kezelési terv is döntően urológiai szemléletet tükröz. Olyan esetekben viszont, amikor szisztémás társbetegség is fennáll, a multidiszciplináris megközelítés megkerül­ hetetlen. Korai stádiumban a daganattól távolabb lévő here­ parenchymából nyert szövettani minta még sok esetben nem mutat eltérést. Ennek lehetséges magyarázata a ká­ rosodás progresszív volta, mely a tumor körüli szövetek­ ben kezdődik, és a távolabbi területek csak később válnak érintetté. Ez alapján érthető, hogy nemcsak a daganatok lokalizációja, hanem azok mérete, illetve az obstrukció időtartama is fontos tényező a gonadális diszfunkció ki­ alakulása szempontjából [26]. Az elváltozás növekedésé­ vel a herében irreverzibilis károsodás alakul ki, mely az endokrin és az exokrin funkciót egyaránt érinti [24, 27]. Számos tanulmány igazolta, hogy az elváltozások ki­ mutatására here-UH-vizsgálat elvégzése a legalkalma­ sabb, mivel ezzel a még nem tapintható (<2 cm) laesiók is jól detektálhatók [24]. A 21-hidroxiláz-deficientia az egyik leggyakoribb mo­ nogénes anyagcsere-betegség; férfi betegek esetében in­ fertilitás elsősorban TART jelenléte, gonadotropin­ szuppresszió és herekárosodás miatt jöhet létre. Mivel a TART előfordulása CAH-ban 10 éves kor után növek­ szik, fontos a páciensek rendszeres, 1-2 évenkénti ultra­ hangos szűrése. A TART már a második stádiumtól könnyen jól elkülöníthető a mediastinum testis területén látható kicsi, jól körülhatárolt, echószegény eltérésként, mely gyakran kétoldali. Az időben megkezdett glüko­ kortikoidkezeléssel, valamint az ACTH-szintek szupp­ ressziójával a TART volumene jelentősen csökkenthető, kialakulása megelőzhető, így a fertilitás megtartható. Amennyiben a TART nem reagál a terápiára, a termé­ kenység megtartására a spermiumnyerés és -tárolás ­megfontolandó [29]. Amennyiben a TART felfedezése a fiatal felnőtt korra tolódik ki, fontos a pontos differenci­ áldiagnózis, hogy a beteget a feleslegesen végzett szemi­ kasztrációtól megóvjuk, amennyiben pedig azoospermia igazolódik, mielőbbi mikrosebészeti herebiopszia elvég­ zése javasolt a sikeres gyermekvállalás reményében. A si­ keres kezelés az endokrinológus, a képalkotást végző szakember, a patológus és az urológus/andrológus szo­ ros együttműködésén és a jó kommunikáción múlik. Számos tanulmány igazolta, hogy az elváltozások ki­ mutatására here-UH-vizsgálat elvégzése a legalkalma­ sabb, mivel ezzel a még nem tapintható (<2 cm) laesiók is jól detektálhatók [24]. Claahsen-van der Grinten és munkacsoportja a TART-ot 5 stádiumba sorolja, figyelembe véve a klinikai és a szö­ vettani megjelenést, valamint a tumor növekedését. Megbeszélés Az első stádiumban az elváltozás képalkotással még nem mutatható ki, az adrenalis rest sejtek a rete testis terüle­ tén helyezkednek el. Az UH-vizsgálatnak a második stá­ diumtól van jelentősége, amelynél a TART már kimutat­ ható, a mediastinum testis területén elhelyezkedő kicsi, echószegény elváltozás formájában. A harmadik stádi­ umtól a rete testis fokozatos kompressziója alakul ki, melyhez a négyes stádiumban már fibrosis társul. Az ötö­ dik stádiumban megjelenik a hereparenchyma károsodá­ sa a tömeges TART mellett [27]. A TART kezelését illetően a korai stádiumban végzett nagy dózisú hormonkezelés vagy a műtéti kezelés csök­ kenti a tumor méretét, eliminálja az obstrukciót, meg­ előzheti a további károsodást. Jóllehet a műtétet követő­ en a TART kiújulását nem figyelték meg, a beavatkozás a már kialakult károsodást nem javítja [2, 26, 28]. Anyagi támogatás: A közlemény megírása anyagi támo­ gatásban nem részesült. Szerzői munkamegosztás: B. Á.: A vizsgálati szempontok kidolgozása a képalkotás szemszögéből, szakirodalmi másodelemzés, adatgyűjtés, a kézirat szövegezése. K. L.: A vizsgálati szempontok kidolgozása a patológus szem­ szögéből, szakirodalmi másodelemzés, adatgyűjtés, a kézirat szövegezése. H. E.: A vizsgálati szempontok ki­ dolgozása az urológus szemszögéből, szakirodalmi má­ sodelemzés, adatgyűjtés, a kézirat szövegezése. M. S.: A vizsgálati szempontok kidolgozása az endokrinológus szemszögéből, szakirodalmi másodelemzés, adatgyűjtés, a kézirat szövegezése. K. Zs. T.: A kézirat szövegezése. Sz. A., Sz. E.: A kézirat szövegezése, rendszerezés, szak­ irodalmi másodelemzés. K. I. E.: Az andrológiai szem­ pontok bemutatása, szakirodalmi elemzés. P. A., F. Zs.: A kézirat szövegezése. A cikk végleges változatát vala­ mennyi szerző elolvasta és jóváhagyta. Szerzői munkamegosztás: B. Á.: A vizsgálati szempontok kidolgozása a képalkotás szemszögéből, szakirodalmi másodelemzés, adatgyűjtés, a kézirat szövegezése. K. L.: A vizsgálati szempontok kidolgozása a patológus szem­ szögéből, szakirodalmi másodelemzés, adatgyűjtés, a kézirat szövegezése. H. E.: A vizsgálati szempontok ki­ dolgozása az urológus szemszögéből, szakirodalmi má­ sodelemzés, adatgyűjtés, a kézirat szövegezése. M. S.: A vizsgálati szempontok kidolgozása az endokrinológus szemszögéből, szakirodalmi másodelemzés, adatgyűjtés, a kézirat szövegezése. K. Zs. T.: A kézirat szövegezése. Sz. A., Sz. E.: A kézirat szövegezése, rendszerezés, szak­ irodalmi másodelemzés. K. I. E.: Az andrológiai szem­ pontok bemutatása, szakirodalmi elemzés. P. A., F. Zs.: A kézirat szövegezése. A cikk végleges változatát vala­ mennyi szerző elolvasta és jóváhagyta. Azon betegeknél, akiknél a kétoldali TART hosszú ideje fennáll, a szervmegtartó sebészi kezelésnek nincs hatása a gonadális funkcióra, a spermaparaméterekben sem találtak javulást, továbbá a műtét okozta további ká­ rosodások veszélye sem zárható ki [24, 25, 27]. ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS 2020 ■ 161. évfolyam, 16. szám Irodalom [16]  Seidenwurm D, Smathers RL, Kan P, et al. Intratesticular adrenal rests diagnosed by ultrasound. Radiology 1985; 155: 479–481. [1]  Fejes Zs, Pásztor N, Karczagi L. The role of ultrasonography in the investigation of male infertility. [Az ultrahangmódszerek ­szerepe a férfimeddőség kivizsgálásában.] Orv Hetil. 2018; 159: 815–822. [Hungarian] [17]  Stikkelbroeck NM, Suliman HM, Otten BJ, et al. Testicular ad­ renal rest tumours in postpubertal males with congenital adrenal hyperplasia: sonographic and MR features. Eur Radiol. 2003; 13: 1597–1603. [18]  Nuffer Z, Lu M, Jefferson, et al. Sonographic spectrum of tes­ ticular adrenal rest tumors. Am J Sonogr. 2018; 1: 1–4. g [2]  Vajda Zs, Borbély G, Jakab Zs, et al. Bilateral testicular tumors in a young male with congenital 11β-hydroxilase deficiency. [11-β-hidroxiláz enzim defektusában szenvedő fiatal férfi kétol­ dali heredaganata.] Orv Hetil. 2010; 151: 1863–1868. [Hun­ garian] [19]  Walker BR, Skoog SJ, Winslow BH, et al. Testis sparing surgery for steroid unresponsive testicular tumors of the adrenogenital syndrome. J Urol. 1997; 157: 1460–1463. [20]  Avila NA, Premkumar A, Merke DP. Testicular adrenal rest tissue in congenital adrenal hyperplasia: comparison of MR imaging and sonographic findings. Am J Roentgenol. 1999; 172: 1003– 1006. g [3]  Doleschall M, Török D, Mészáros K, et al. Steroid 21-hydroxy­ lase deficiency, the most frequent cause of congenital adrenal hyperplasia. [Szteroid-21-hidroxiláz-deficiencia, a congenitalis adrenalis hyperplasia leggyakoribb oka.] Orv Hetil. 2018; 159: 269–277. [Hungarian] [21]  Yılmaz R, Şahin D, Aghayev A, et al. Sonography and magnetic resonance imaging characteristics of testicular adrenal rest tu­ mors. Pol J Radiol. 2017; 82: 583–588. [4]  White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev. 2000; 21: 245–291. [Correction: Endocr Rev. 2000; 21: 550.] [22]  Ozisik, H, Yurekli SB, Simsir YI, et al. Testicular adrenal rest ­tumor (TART) in congenital adrenal hyperplasia. Eur J Med Genet. 2017; 60: 489–493. [5]  Giacaglia LR, Mendonca BB, Madureira G, et al. Adrenal nod­ ules in patients with congenital adrenal hyperplasia due to 21-hy­ droxylase deficiency: regression after adequate hormonal con­ trol. J Pediatr Endocrinol Metab. 2001; 14: 415–419. [23]  Parenti GC, Albarello F, Campioni P. Role of MR spectroscopy (H1-MRS) of the testis in men with semen analysis altered. ­Reprod Syst Sex Disord. 2016; 5: 182. [6]  Bonaccorsi AC, Adler I, Figueiredo JG. Male infertility due to congenital adrenal hyperplasia: testicular biopsy findings, hor­ monal evaluation, and therapeutic results in three patients. Fertil Steril. 1987; 47: 664–670. Irodalom [24]  Dumic M, Duspara V, Grubic Z, et al. Testicular adrenal rest tumors in congenital adrenal hyperplasia – cross-sectional study of 51 Croatian male patients. Eur J Pediatr. 2017; 176: 1393– 1404. [7]  Smeets EE, Span PN, van Herwaarden AE, et al. Molecular char­ acterization of testicular adrenal rest tumors in congenital adre­ nal hyperplasia: lesions with both adrenocortical and Leydig cell features. J Clin Endocrinol Metab. 2005; 100: E524–E530. [25]  Sha YK, Sha YW, Ding L, et al. A case of bilateral testicular tu­ mors subsequently diagnosed as congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Int J Fertil Steril. 2016; 9: 574–580. [8]  Ashley RA, McGee SM, Isotaolo PA, et al. Clinical and patho­ logical features associated with the testicular tumor of the adre­ nogenital syndrome. J Urol. 2007; 177: 546–549. [26]  Claahsen-van der Grinten HL, Otten BJ, Hermus AR, et al. Tes­ ticular adrenal rest tumors in patients with congenital adrenal hyperplasia can cause severe testicular damage. Fertil Steril. 2008; 89: 597–601. [9]  Naouar S, Braiek S, El Kamel R. Testicular tumors of adreno­ genital syndrome: from physiopathology to therapy. Presse Med. 2017; 46: 572–578. [27]  Claahsen-van der Grinten HL, Hermus AR, Otten BJ. Testicular adrenal rest tumours in congenital adrenal hyperplasia. Int J Pediatr Endocrinol. 2009; 2009: 624823. [10]  Wang Z, Yang S, Shi H, et al. Histopathological and immu­ nophenotypic features of testicular tumour of the adrenogenital syndrome. Histopathology 2011; 58: 1013–1018. [28]  El-Maouche D, Arlt W, Merke DP. Congenital adrenal hyperpla­ sia: the Lancet seminar series. Lancet 2017; 390: 2194–2210. [11]  Gutjahr P, Humpl T. Testicular lymphoblastic leukemia/lym­ phoma. World J Urol. 1995; 13: 230–232. [29]  Speiser PW, Arlt W, Auchus RJ, et al. Congenital adrenal hyper­ plasia due to steroid 21-hydroxylase deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018; 103: 4043–4088. [Correction: J Clin Endocrinol Metab. 2019; 104: 39–40.] [12]  Zequi Sde C, Da Costa WH, Santana TB, et al. Bilateral testicu­ lar germ cell tumours: a systematic review. BJU Int. 2012; 110: 1102–1109. [13]  Cheah CY, Wirth A, Seymour JF. Primary testicular lymphoma. Blood 2014; 123: 486–493. [14]  Bart J, Groen, HJ, van der Graaf WT, et al. An oncological view on the blood–testis barrier. Lancet Oncol. 2002; 3: 357–363. (Fejes Zsuzsanna dr., Szeged, Semmelweis u. 6/A, 6725 e-mail: dr.fejes.zsuzsanna@gmail.com) [15]  Miskin M, Buckspan M, Bain J. Ultrasonographic examination of scrotal masses. J Urol. 1977; 117: 185–188. Megbeszélés Ezért ezekben az esetekben a sebészi kezelés már csak fájdalom és a diszkomfort csökkentése céljából indokolt [27]. A fertilitási ráta igen alacsony. Korai stádiumban, a tu­ mor eltávolításával egy ülésben végzett sebészi spermi­ umnyerés, illetve az intracitoplazmatikus spermiumin­ jekció lehetséges megoldást jelent [24, 26, 28]. A fertilitás biztosítása és a további irreverzibilis here­ károsodás megelőzése céljából a minél korábban, a pu­ bertás kezdetén, illetve a pubertáskorban végzett here- UH-vizsgálat, a sebészi spermiumnyerés és a páciensek szoros követése elengedhetetlen. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. 2020 ■ 161. évfolyam, 16. szám 630 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS Irodalom A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) Irodalom 2020 ■ 161. évfolyam, 16. szám 631 ORVOSI HETILAP
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Virtual Analog Models of the Lockhart and Serge Wavefolders
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Virtual Analog Model of the Lockhart and Serge Wave Folders Citation for published version: Esqueda, F, Pontynen, H, Parker, J & Bilbao, S 2017, 'Virtual Analog Model of the Lockhart and Serge Wave Folders', Applied Sciences, vol. 7, no. 12. https://doi.org/10.3390/app7121328 Citation for published version: General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Digital Object Identifier (DOI): 10.3390/app7121328 Link: Link to publication record in Edinburgh Research Explorer Edinburgh Research Explorer Article Virtual Analog Models of the Lockhart and Serge Wavefolders † Fabián Esqueda 1,*, Henri Pöntynen 1, Julian D. Parker 2 and Stefan Bilbao 3 Fabián Esqueda , Henri Pöntynen , Julian D. Parker and Stefan Bilbao 1 Aalto Acoustics Lab, Aalto University, 02150 Espoo, Finland; henri.pontynen@aalto.fi 2 Native Instruments GmbH, D-10997 Berlin, Germany; julian.parker@native-instruments.de 3 Acoustics and Audio Group, University of Edinburgh, Edinburgh EH9 3FD, Scotland, UK; s.bilbao@ed.ac.uk * Correspondence: fabian.esqueda@aalto.fi; Tel.: +358-50-4646-041 † This paper is an extended version of our paper published in the 14th Sound and Music Computing Conference (SMC-17), Espoo, Finland, 5–8 July 2017. q , y , J 1 Aalto Acoustics Lab, Aalto University, 02150 Espoo, Finland; henri.pontynen@aalto.fi 2 Native Instruments GmbH, D-10997 Berlin, Germany; julian.parker@native-instruments.de 3 Acoustics and Audio Group, University of Edinburgh, Edinburgh EH9 3FD, Scotland, UK; s.bilbao@ed.ac.uk * Correspondence: fabian.esqueda@aalto.fi; Tel.: +358-50-4646-041 † This paper is an extended version of our paper published in the 14th Sound and Music Computing Conference (SMC-17), Espoo, Finland, 5–8 July 2017. † This paper is an extended version of our paper published in the 14th Sound and Music Computing Conference (SMC-17), Espoo, Finland, 5–8 July 2017. Academic Editor: Stefania Serafin Received: 12 October 2017; Accepted: 13 December 2017; Published: 20 December 2017 Received: 12 October 2017; Accepted: 13 December 2017; Published: 20 December 2017 Abstract: Wavefolders are a particular class of nonlinear waveshaping circuits, and a staple of the “West Coast” tradition of analog sound synthesis. In this paper, we present analyses of two popular wavefolding circuits—the Lockhart and Serge wavefolders—and show that they achieve a very similar audio effect. We digitally model the input–output relationship of both circuits using the Lambert-W function, and examine their time- and frequency-domain behavior. To ameliorate the issue of aliasing distortion introduced by the nonlinear nature of wavefolding, we propose the use of the first-order antiderivative method. This method allows us to implement the proposed digital models in real-time without having to resort to high oversampling factors. The practical synthesis usage of both circuits is discussed by considering the case of multiple wavefolder stages arranged in series. Keywords: acoustic signal processing; circuit modeling; nonlinear waveshaping; antialiasing; synthesis; music 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 applied sciences Appl. Sci. 2017, 7, 1328; doi:10.3390/app7121328 1. Introduction In recent years, West Coast synthesis has become increasingly popular, with contemporary manufacturers such as Make Noise and Verbos Electronics releasing their own takes on classic Buchla circuits. This study presents virtual analog (VA) models for two analog synthesizer circuits: the Lockhart wavefolder and the wavefolder used in the middle section of the Serge Wave Multipliers. Wavefolding is a type of nonlinear waveshaping common in West Coast synthesis where portions of the input signal that exceed certain threshold are inverted or “folded back”, hence the name of the effect. The two circuits considered in this study were chosen because of the strong similarities between their general behavior. In a similar way to guitar distortion pedals, both wavefolders exploit the saturating behavior of semiconductor p–n junctions (i.e., transistors/diodes) to implement a folding function. Wavefolders are amongst the most emblematic building blocks of West Coast synthesis. In spite of that, they have been mostly overlooked by both VA and digital waveshaping research. We have recently begun to fill this research gap in [17], which presents a VA model of the wavefolder circuit in the seminal Buchla 259 module. Previous work on circuit-based VA modeling has researched the filters found in vintage synthesizers such as those produced by Moog [19–22], Electronic Music Studios (EMS) [23,24], Korg [25,26] and Buchla [16]. Extensive work has also been done on modeling guitar distortion pedals [13,27], tube amplifiers [11,28,29], modulation effects [30–33] and the Roland TR-808 drum machine [34,35]. Measurement-based VA modeling, commonly known as “black-box modeling”, has also been thoroughly studied within the context of guitar amplifiers and pedals [36–38]. This approach is particularly useful when the original circuit schematics are not available. A major challenge in VA modeling of nonlinear circuits, and digital waveshaping in general, is aliasing suppression. Early research on waveshaping synthesis addressed this issue by using low-order polynomial transfer functions, which not only allowed full parametric control of the produced spectrum but also ensured that the output waveform was bandlimited [4]. In VA modeling, high oversampling factors are usually necessary to prevent harmonics introduced by nonlinearities from reflecting into the baseband as aliases [13]. Oversampling increases the computational requirements of the model, by introducing additional filtering stages and scaling the number operations required to compute each output sample. 1. Introduction Nonlinear waveshaping is a technique used in sound synthesis to generate complex harmonic spectra. It consists of processing a signal with low harmonic content (typically a sinusoid) using a nonlinear mapping function designed to introduce harmonic overtones to the output signal [1]. The first documented use of waveshaping in the digital domain can be traced back to 1969, when Jean-Claude Risset emulated the sound of a clarinet by distorting a sinusoid with a clipping function [2]. Waveshaping techniques were extensively researched within the context of computer music in the 1970s, with several authors exploring the use of Chebyshev polynomials in particular, as an accurate and computationally cheap alternative to additive synthesis [1,3–5]. The underlying principles behind waveshaping synthesis are closely related to other well-known synthesis techniques, such as frequency modulation (FM) and phase distortion (PD) synthesis [6,7]. These two techniques rely on distorting the frequency and phase, respectively, of sinusoidal oscillators. Recent research on the topic of distortion-based synthesis has explored the use of logic operators in lieu of traditional polynomial waveshapers [8], and proposed extensions to both FM and PD synthesis [9,10]. The use of waveshaping in the analog domain began in the 1950s, when guitar players started deliberately overdriving their tube amplifiers to alter the timbre of their instrument [11]. In 1961, Gibson released the “Maestro FZ-1 Fuzz Tone”, the first commercially available fuzz distortion pedal, which exploited the saturating behavior of transistors to introduce harmonic distortion [12]. Most guitar Appl. Sci. 2017, 7, 1328; doi:10.3390/app7121328 www.mdpi.com/journal/applsci www.mdpi.com/journal/applsci Appl. Sci. 2017, 7, 1328 2 of 23 distortion pedals, including popular designs such as the Ibanez Tube Screamer and Electro-Harmonix Big Muff Pi, operate under this same basic principle [13,14]. distortion pedals, including popular designs such as the Ibanez Tube Screamer and Electro-Harmonix Big Muff Pi, operate under this same basic principle [13,14]. In analog synthesizers, the use of distortion-based methods is one of the cornerstones of “West Coast” synthesis, a paradigm pioneered by California-native Don Buchla during the 1960s. Buchla’s instruments focused on timbre manipulation at oscillator level by employing a variety of techniques such as nonlinear waveshaping, oscillator synchronization and pitch modulation [15–17]. This approach to sound synthesis contrasts that of traditional subtractive synthesis, where timbre is typically controlled by filtering harmonically-rich oscillator waveforms, like sawtooth and square waves, using resonant filters [18]. 1. Introduction For VA models that require evaluating transcendental functions, as is the case with the proposed Lockhart and Serge models, these added costs could compromise the integration of the system within a larger, real-time computer music system. A sizable portion of VA research has concentrated on designing efficient algorithms to generate alias-free geometric waveforms like those used in analog subtractive synthesizers, the so-called classic analog waveforms. Well-known techniques include the bandlimited impulse train (BLIT) family of methods, which involves the use of bandlimited basis functions and their integrated forms [39–41], and the use of differentiated polynomial waveforms (DPW) [42–44]. Moreover, Välimäki and Franck have applied the antialiasing principle behind the DPW algorithm to tackle aliasing in wavetable oscillators [45]. Recent work on antialiasing techniques has extended the use of the bandlimited ramp (BLAMP) method, originally proposed to antialias triangular oscillators in [25,41], to special cases of linear piecewise nonlinearities such as signal rectification, and inverse/hard clipping [17,46,47]. In this work, we propose the use of the antiderivative antialiasing method introduced in [48,49]. This approach can be used to reduce the aliasing caused by arbitrary nonlinear waveshaping functions and is applicable to the proposed wavefolder models. In its first-order form, the method can be Appl. Sci. 2017, 7, 1328 3 of 23 derived by analytically convolving a linear continuous-time representation of the input signal with a rectangular lowpass kernel [48]. As shown in this work, the use of the antiderivative method reduces the oversampling requirements of the proposed wavefolder models. A VA model of the Lockhart wavefolder was originally presented in [50]. This paper extends that work by introducing a second wavefolding circuit and studying the similarities between both systems. Additionally, we present a different treatment of the required Lambert-W function and an extended evaluation of the proposed antialiasing method in terms of computational costs. This paper is organized as follows. Sections 2 and 3 describe the model derivation of the Lockhart and Serge wavefolders, respectively. Time-domain simulations of the circuits are also presented in these two sections. Section 4 deals with two implications of VA wavefolding in the digital domain, namely aliasing suppression and evaluation of the Lambert-W function. Section 5 presents frequency-domain results of the Lockhart and Serge wavefolders, as well as an evaluation of the proposed antialiasing method in terms of perceived sound quality and computational costs. 1. Introduction Section 6 discusses the practical synthesis usage of both circuits and compares the behavior of the middle Serge Wave Multiplier with a recommended four-stage topology built around the Lockhart wavefolder. Concluding remarks and perspectives appear in Section 7. 2.1. Circuit Analysis The Lockhart wavefolder consists of an NPN and a PNP bipolar junction transistors connected at their base and collector terminals. In order to model the large-signal behavior of the circuit, we replace transistors Q1 and Q2 with their corresponding Ebers-Moll large-signal models [23,24]. Figure 1b shows the large-signal equivalent circuit of the Lockhart wavefolder. We use a double subscript notation to distinguish the voltages and currents in transistor Q1 from those in Q2. For example, IED,2 denotes the current through the base-emitter diode in Q2. Component values for the circuit have been compiled in Table 1. Table 1. Component values for the Lockhart wavefolder circuit. Component Value (kΩ) Component Value (V) R 15 VCC 15 RL 1–50 VEE −15 Table 1. Component values for the Lockhart wavefolder circuit. Component Value (kΩ) Component Value (V) R 15 VCC 15 RL 1–50 VEE −15 We begin the analysis of the circuit by assuming that the supply voltages are equal but opposite in sign (i.e., VCC = −VEE), and that |Vin| ≪VCC. This assumption means that the base-emitter junctions of both Q1 and Q2 will always be forward-biased and their voltage drops will remain approximately constant for all expected values of Vin. In Ken Stone’s version of the circuit, Vin is assumed to be bounded between approximately ±1.2 V [53]. Applying KVL around both input–emitter loops gives us Vin = VCC −RIE,1 −VBE,1 (1) Vin = VBE,2 + RIE,2 + VEE, (2) (1) (1) (2) (2) where IE,1 and IE,2 are the emitter currents, and VBE,1 and VBE,2 are the voltages across the base–emitter junctions of Q1 and Q2, respectively. Solving Equations (1) and (2) for IE,1 and IE,2 gives us: where IE,1 and IE,2 are the emitter currents, and VBE,1 and VBE,2 are the voltages across the base–emitter junctions of Q1 and Q2, respectively. Solving Equations (1) and (2) for IE,1 and IE,2 gives us: IE,1 = VCC −VBE,1 −Vin R (3) IE,2 = Vin −VBE,2 −VEE R . (4) IE,1 = VCC −VBE,1 −Vin R (3) IE,2 = Vin −VBE,2 −VEE R . (4) (3) (4) Next, we apply KCL at the collector node, which gives us Next, we apply KCL at the collector node, which gives us Next, we apply KCL at the collector node, which gives us Iout = IC,1 −IC,2, (5) where IC,1 = αFIED,1 −ICD,1 (6) IC,2 = αFIED,2 −ICD,2. 2. The Lockhart Wavefolder Figure 1a shows a simplified circuit diagram of the Lockhart wavefolder. This circuit was designed in 1973 by R. Lockhart Jr., who intended it to be used as a general-purpose frequency tripler [51]. Following its publication in Bernie Hutchin’s Electronotes [52], Lockhart’s design was repurposed as a wavefolder by Ken Stone, who realized its potential as a simple yet interesting waveshaper [53,54]. The Lockhart wavefolder has become ubiquitous in the music synthesizer do-it-yourself (DIY) community. For example, it is the core processor in Yves Usson’s “Metalizer” module [55]. (a) (a) IE,2 IE,2 IED,2 IED,2 ↵RICD,2 ↵RICD,2 ↵FIED,2 ↵FIED,2 IE,1 IE,1 IC,2 IC,2 IC,1 IC,1 ICD,1 ICD,1 VBC,2 VBC,2 VBC,1 VBC,1 IED,1 IED,1 VBE,1 VBE,1 VBE,2 VBE,2 Iout Iout RL RL Vout Vout R R Vin Vin Q1 Q1 Q2 Q2 VCC VCC VEE VEE ↵RICD,1 ↵RICD,1 ↵FIED,1 ↵FIED,1 (b) (b) Vin Vin Q2 Q2 Vout Vout RL RL R R VCC VCC VEE VEE Q1 Q1 PNP PNP NPN NPN VBE,1 VBE,1 VBE,2 VBE,2 ICD,2 ICD,2 Figure 1. (a) Simplified schematic of the Lockhart wavefolder circuit (adapted from [53]); and (b) its Ebers–Moll large-signal equivalent model. VBE,1 VBE,1 ↵RICD,1 ↵RICD,1 VBC,1 VBC,1 ↵FIED,1 ↵FIED,1 ↵FIED,2 ↵FIED,2 (a) (a) (b) (b) Figure 1. (a) Simplified schematic of the Lockhart wavefolder circuit (adapted from [53]); and (b) its Ebers–Moll large-signal equivalent model. Appl. Sci. 2017, 7, 1328 4 of 23 The main modifications made by Stone to Lockhart’s original design were the addition of an input potentiometer to attenuate the amplitude of the input waveform, and an inverting amplifier at the output of the circuit [54]. For the sake of simplifying the analysis, these are not shown in Figure 1a. The inverting stage at the output is reintroduced in Section 2.3. In our treatment of the circuit, we introduce the load resistance RL as an additional parameter which can be used to further control the timbre of the folded waveform. 2.1. Circuit Analysis (7) Iout = IC,1 −IC,2, (5) (5) where IC,1 = αFIED,1 −ICD,1 (6) IC,2 = αFIED,2 −ICD,2. (7) (6) (6) (7) (7) If we then assume that the contributions of the reverse currents αRICD1 and αRICD2 to the total currents associated with the emitter nodes are negligible (i.e., αR ≈0), we can establish that 28 5 of 23 Appl. Sci. 2017, 7, 1328 5 of 23 IED,1 ≈ IE,1 (8) IED,2 ≈ IE,2. (9) (8) (8) (9) (9) Assuming αF = 1, as suggested in [23], and inserting Equations (8) and (9) into Equations (6) and (7), respectively, yields a new expression for the total output current of the circuit: Assuming αF = 1, as suggested in [23], and inserting Equations (8) and (9) into Equations (6) and (7), respectively, yields a new expression for the total output current of the circuit: Iout = IE,1 −IE,2 −ICD,1 + ICD,2. (10) (10) We then combine Equations (3) and (4) to derive an expression for the difference between emitter currents: V + V 2V + V V We then combine Equations (3) and (4) to derive an expression for the difference between emitter currents: IE,1 −IE,2 = VCC + VEE −2Vin + VBE,2 −VBE,1 R . (11) (11) Since VCC + VEE = 0, and voltage drops VBE,1 and VBE,2 are assumed to be constant and equal, their contribution to this expression disappears. Therefore, we can simplify this result as: Since VCC + VEE = 0, and voltage drops VBE,1 and VBE,2 are assumed to be constant and equal, their contribution to this expression disappears. Therefore, we can simplify this result as: IE,1 −IE,2 = −2 RVin. (12) (12) Substituting Equation (12) into Equation (10) produces an expression for the total output current Iout in terms of the input voltage and the currents through the collector diodes: Substituting Equation (12) into Equation (10) produces an expression for the total output current Iout in terms of the input voltage and the currents through the collector diodes: Iout = −2 RVin −ICD,1 + ICD,2. 2.1. Circuit Analysis (13) (13) The current–voltage (I–V) relationship of diodes can be modeled using Shockley’s ideal diode equation, defined as     The current–voltage (I–V) relationship of diodes can be modeled using Shockley’s ideal diode equation, defined as     ID = Is  exp  VD ηVT  −1  , (14) (14) where ID is the current through the diode, Is is the reverse bias saturation current, VD is the voltage across the diode, VT is thermal voltage and η is the ideality factor of the diode [56]. For the p–n junctions inside transistors we can assume a reverse saturation current value Is = 10−17 A and an ideality factor η = 1. A thermal voltage value VT = 25.864 mV is used throughout this study. where ID is the current through the diode, Is is the reverse bias saturation current, VD is the voltage across the diode, VT is thermal voltage and η is the ideality factor of the diode [56]. For the p–n junctions inside transistors we can assume a reverse saturation current value Is = 10−17 A and an ideality factor η = 1. A thermal voltage value VT = 25.864 mV is used throughout this study. pplying Shockley’s diode equation to the collector diodes and substituting into Equation (13) Applying Shockley’s diode equation to the collector diodes and substituting into Equation (13) gives us: Applying Shockley’s diode equation to the collector diodes and substituting into Equati gives us: Iout = −2Vin R −Is  exp VCD,1 ηVT  −1  | {z } + Is  exp VCD,2 ηVT  −1  | {z } . (15) (15) {z ICD,2 {z ICD,1 {z ICD,2 ICD,1 Next, we use KVL to derive expressions for VCD,1 and VCD,2 in terms of Vin and Vout: Next, we use KVL to derive expressions for VCD,1 and VCD,2 in terms of Vin and Vout: VCD,1 = Vout −Vin (16) VCD,2 = Vin −Vout. (17) (16) (17) (16) (17) (16) (17) (17) Now, the collector diodes in the large-signal model are antiparallel. Therefore, we can make the further assumption that only one of them will conduct at a time depending on the polarity of Vin. A similar treatment is presented in [57] for the case of diode pairs in guitar distortion circuits. This means that ICD,1 ≈0 for Vin ≥0 and ICD,2 ≈0 for Vin < 0. 2.1. Circuit Analysis By combining these new assumptions with Equations (15)–(17) we arrive at the piecewise expres Iout = −2 RVin + λIs  exp λ (Vin −Vout) ηVT  −1  , (18) (18) Appl. Sci. 2017, 7, 1328 6 of 23 where λ = sgn (Vin) and sgn () is the signum function where λ = sgn (Vin) and sgn () is the signum function where λ = sgn (Vin) and sgn () is the signum function where λ = sgn (Vin) and sgn () is the signum function sgn(x) :=      −1 if x < 0 0 if x = 0 1 if x > 0. (19) (19) Equation (18) can be further simplified if we consider that the independent constant factor λIs that results from its expansion will be very small (±10−17 A) and can therefore be neglected. This gives us: Equation (18) can be further simplified if we consider that the independent constant factor λIs that results from its expansion will be very small (±10−17 A) and can therefore be neglected. This gives us: Iout = −2 RVin + λIs exp λ (Vin −Vout) ηVT  . (20) (20) Finally, we multiply both sides of this expression by RL to derive an input–output voltage relationship for the Lockhart wavefolder: Finally, we multiply both sides of this expression by RL to derive an input–output voltage relationship for the Lockhart wavefolder: Vout = −2RL R Vin + λRLIs exp λ (Vin −Vout) ηVT  . (21) (21) Table 2. Parameter values for the Lockhart wavefolder described by Equation (26). Name Value Name Value Name Value R 15 kΩ Is 10−17 A VT 25.864 mV RL 1–50 kΩ η 1 – – Table 2 summarizes all parameter values for the proposed Lockhart wavefolder model. Table 2 summarizes all parameter values for the proposed Lockhart wavefolder model. Table 2 summarizes all parameter values for the proposed Lockhart wavefolder mode 2.3. Model Discretization and Evaluation Model Discretization and Evaluation 2.3. Model Discretization and Evaluation Th l d h L kh f ld d d Th f d b h The voltages inside the Lockhart wavefolder are time-dependent. Therefore, we can describe the continuous-time model defined by Equation (26) as being of the form Vout(t) = f (Vin(t)), (27) (27) where f () is the transfer function of the system and t is time. In the synthesis literature, the term “transfer function” is commonly used to denote the waveshaping function [4]. It should not be confused with the s- and z-domain transfer functions used in linear system analysis. As previously mentioned, Ken Stone’s circuit features an inverting stage before the output which can be modeled by inverting the polarity of the right-hand side of Equation (26): Vout = αVin −ληVTW (∆exp (λβVin)) . (28) (28) Vout = αVin −ληVTW (∆exp (λβVin)) . While including this step is not strictly necessary, we have chosen to do so, as it will facilitate the evaluation of the model. Now, given the form described by Equation (27), the Lockhart model can be discretized trivially by replacing all continuous-time signals with their discrete-time equivalents, i.e., (29) Vout[n] = f (Vin[n]), (29) where n is the sample index. 2.2. Explicit Formulation Equation (21) describes an implicit relationship between the input and output voltages of the circuit; it cannot be solved algebraically. Instead, a closed-form solution for Vout can be derived with the help of the Lambert-W function. The use of the Lambert-W function W() has been previously researched within the context of VA modeling. Several authors have used it to solve the implicit I–V relationship of diodes [56,58,59]. Parker and D’Angelo used W() to model the Buchla Lowpass-Gate, a synthesizer circuit that employs a resistive opto-isolator (also known as a vactrol) in its control path [16]. Strictly speaking, W() is multivalued; however, in this work, we only utilize the upper branch of the function. This branch is known as W0() in the literature [56,60]. The Lambert-W function is used to solve equations of the form (A + Bx) exp (Cx) = D, (22) (22) which have the explicit solution which have the explicit solution x = 1 CW CD B exp  AC B  −A B , (23) (23) where A, B, C and D ∈R [58]. where A, B, C and D ∈R [58]. Equation (21) can be arranged in the form described by Equation (22) by first rewriting it as Vout + 2RL R Vin = λRLIs exp λVin ηVT  exp −λVout ηVT  , (24) (24) and dividing both sides by exp (−λVout/ηVT), which gives us and dividing both sides by exp (−λVout/ηVT), which gives us and dividing both sides by exp (−λVout/ηVT), which gives us and dividing both sides by exp (−λVout/ηVT), which gives us  Vout + 2RL R Vin  exp λVout ηVT  = λRLIs exp λVin ηVT  . (25) (25) Solving for Vout as defined in Equation (23) yields an explicit model for the Lockhart wavefolder Solving for Vout as defined in Equation (23) yields an explicit model for the Lockhart wavefolder Vout = ληVTW (∆exp (λβVin)) −αVin, Vout = ληVTW (∆exp (λβVin)) −αVin, (26) (26) where where α = 2RL R , β = 2RL + R ηVTR and ∆= RLIs ηVT . α = 2RL R , β = 2RL + R ηVTR and ∆= RLIs ηVT . 7 of 23 Appl. Sci. 2017, 7, 1328 where n is the sample index. p The time-domain behavior of the proposed circuit model was validated by comparing it against a reference simulation obtained using the SPICE (Simulation Program with Integrated Circuit Emphasis) software LTspice (Version IV, Linear Technology, Milpitas, CA, USA, 2016) [61]. The results of this simulation are shown in Figure 2a for values of Vin between −1.5 and 1.5 V. Figure 2b shows the transfer function of the proposed model implemented in MATLAB (Version R2017a, MathWorks, Natick, MA, USA, 2017) using Equation (28) and MATLAB’s native “lambertw” function. In both cases, four different values of RL were simulated: 1, 5, 10 and 50 kΩ. From these figures, we can observe the general behavior of the Lockhart wavefolder. At low input values, the system behaves linearly, whereas for high input values the circuit inverts the slope of the driving signal. The transition between non-folded and folded portions of the signal is gradual, which responds to the characteristic soft saturating behavior of p–n junctions. The region where the transfer function folds the input signal is indicated with a blue arrow in Figure 2b for the case when RL = 50 kΩ. As shown in these Figures, increasing the value of RL sharpens the shape of the transfer function. The curves shown in Figure 2a,b indicate a good match between the SPICE simulations and the proposed digital model. Figure 3 shows the absolute value (in mV) of the difference between both simulations. From this plot, we can observe that the difference between the curves is indeed very small, below 1 mV for all values of Vin measured. These small differences are perceptually insignificant and can be attributed to the simplifications made during the analysis of the circuit and to the way in which SPICE computes currents flowing through semiconductor devices. For example, SPICE introduces a small fictitious conductance in parallel with each p–n junction in order to aid the convergence of its iterative solvers. Additionally, the SPICE diode model will account for the small reverse current that flows when the voltage across the diode is negative [61]. Appl. Sci. 2017, 7, 1328 8 of 23 8 of 23 1k 5k 10k 50k Fold Fold Figure 2. Transfer function of the Lockhart wavefolder simulated using: (a) SPICE (Simulation Program with Integrated Circuit Emphasis); and (b) the proposed virtual analog (VA) model. Different colors indicate different values of RL. Fold Fold 1k 5k 10k 50k Figure 2. where n is the sample index. Transfer function of the Lockhart wavefolder simulated using: (a) SPICE (Simulation Program with Integrated Circuit Emphasis); and (b) the proposed virtual analog (VA) model. Different colors indicate different values of RL. 1k 5k 10k 50k Figure 3. Absolute value of the difference between a SPICE simulation of the Lockhart wavefolder and its proposed VA model. Figure 3. Absolute value of the difference between a SPICE simulation of the Lockhart wavefolder and its proposed VA model. Figure 4 shows a time-domain view of the output of the proposed model when driven by a 500-Hz sinusoidal waveform with a peak amplitude of 1 V for two different load resistance values, RL = 10 and 50 kΩ. A sampling rate Fs = 44.1 kHz was used to generate these figures, which are plotted against their corresponding SPICE simulations. From these results, we can once again observe the effect of wavefolding and the impact of RL on the overall shape of the output. For high values of RL the transition region between folded and non-folded values becomes very small and the resulting waveform is almost discontinuous (see Figure 4b). In the frequency domain, this will translate to higher harmonic content, similar to that of a square wave oscillator. A more detailed frequency-domain analysis of the Lockhart circuit is presented in Section 5 of this study. Figure 4. Time-domain view of the proposed Lockhart wavefolder model plotted against its SPICE simulation for a 500-Hz sinusoidal input (peak amplitude 1 V) with load resistance: (a) RL = 10 kΩ; and (b) RL = 50 kΩ. Figure 4. Time-domain view of the proposed Lockhart wavefolder model plotted against its SPICE simulation for a 500-Hz sinusoidal input (peak amplitude 1 V) with load resistance: (a) RL = 10 kΩ; and (b) RL = 50 kΩ. 3. The Serge Middle Wave Multiplier The second circuit considered in this study is the middle section of the Serge Wave Multipliers (often abbreviated as the Serge VCM). The Serge VCM is a synthesizer module designed in 1977 by West Coast designer Serge Tcherepnin, founder of Serge Modular Music Systems. It offered three separate and independent analog sound processors, namely the “top”, “middle” and “bottom” sections. As described in an original 1980 Serge product catalog, “The middle section generates a sweep of the odd harmonics (1, 3, 5, 7, 9, 11 and 13th) when a triangle wave is applied to its input... This module can be used to explore timbral areas beyond the range of ring modulation because there are more varied harmonics than the sum and difference tones” [62]. Appl. Sci. 2017, 7, 1328 9 of 23 The middle Serge VCM is essentially a waveshaping circuit consisting of six identical wavefolding stages arranged in series. An amplifier at the input of the circuit is used to modulate the gain of the input waveform and control the amount of folds introduced [63]. In this section we focus on the analysis of a single folding stage. The transfer function and frequency-domain behavior of the complete system are presented in Section 6. Figure 5 shows the schematic of a single wavefolding stage in the circuit. Component information is given in Table 3. - + Vin Vin Vout Vout R1 R1 R2 R2 Vx Vx R3 R3 Figure 5. Schematic of a single folding cell in the middle section of the Serge Wave Multipliers (VCM). Figure adapted from [63]. Figure 5. Schematic of a single folding cell in the middle section of the Serge Wave Multipliers (VCM). Figure adapted from [63]. Table 3. Component information for the Serge wavefolder circuit shown in Figure 5. Table 3. Component information for the Serge wavefolder circuit shown in Figure 5. Table 3. Component information for the Serge wavefolder circuit shown in Figure 5 Component Value (kΩ) Component Description R1 33 Diodes 1N4148 or similar R2 100 Op-Amp TL072 or similar R3 100 – – To derive the transfer function for the Serge wavefolding circuit, we first assume ideal op-amp behavior and derive an expression for Vout in terms of Vin and Vx, the voltage at the non-inverting input of the amplifier. This gives us: Vout = Vx −R3 R2 (Vin −Vx) . 3. The Serge Middle Wave Multiplier (30) (30) Since in this case R3 = R2, we can further simplify this result as: Since in this case R3 = R2, we can further simplify this result as: Since in this case R3 = R2, we can further simplify this result as: (31) Vout = 2Vx −Vin. (31) Next, we derive an expression for Vx by considering the subcircuit shown in Figure 6, which is essentially a diode pair similar to those found in guitar distortion circuits [13,57,58]. Vin Vin Vx Vx IF IF IR IR R1 R1 I Figure 6. Equivalent view of the diode saturator at the non-inverting input of the op-amp in Figure 5. Figure 6. Equivalent view of the diode saturator at the non-inverting input of the op-amp in Figure 5. gure 6. Equivalent view of the diode saturator at the non-inverting input of the op-amp in Figure 5. Applying KVL around the outer loop of the circuit yields the relation Applying KVL around the outer loop of the circuit yields the relation Vin = R1I + Vx, (32) (32) Vin = R1I + Vx, Appl. Sci. 2017, 7, 1328 10 of 23 where I is the current through resistor R1. Then, we apply KCL at the output node of the circuit, which gives us I = IF −IR. (33) I = IF −IR. (33) ombining Equation (32) with Equation (33) and applying Shockley’s diode equation gives us uation (32) with Equation (33) and applying Shockley’s diode equation gives us Vin −Vx R1 = Is  exp  Vx ηVT  −1  | {z } IF −Is  exp −Vx ηVT  −1  | {z } IR . (34) (34) As before, we assume the diodes will not conduct simultaneously and arrive at the piecewise relationship As before, we assume the diodes will not conduct simultaneously and arrive at the piecewise relationship   λV   Vin −Vx = λR1Is  exp  λVx ηVT  −1  , (35) (35) where once again λ = sgn(Vin). To further simplify this expression we neglect the constant factor λR1Is that results from its expansion. This gives us: Vin −Vx = λR1Is exp  λVx ηVT  . (36) (36) Next, we rearrange this equation in the Lambert-W form described by Equation (22) by dividing both sides by exp (λVx/ηVT). 3. The Serge Middle Wave Multiplier This yields Next, we rearrange this equation in the Lambert-W form described by Equation (22) by dividing both sides by exp (λVx/ηVT). This yields (Vin −Vx) exp  −λVx ηVT  = λR1Is, (37) (37) which can be solved for Vx as: which can be solved for Vx as: Vx = Vin −ληVTW  R1Is ηVT exp λVin ηVT  . (38) (38) As a final step, we insert Equation (38) into Equation (31) to derive a complete expression for the transfer function of a single wavefolding stage in the Serge middle VCM: As a final step, we insert Equation (38) into Equation (31) to derive a complete expression for the transfer function of a single wavefolding stage in the Serge middle VCM: Vout = Vin −2ληVTW  R1Is ηVT exp λVin ηVT  . (39) (39) Figure 7 shows the transfer function of the circuit, evaluated in MATLAB for values of Vin between −1.5 and 1.5 V. As before, the model was discretized trivially and is presented against its corresponding SPICE simulation. Parameter values used in this simulation are compiled in Table 4. The value of parameters Is and η for the 1N4148 diode were matched to those of its corresponding SPICE model [61]. Figure 7b shows the absolute difference between both simulations. These results indicate a good match between the models, as the maximum difference was once again found to be below 1 mV. -1.5 -1 -0.5 0 0.5 1 1.5 -1 -0.5 0 0.5 1 SPICE Model -1.5 -1 -0.5 0 0.5 1 1.5 0 0.1 0.2 Figure 7. (a) Transfer function of a single wavefolding stage in the Serge middle VCM measured using SPICE and the proposed model; and (b) the absolute difference between these two curves. -1.5 -1 -0.5 0 0.5 1 1.5 0 0.1 0.2 -1.5 -1 -0.5 0 0.5 1 1.5 -1 -0.5 0 0.5 1 SPICE Model Figure 7. (a) Transfer function of a single wavefolding stage in the Serge middle VCM measured using SPICE and the proposed model; and (b) the absolute difference between these two curves. 11 of 23 Appl. Sci. 2017, 7, 1328 Table 4. Simulation parameters for a single folding stage in the middle Serge Wave Multiplier. Name Value Name Value Is 2.52 nA R1 33 kΩ η 1.752 VT 25.864 mV Finally, Figure 8 shows the output of the Serge wavefolder for a 500-Hz sinusoidal input. 3. The Serge Middle Wave Multiplier As expected, the circuit behaves as a wavefolder, folding portions of the input waveform whose absolute value exceeds approximately 0.3 V. This behavior is similar to that of the Lockhart wavefolder (cf. Figure 4a). Figure 8. Time-domain view of the Serge wavefolder model plotted against its SPICE simulation for a 500-Hz sinusoidal input with peak amplitude if 1 V. Figure 8. Time-domain view of the Serge wavefolder model plotted against its SPICE simulation for a 500-Hz sinusoidal input with peak amplitude if 1 V. 3.1. Model Equivalence Equation (39) shares a close resemblance with Equation (28), the proposed Lockhart wavefolder model. In fact, both expressions have the same form, which consists of the difference between a portion of the input signal and an input-dependent nonlinear element. In the case of the Lockhart wavefolder, when the RL = R/2 Equation (28) simplifies to Vout = Vin −ληVTW  RLIs ηVT exp λVin ηVT  , (40) (40) which is remarkably close to Equation (39), with the only difference being the missing factor of two outside the Lambert-W function. This factor accounts for the difference between physical parameters Is and η in each circuit. Figure 9 shows a comparison of the transfer functions for the Lockhart (RL = 7.5 kΩ) and Serge wavefolders implemented using the parameter values in Tables 2 and 4, respectively. From this figure, we can observe that the only significant difference between both transfer functions is in their sharpness at the folding points. This means the Lockhart wavefolder will introduce sharper folds which will translate into brighter sounds at the output. From this analysis, it is clear that both circuits result in a similar audio effect, even though they are produced using different architectures. Figure 9. Transfer functions for the proposed Serge and Lockhart (RL = 7.5 kΩ) wavefolder models. Figure 9. Transfer functions for the proposed Serge and Lockhart (RL = 7.5 kΩ) wavefolder models. 4.1. Evaluating the Lambert-W Function A particular challenge of using the Lambert-W function in VA modeling, where real-time operation is paramount, is that of computational efficiency. Optimizing the evaluation of W() is an active research topic (see, e.g., [64]). For the case of guitar distortion circuits, Paiva et al. proposed the use of a simplified iterative method which relies on a lookup table for its initial guess [57]. In this work, we propose approximating the value of W() directly using Fritsch’s iteration, as suggested in [60]. In order to compute wm, an approximation to W(x), where x ∈R>0, we iterate over wm+1 = wm(1 + εm), (41) (41) where where where εm =  rm 1 + wm   qm −rm qm −2rm  (42) rm = ln  x wm  −wm (43) qm = 2 (1 + wm)  1 + wm + 2 3rm  , (44) εm =  rm 1 + wm   qm −rm qm −2rm  (42) (42) rm = ln  x wm  −wm (43) (43) qm = 2 (1 + wm)  1 + wm + 2 3rm  , (44) (44) and m = 0, 1, 2, . . . , M −1. The value w0 is an initial guess and M is the number of iterations required for εm to approximate zero within machine-size floating point precision. The special case W(0) = 0 is defined separately. The efficiency of Fritsch’s iteration will depend on the choice of initial guess. As explained in [60], an initial guess within 10−4 of the solution will yield an approximation to W() accurate to within 10−16 in just one iteration. Figure 10 shows the approximate times required to compute W(x) for a set of values of x between 10−24 and 10300 using Fritsch’s iteration and the previously-proposed Halley’s method [50]. This range was chosen as it covers all values of interest. For instance, when Vin = 5 V, the argument of W() in the Serge wavefolder model will be approximately 1.52 × 1044. All times were computed by averaging the result of 30 iterations implemented under identical circumstances. A piecewise approximation was used to compute the initial guess, as described in [60]. From this plot, we can observe that Fritsch’s iteration outperforms Halley’s method by up to approximately 11 times. 4.1. Evaluating the Lambert-W Function A MATLAB implementation of the Lambert-W function used to perform these measurements can be found in the accompanying website for this article. Halley Fritsch Figure 10. Averaged processing times required to compute W(x) using Halley’s method and Fritsch’s iteration. ure 10. Averaged processing times required to compute W(x) using Halley’s method and Fritsch’s iteration 4. Wavefolding in the Digital Domain In the previous sections, the time-domain behavior of the Lockhart and Serge wavefolder models was examined via trivial discretization of their characteristic transfer functions. In this section, we move Appl. Sci. 2017, 7, 1328 12 of 23 on to consider two implications of virtual analog wavefolding: evaluation of the Lambert-W function and aliasing. on to consider two implications of virtual analog wavefolding: evaluation of the Lambert-W function and aliasing. 5. Results This section examines the frequency-domain behavior of the Lockhart and Serge wavefolders and their proposed antialiased forms. Next, we evaluate the computational costs of the antiderivative method with respect to oversampling for the case of the Lockhart model. 4.2. Aliasing Considerations when |Vin[n] −Vin[n −1]| is smaller than a predetermined threshold [48]. This special case simply bypasses the antialiased form while compensating for the half-sample latency of the method. 4.2. Aliasing Considerations 4.2. Aliasing Considerations As discussed in Section 1, nonlinear waveshaping in the digital domain is susceptible to aliasing distortion due to its frequency-expanding nature. Wavefolding is no exception to this problem. As an arbitrary input waveform is folded, new harmonic overtones will be added to the frequency spectrum. Harmonics at frequencies exceeding half of the sampling rate, or the Nyquist limit, will be reflected into Appl. Sci. 2017, 7, 1328 13 of 23 the audio baseband as aliases. Aliasing is known to cause unpleasant artifacts—such as beating and inharmonicity—that cannot be tolerated in a music computing scenario. Oversampling is commonly employed to mitigate this issue; however, this approach increases the computational requirements of the system by introducing additional operations. We propose the use of the first-order antialiasing method presented in [48,49]. This method is designed to reduce aliasing caused by memoryless waveshaping functions with the form described by Equation (29). The antialiased output of the waveshaping function is defined as Vout[n] = F(Vin[n]) −F(Vin[n −1]) Vin[n] −Vin[n −1] , (45) (45) where F() is the antiderivative of f (), the original transfer function. For the case of the Lockhart wavefolder defined by Equation (28), the integrated transfer function is defined as F(Vin) = α 2V2 in −ηVT 2β [Ψ1(Ψ1 + 2)] , (46) (46) where Ψ1 = W (∆exp (λβVin)) , Ψ1 = W (∆exp (λβVin)) , (47) (47) and α, β and ∆remain as before. This result showcases an advantageous property of the Lambert-W function W(); its antiderivative is defined in terms of W() itself. Therefore, computing F() does not pose a major increase in computational costs with respect to evaluating simply f (). For the case of the Serge wavefolder defined by Equation (39), the required antiderivative is given by F(Vin) = V2 in 2 −(ηVT)2 [Ψ2(Ψ2 + 2)] , (48) (48) where where Ψ2 = W  R1Is ηVT exp λVin ηVT  . (49) (49) When Vin[n] ≈Vin[n −1], Equation (45) can become ill-conditioned. This is avoided by defining the special case When Vin[n] ≈Vin[n −1], Equation (45) can become ill-conditioned. This is avoided by defining the special case Vout[n] = f Vin[n] + Vin[n −1] 2  , (50) (50) when |Vin[n] −Vin[n −1]| is smaller than a predetermined threshold [48]. This special case simply bypasses the antialiased form while compensating for the half-sample latency of the method. 5.1. Frequency-Domain Behavior The spectrogram in Figure 11 shows the effect of increasing the value of RL in the Lockhart wavefolder model for a constant 150-Hz sinusoidal input. As expected, the level of harmonic distortion introduced by the circuit is proportional to the value of this resistance. Therefore, this parameter can be used for additional timbral control. It should be noted that due to the antisymmetric nature of the folding function, the system introduces odd harmonics only for input signals centered around zero. Since the level of harmonics introduced by the Lockhart wavefolder depends on the choice of RL, we consider the highest recommended case RL = 50 kΩas a worst-case scenario in terms of aliasing distortion. pl. Sci. 2017, 7, 1328 14 o Frequency (Hz) -80 -60 -40 -20 0 Figure 11. Spectrogram of the Lockhart wavefolder under 150-Hz sinusoidal input for values of RL between 1 and 50 kΩ. 14 of 23 Appl. Sci. 2017, 7, 1328 Frequency (Hz) -80 -60 -40 -20 0 Figure 11. Spectrogram of the Lockhart wavefolder under 150-Hz sinusoidal input for values of RL between 1 and 50 kΩ. Figure 12 shows the spectrograms of a linear sweep from 20 Hz–5 kHz with peak amplitude of 1 V processed by the proposed Lockhart and Serge wavefolder models. This frequency range was chosen as it covers all fundamental frequencies of musical interest. A sample rate Fs = 1 MHz was used to generate these figures in order to simulate an ideal alias-free continuous-time behavior. These results will be used as a reference when evaluating the performance of the proposed antialiased forms. From these spectrograms we can observe how the Lockhart wavefolder is capable of generating brighter sounds. This perceptual attribute can be varied by changing the value of RL. Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Figure 12. Spectogram for a linear sweep from 20 Hz to 5 kHz processed using: (a) the proposed Lockhart wavefolder model (RL = 50 kΩ); and (b) the proposed Serge wavefolder model. A sample rate Fs = 1 MHz was used to simulate analog behavior. -80 -60 -40 -20 0 Figure 12. Spectogram for a linear sweep from 20 Hz to 5 kHz processed using: (a) the proposed Lockhart wavefolder model (RL = 50 kΩ); and (b) the proposed Serge wavefolder model. 5.1. Frequency-Domain Behavior A sample rate Fs = 1 MHz was used to simulate analog behavior. Figure 13a shows the result of processing the same linear sweep using the trivial (i.e., non-antialiased) Lockhart model at a standard audio rate of Fs = 44.1kHz. When compared to Figure 12a, we can clearly observe the high levels of aliasing distortion introduced by the model. This is somewhat ameliorated in Figure 13b, where the sweep has been processed at a sample rate of Fs = 88.2kHz (i.e., two-times oversampling). Figure 13c,d shows the result of processing the sweep using the proposed antialiasing method at audio rate and with two-times oversampling, respectively. As shown in these spectrograms, there is a significant reduction in aliasing, particularly below the fundamental frequency. This behavior is advantageous in music applications because at low frequencies the audibility of aliasing distortion is only limited by the hearing threshold. On the other end of the spectrum, the masking effects of harmonics will help suppress the audible effects of high-frequency aliases [65]. The spectrograms in Figure 14a,b show the outcome of processing the 1 V linear sweep with the proposed Serge wavefolder model at audio rate and with two-times oversampling, respectively. When compared with Figure 13a,b it is evident that the Serge wavefolder model is less susceptible to aliasing distortion. This can be attributed to the fact that its transfer function is not as sharp as that of the Lockhart, particularly when RL = 50 kΩ. Figure 14c,d shows the result of processing the linear sweep using the antiderivative method at audio rate and with oversampling by two. In this case, operating at audio rate yields very effective results as there are very few visible aliases left below the fundamental. When combined with oversampling by two the antiderivative method produces a nearly alias-free spectrum for the measured frequency range. Appl. Sci. 2017, 7, 1328 15 of 23 15 of 23 Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Figure 13. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Lockhart wavefolder (RL = 50 kΩ) model: (a) at audio rate; (b) using two times oversampling; (c) with antialiasing at audio rate; and (d) with antialiasing and oversampling by two. 5.1. Frequency-Domain Behavior 2017, 7, 1328 16 of 23 16 of 23 signal quality obtained by the proposed antialiasing method when applied to the Lockhart wavefolder, even when operating at audio rate. Moreover, these measurements show that the performance of the proposed method, when combined with two-times oversampling, is on par with oversampling by a factor of 8. For all fundamental frequencies below approximately 4.2 kHz, the ANMR lies below the −10 dB line. This range can be regarded as sufficient for musical applications if we consider that the highest fundamental frequency on a standard grand piano is 4186.01 Hz (MIDI note C8). Figure 15. Measured A-weighted noise-to-mask ratios (ANMRs) for a range of sinusoidal waveforms processed: (a) using the Lockhart wavefolder model (RL = 50 kΩ) under six different sampling rates; and (b) using the Serge wavefolder model under two different sampling rates, with and without the proposed antialiasing method. Values below the −10 dB threshold indicate lack of perceivable aliasing. Figure 15. Measured A-weighted noise-to-mask ratios (ANMRs) for a range of sinusoidal waveforms processed: (a) using the Lockhart wavefolder model (RL = 50 kΩ) under six different sampling rates; and (b) using the Serge wavefolder model under two different sampling rates, with and without the proposed antialiasing method. Values below the −10 dB threshold indicate lack of perceivable aliasing. Figure 15b shows the measured ANMRs for the Serge wavefolder. When implemented at audio rate, the output is free from perceivable aliasing for fundamental frequencies up to approximately 2 kHz. These measurements go in accordance with the Spectrogram in Figure 14a, which shows aliasing is significantly more evident above this frequency. The use of the antiderivative method yields results comparable to those of oversampling by a factor of two, with all measured fundamental frequencies below approximately 4.6 kHz lying below the −10 dB aliasing threshold. Overall, these results indicate the proposed Serge wavefolder model can operate at audio rate with the help of the antiderivative method, therefore avoiding the need for oversampling. 5.1. Frequency-Domain Behavior Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Figure 13. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Lockhart wavefolder (RL = 50 kΩ) model: (a) at audio rate; (b) using two times oversampling; (c) with antialiasing at audio rate; and (d) with antialiasing and oversampling by two. Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Frequency (Hz) -80 -60 -40 -20 0 Figure 14. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Serge wavefolder model: (a) at audio rate; (b) using two times oversampling; (c) with antialiasing at audio rate; and (d) with antialiasing and oversampling by two. Figure 14. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Serge wavefolder model: (a) at audio rate; (b) using two times oversampling; (c) with antialiasing at audio rate; and (d) with antialiasing and oversampling by two. The performance of the proposed antialiasing method was further evaluated by computing the A-weighted noise-to-mask ratio (ANMR) for a set of sinusoidal input signals processed by both wavefolder models. The ANMR has been previously researched as a perceptually-informed measure to evaluate the audibility of aliasing distortion [41,65]. The algorithm computes the power ratio in decibels between the wanted harmonics and aliased components, but takes into account the masking effects of the former. An A-weighting filter is applied to all signals prior to the evaluation of the ANMR in order to account for the frequency-dependent sensitivity of hearing for low-level sounds. Signals with an ANMR value below −10 dB are considered to be completely free from perceivable aliasing. A detailed account of this method can be found in [65]. Figure 15a compares the measured ANMRs for a set of sinusoidal inputs with fundamental frequencies between 1 and 5 kHz processed by the Lockhart model at different sampling rates. The ideal alias-free signals required to compute these values were synthesized using Fourier analysis and additive synthesis, as suggested in [41]. All signals were downsampled back to audio rate (i.e., 44.1 kHz) prior to evaluation. A dashed horizontal line has been used to indicate the −10 dB hearing threshold for aliasing distortion. In Figure 15a we can observe the significant increase in Appl. Sci. 5.2. Computational Costs The computational costs of the antialiased Lockhart wavefolder model were measured by porting the algorithms into C code using the 128-bit long double data type. Table 5 shows the computation times for a 1-s 100-Hz sinusoidal input processed using the proposed model for different peak amplitude values. These results were computed by averaging the processing times of one hundred implementations. All tests were performed under identical circumstances, using a fixed resistance value of RL = 50 kΩ, the highest recommended value. From these results we can observe that the complexity of the model does not depend on the input and that the overhead of implementing the antialiasing method is minimal. When operating at audio rate, the added computation time is approximately 1 ms for a 1-second simulation. Moreover, these time measurements show that the antialiased Lockhart model, implemented at a sample rate Fs = 88.2 kHz, is approximately 3.5 times faster than oversampling by a factor of 8 (i.e., Fs = 352.8 kHz) and nearly twice as fast as oversampling by a factor of 4. Changing the value of RL did not affect the execution times of the algorithms. 17 of 23 Appl. Sci. 2017, 7, 1328 Table 5. Averaged computation times (in milliseconds) for the proposed Lockhart wavefolder model (RL = 50 kΩ) implemented in C for a 1-s 100-Hz sinusoidal input sampled at different oversampling (OS) rates and with different peak amplitude levels. Amplitude (V) Audio Rate Audio Rate OSx2 OSx4 OSx8 OSx2 (ms) w/Antialiasing (ms) (ms) (ms) (ms) w/ Antialiasing (ms) 1 11.5 12.5 23.4 46.6 92.9 25.4 5 11.6 12.6 23.3 46.8 92.7 25.3 10 11.5 12.6 23.7 46.7 92.9 25.5 15 11.5 12.7 23.5 46.5 92.9 25.5 For high values of RL, long double representation is necessary to account for the large values that will result at the argument of the Lambert-W function. For smaller values, 64-bit precision will be sufficient to accommodate most input levels of interest. For instance, when RL = 7.5 kΩthe signal at the input of the proposed Lockhart wavefolder model can have a peak amplitude of up to 9 V. The measurements in Table 5 were conducted by synthesizing all input signals at the target rates. In practical implementations, oversampling will require additional pre- and post-filtering stages that will further increase the computational costs of the system. 5.2. Computational Costs The complexity and costs of these filtering stages will be directly proportional to the required oversampling factor. This constitutes another advantage of the proposed antialiasing method. 6. Practical Synthesis Usage For simplicity, in this section, we assume the range of Vin to be fixed at ±1 V; therefore, all gain modulation is done using GS only. Figure 17. (a) Transfer function of the proposed Serge middle VCM; and (b) its output when driven by a 100-Hz sinewave for GS = 6 and zero dc offset. Figure 17. (a) Transfer function of the proposed Serge middle VCM; and (b) its output when driven by a 100-Hz sinewave for GS = 6 and zero dc offset. The spectrogram in Figure 18a shows the effect of increasing GS from 0 to 6 for a 150-Hz sinusoidal input. This plot effectively depicts the rich harmonic patterns introduced by the system, which are far more complex than those introduce by traditional waveshaping methods and offer a wide timbral palette for sound synthesis. The fluctuations in energy at the fundamental and first few harmonics indicate the gain values at which each new fold is introduced. Figure 17b shows the effect of introducing a dc offset at the input of the system for a constant 200-Hz sinusoidal input. This result shows how the use of a dc offset can extend the timbral possibilities of the system even further, by introducing complex patterns consisting of both even and odd harmonics. Frequency (Hz) -80 -70 -60 -50 -40 -30 -20 -10 0 (a) (a) Frequency (Hz) -80 -70 -60 -50 -40 -30 -20 -10 0 0 3 1 2 dc o↵set (V) dc o↵set (V) (b) (b) Figure 18. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed Serge middle VCM with varying gain GS from 0–6; and (b) a 200-Hz sinewave processed with varying gain GS from 0–3 and dc offset from 0–3 V. Figure 18. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed Serge middle VCM with varying gain GS from 0–6; and (b) a 200-Hz sinewave processed with varying gain GS from 0–3 and dc offset from 0–3 V. Now, although the Lockhart wavefolder was originally designed to operate as a standalone unit, it can be adapted into a series topology with relative ease. Here, we propose using the wavefolding structure shown in Figure 19 to expand the synthesis capabilities of the Lockhart wavefolder. 6. Practical Synthesis Usage In practical sound synthesis applications, a single folding stage is rarely used, as the timbral variety it can produce is quite limited. Most analog designs, for example the Intellijel µFold and the aforementioned Yusynth Metalizer, employ several wavefolding stages arranged in series. The number of stages varies according to the design, but typically cascades of two to six stages are used. As mentioned in Section 3, the Serge middle VCM utilizes six identical folding stages. Figure 16 shows a simplified block diagram representation of the Serge middle VCM based on the original design [63]. Blocks labeled “SWF” correspond to the proposed Serge wavefolder model. An ad hoc gain factor of four, not present in the original circuit, has been added to compensate for the scaling of the signal introduced by the cascade of wavefolders. Vin[n] Vin[n] SWF SWF SWF SWF GS GS SWF SWF dc o↵set dc o↵set SWF SWF SWF SWF SWF SWF Vout[n] Vout[n] 4 Figure 16. Block diagram representation of the Serge middle VCM. Blocks labeled “SWF” indicate the Serge wavefolder model. Figure 16. Block diagram representation of the Serge middle VCM. Blocks labeled “SWF” indicate the Serge wavefolder model. In cascaded wavefolder structures like the one shown in Figure 16, timbral control can be achieved in two manners. The first is by adjusting the gain of the input waveform (using GS in this case). This parameter controls the amount of folds introduced, allowing the overall brightness of the sound to be varied. It can be modulated in real-time to provide articulation to a sound similar to filtering in subtractive synthesis or modulation index in FM synthesis. The second way to control timbre is by adding a dc offset to the input of the wavefolder. This breaks the aforementioned symmetry of the folding function and introduces even harmonics. When modulated by using, for example, a low-frequency oscillator, this parameter provides an effect reminiscent of pulse-width modulation. Figure 17a shows the transfer function of the Serge middle VCM model for the case of zero dc offset. This plot was generated by defining Vin to have a constant value of 1 V and sweeping through values of GS between −8 and 8. Figure 17b shows the output of the Serge middle VCM when driven by a Appl. Sci. 2017, 7, 1328 18 of 23 18 of 23 100-Hz sinusoid with GS = 6. 6. Practical Synthesis Usage This design, while not based on any existing circuit, is comparable to that of the Yusynth Metalizer which also utilizes four Lockhart circuits in series [55]. The following paragraphs describe the sections of this proposed topology. Its frequency-domain behavior is then examined and compared with that of the Serge middle VCM. The blocks labeled “LWF” in Figure 19 correspond to the proposed Lockhart wavefolder model. In order for this cascade of Lockhart wavefolders to behave as expected, we need to make sure that the individual folding stages satisfy two criteria. Firstly, the individual folders must provide approximately unity gain for small input values, i.e., below the folding point, and approximately negative unity gain Appl. Sci. 2017, 7, 1328 19 of 23 19 of 23 beyond the folding point. Secondly, each stage should start folding at the same point with respect to its individual input. beyond the folding point. Secondly, each stage should start folding at the same point with respect to its individual input. tanh() tanh() LPF LPF Vin[n] Vin[n] Vout[n] Vout[n] 1/3 1/3 3 fc fc RL RL LWF LWF LWF LWF LWF LWF LWF LWF GL GL dc o↵set dc o↵set Figure 19. Block diagram for the proposed VA cascaded Lockhart wavefolder topology. Blocks labeled “LWF” and “LPF” indicate the Lockart wavefolder model and lowpass filtering, respectively. Figure 19. Block diagram for the proposed VA cascaded Lockhart wavefolder topology. Blocks labeled “LWF” and “LPF” indicate the Lockart wavefolder model and lowpass filtering, respectively. We can meet these criteria with the proposed Lockhart model by selecting an appropriate value for RL and adding static gain stages before and after the folding stages. These gain blocks will also help compensate for the attenuation introduced by the folding operation. First, we choose a value of RL for which the Lockhart wavefolder exhibits unity gain for small input values. Having found this resistance value, the pre- and post-gain stages can be determined by measuring the value of |Vout| at exactly the folding point. The pre-gain is taken to be approximately this value, and the post-gain is taken to be its inverse. In Section 3.1, it was shown that for RL = 7.5 kΩthe Lockhart wavefolder exhibits approximately unity gain below the folding point. This value leads to pre- and post-gains of approximately 1/3 and 3, respectively. 6. Practical Synthesis Usage Figure 20a shows the transfer function of the proposed structure measured at the output of the post-gain block. We can observe how the folds introduced by this structure are evenly distributed, unlike those in Figure 17a. As with the Serge middle VCM, timbral control is achieved by modulating the value of GL and by adding a dc offset. The static gain blocks ensure the amplitude of the folded output is bounded between approximately ±1 V for values of GL between −10 and 10 (assuming once more that Vin has a peak amplitude of 1 V). Figure 20b shows the time-domain result of processing a 100-Hz sinusoidal input with the proposed structure for GL = 10 and zero dc offset. In this particular design, additional timbral control can be achieved by modulating the value of RL. Figure 20. (a) Transfer function of the proposed cascaded Lockhart wavefolder structure measured after the post-gain block; and (b) its output when driven by a 100-Hz sinusoidal input for GL = 10 and zero dc offset. Figure 20. (a) Transfer function of the proposed cascaded Lockhart wavefolder structure measured after the post-gain block; and (b) its output when driven by a 100-Hz sinusoidal input for GL = 10 and zero dc offset. Lastly, we add two optional blocks. The first is a tanh() function after the post-gain block to model the behavior of an output buffering stage and to limit the range of the output waveform. This tanh() block can also be antialiased using the antiderivative method described by Equation (45). The antiderivative of the tanh() function is given by log(cosh()) [48]. The second optional block is a static one-pole lowpass filter with a cutoff at fc = 1.3 kHz whose purpose is to act as a simple tone control. A similar static filtering stage can be found at the output of the Buchla 259 wavefolder [17]. The s-domain transfer function of this filtering stage is defined as H(s) = wc s + wc , (51) H(s) = wc s + wc , (51) where wc = 2π fc. where wc = 2π fc. Appl. Sci. 2017, 7, 1328 20 of 23 20 of 23 Finally, we examine the time-varying behavior of the proposed structure by considering the case of a 150-Hz input sinewave with variable gain GL and dc offset. Figure 21 shows the spectrogram that results from varying GL from 0 to 15. 6. Practical Synthesis Usage As expected, the system introduces complex harmonic patterns similar to those shown in Figure 18a. Likewise, Figure 18b demonstrates the effect of varying GL from 0 to 10 while simultaneously increasing the level of dc offset from 0 to 5 V. This response is comparable to that of the Serge middle VCM (see Figure 18b). Frequency (Hz) -80 -70 -60 -50 -40 -30 -20 -10 0 0 1 2 3 4 5 -80 -70 -60 -50 -40 -30 -20 -10 0 dc o↵set (V) dc o↵set (V) (b) (b) Figure 21. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed cascaded Lockhart topology with varying gain GL from 0 to15; and (b) a 200-Hz sinewave processed with varying gain GS from 0 to 0 and dc offset from 0 to 5 V. -80 -70 -60 -50 -40 -30 -20 -10 0 0 1 2 3 4 5 -80 -70 -60 -50 -40 -30 -20 -10 0 dc o↵set (V) dc o↵set (V) (b) (b) Figure 21. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed cascaded Lockhart topology with varying gain GL from 0 to15; and (b) a 200-Hz sinewave processed with varying gain GS from 0 to 0 and dc offset from 0 to 5 V. A real-time demo of the proposed Lockhart wavefolder topology implemented using Max/MSP and Gen is available at http://research.spa.aalto.fi/publications/papers/smc17-wavefolder. A real-time demo of the proposed Lockhart wavefolder topology implemented using Max/MSP and Gen is available at http://research.spa.aalto.fi/publications/papers/smc17-wavefolder. Supplementary Materials: The following are available online at http://research.spa.aalto.fi/publications/ papers/smc17-wavefolder: a real-time Max/MSP demo of the proposed Lockhart wavefolder topology implemented using Gen~and a MATLAB implementation of the Lambert-W function using Fritsch’s iteration. 7. Conclusions In this work, we have explored the behavior of two West Coast synthesizer circuits: the Lockhart and Serge wavefolders. By means of circuit analysis, we have derived closed-form expressions for the characteristic transfer functions of both systems. These transfer functions were validated against SPICE simulations implemented using LTspice. The results obtained indicate a good match between the proposed models and their corresponding SPICE simulations. In addition to this, we observed that the behavior of both circuits is very similar, despite the fact that their designs are fundamentally different. The issue of aliasing caused by wavefolding in the digital domain was treated by incorporating the first-order antiderivative method. Within the context of the Lockhart wavefolder, it was shown that the proposed antialiased model is perceptually free from the effects of aliasing distortion when implemented at a sampling rate of Fs = 88.2 kHz. A thorough evaluation of the proposed Lockhart model indicates that this configuration yields a signal quality equivalent to that of oversampling by a factor of eight (i.e., Fs = 352.8 kHz) at nearly a fourth of the computational expenses. For the case of the Serge wavefolder, the use of the antiderivative method produces an increase in signal quality equivalent to that of oversampling by a factor of two (i.e., Fs = 88.2 kHz). Furthermore, a recommended synthesis topology built around the Lockhart model consisting of four cascaded wavefolding stages, a saturator and a lowpass filter was presented. This topology was compared against a model of the Serge middle VCM built using six wavefolding stages. These structures illustrate the capabilities of wavefolding in a synthesis environment. However, it should be noted that the discussed topologies are not unique, as they can be modified according to the needs of the particular application. This effectively showcases the flexibility of VA models. Supplementary Materials: The following are available online at http://research.spa.aalto.fi/publications/ papers/smc17-wavefolder: a real-time Max/MSP demo of the proposed Lockhart wavefolder topology implemented using Gen~and a MATLAB implementation of the Lambert-W function using Fritsch’s iteration. 21 of 23 Appl. Sci. 2017, 7, 1328 21 of 23 Acknowledgments: The authors would like to thank Ken Stone for the valuable correspondence about the origins of the Lockhart circuit and Geoffrey Gormond for fruitful discussions on the behavior of the Serge VCM. 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Author Contributions: Fabián Esqueda and Henri Pöntynen conceived the idea for the paper and oversaw its development. Henri Pöntynen and Julian D. Parker wrote Sections 2.1 and 6, respectively, with additional work by Fabián Esqueda. Fabián Esqueda wrote the rest of the paper, conducted all measurements and generated all figures, with additional comments and corrections from the other three co-authors. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References In Proceedings of the International Conference on Digital Audio Effects (DAFx), Trondheim, Norway, 30 November–3 December 2015. 15. Buchla Electronic Musical Instruments. The History of Buchla. Available online: https://buchla.com/ history/ (accessed on 3 October 2017). 16. Parker, J.; D’Angelo, S. A digital model of the Buchla lowpass-gate. 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Audibility of aliasing distortion in sawtooth signals and its implications for oscillator algorithm design. J. Acoust. Soc. Am. 2012, 132, 2721–2733. c⃝2017 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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Prevalence, risk factors and health outcomes associated with polypharmacy among urban community-dwelling older adults in multi-ethnic Malaysia
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RESEARCH ARTICLE Abstract Citation: Lim LM, McStea M, Chung WW, Nor Azmi N, Abdul Aziz SA, Alwi S, et al. (2017) Prevalence, risk factors and health outcomes associated with polypharmacy among urban community-dwelling older adults in multi-ethnic Malaysia. PLoS ONE 12(3): e0173466. doi:10.1371/journal.pone.0173466 Objectives The aim of this study was to determine the prevalence, risk factors and health outcomes associated with polypharmacy in a cohort of urban community-dwelling older adults receiv- ing chronic medications in Malaysia. Received: November 24, 2016 Accepted: February 22, 2017 Published: March 8, 2017 Prevalence, risk factors and health outcomes associated with polypharmacy among urban community-dwelling older adults in multi- ethnic Malaysia Li Min Lim1, Megan McStea2,3, Wen Wei Chung4, Nuruljannah Nor Azmi3, Siti Azdiah Abdul Aziz3,5, Syireen Alwi1, Adeeba Kamarulzaman3,6, Shahrul Bahyah Kamaruzzaman2,6, Siew Siang Chua1, Reena Rajasuriar1,3,7* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 2 The Malaysian Elders Longitudinal Research (MELOR) Group, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 3 Centre of Excellence for Research in AIDS (CERiA), University of Malaya, Kuala Lumpur, Malaysia, 4 Pharmacy Department, University Malaya Medical Centre, Kuala Lumpur, Malaysia, 5 Faculty of Pharmacy, National University of Malaysia (UKM), Kuala Lumpur, Malaysia, 6 Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 7 Peter Doherty Institute for Infection and Immunity, Melbourne University, Melbourne, Australia Methods Copyright: © 2017 Lim 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. This was a baseline study in the Malaysian Elders Longitudinal Research cohort. The inclu- sion criteria were individuals aged 55years and taking at least one medication chronically (3 months). Participants were interviewed using a structured questionnaire during home visits where medications taken were reviewed. Health outcomes assessed were frequency of falls, functional disability, potential inappropriate medication use (PIMs), potential drug- drug interactions (PDDIs), healthcare utilisation and quality of life (QoL). Risk factors and health outcomes associated with polypharmacy (5 medications including dietary supple- ments) were determined using multivariate regression models. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the University of Malaya (UM) High Impact Research (HIR) Grant from the Department of Higher Education, Ministry of Education, Malaysia [UM.C/ 625/1/HIR/MOHE/ASH/02] to SBK and the University Malaya Research Grant from UM [UMRG RP029-14HTM] to RR. Background Polypharmacy has been associated with increased morbidity and mortality in the older population. Editor: Alessandra Marengoni, University of Brescia, ITALY * reena@um.edu.my Polypharmacy among community-dwelling older adults 56.9% of the cohort. The risk factors associated with increasing medication use were increasing age, Indian ethnicity, male, having a higher number of comorbidities specifically those diagnosed with cardiovascular, endocrine and gastrointestinal disorders, as well as supplement use. Health outcomes significantly associated with polypharmacy were PIMS, PDDIs and increased healthcare utilisation. Competing interests: The authors have declared that no competing interests exist. Conclusion A significant proportion of older adults on chronic medications were exposed to polyphar- macy and use of dietary supplements contributed significantly to this. Medication reviews are warranted to reduce significant polypharmacy related issues in the older population. Introduction The older population aged 60 years and above is expected to grow by 56% worldwide between 2015 and 2030, with the fastest growth in urban settings of developing regions [1]. In Malaysia, older populations have been projected to grow from 9.2% in year 2015 to almost a quarter of the total population (23.6%) by the year 2050 and this has been anticipated to add considerable demands on the countries’ health services [1, 2]. Among others, age related multi-morbidity and disability in older adults will increase the use of medications [3, 4] and subsequently the risk of polypharmacy which may be conceptually defined as the use of more medications than is medically necessary, though there is currently no universal definition for this in the literature [5]. Polyphar- macy is a concern especially in older adults as they are prone to age- and disease-related pharmaco- kinetic and pharmacodynamic changes, and are thus more sensitive to drug therapy [6]. Polypharmacy in older individuals is strongly associated with multiple negative health out- comes which include increased healthcare costs [7], functional impairment, falls and fractures [8], harmful drug interactions [9] and adverse drug reactions [10, 11]. Intervention studies which focused on reducing polypharmacy among older persons either with the involvement of a pharmacist or a multidisciplinary team, have largely reported significant improvements in the quality of medication use [12–15]. Few studies have extended such observation to include health outcomes, but the findings have not been consistent [15, 16]. Despite a growing aging population and the known risks of increased medication related issues in this group, studies on polypharmacy among the older population in Malaysia have been limited to hospital settings and institutions of aged care [17–19]. Thus far, no studies have been found on older community dwelling individuals. In addition, only a few studies have included the use of dietary supplements in its assessment of polypharmacy despite their potential to cause clinically significant drug interactions and adverse drug events [9]. In multi-ethnic Malaysia, the use of herbal and dietary supplements are highly prevalent as reported in other Asian settings [20, 21] and these are often utilised as adjunct therapies for chronic health conditions [22–25]. Therefore, the present study aimed to determine the prevalence, risk factors and health outcomes associated with polypharmacy among urban community-dwelling older adults receiving chronic medications in Malaysia and further examined the role of dietary supplements in this context. Materials and methods Study design, setting and population Results A total of 1256 participants were included with a median (interquartile range) age of 69(63– 74) years. The prevalence of polypharmacy was 45.9% while supplement users made up 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Data collection Participants’ baseline socio-demographic data and medical history were extracted from the MELoR database. Information on all medications used including dietary supplements (brand names and dosages) were verified by reviewing prescriptions and the actual medications dur- ing the home visits. Medications were classified according to the WHO Anatomical Therapeu- tic Chemical (ATC) classification system [27]. Dietary supplements which could not be classified according to the ATC system were coded as “others”. Two researchers independently classified the medications and any discrepancies were resolved by a third researcher, with the consensus of the initial two researchers. Polypharmacy among community-dwelling older adults aspects of aging faced by the urban community-dwelling older adults in Malaysia. Details of this cohort have been published previously [26]. Briefly, participants were individuals ran- domly identified through the Malaysian electoral roll at three representative urban parliamen- tary constituencies (Petaling Jaya South, Petaling Jaya North and Lembah Pantai), aged 55 years and above in 2013 and stratified by the three main ethnicities in Malaysia; Malays, Chi- nese and Indians. 8769 invitations were sent to the main ethnic groups and volunteers were invited through aged focused community groups. A total of 5815 participants with contactable addresses were identified through these efforts. Of these, only 3334 were eligible for the study (cri- teria below), had not moved to a new address or passed away. An age cut-off of 55 years was cho- sen because this age range enabled the analysis of issues in older individuals as they approached retirement. In Malaysia, the voluntary retirement age is 55 years and mandatory retirement age is 60 years. Individuals who were bed-bound, unable to be transported to the research centre for assessment and unable to communicate or answer questions due to advanced dementia or severe speech impediments, were excluded from the MELoR study. In addition, only individuals who were using at least one chronic medication, whether prescription or non-prescription and dietary supplements, were included in this study. Dietary supplements were defined as vitamins, miner- als, amino acids, herbs and other botanicals as described previously [22]. Following written informed consent to participate in the study, all participants answered a questionnaire (either in English, Malay, Chinese or Tamil language) executed in the setting of their homes by trained interviewers. Subsequently, participants attended a study clinic in the University Malaya Medical Centre (UMMC) where further health assessments and biochemi- cal screenings were performed. The questionnaire covered aspects of socio-demographic, gen- eral health, medical and medication history, healthcare utilisation and a series of validated questionnaires to assess falls, functional disability and quality of life (QoL). Recruitment for MELoR is ongoing and participants for the present study were selected among participants recruited consecutively from October 2013 till June 2015. Study design, setting and population This was a baseline study conducted in the Malaysian Elders Longitudinal Research (MELoR) cohort. MELoR is a multi-disciplinary study conducted to explore the multi-dimensional 2 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Polypharmacy among community-dwelling older adults used as it provides improved guidance on drug issues related to drug-drug interactions and medications to be avoided in older individuals with impaired renal function. Two researchers independently assessed participants’ medications for PIMs use and any discrepancies were resolved by a third researcher with their consensus. Assessment of kidney function were based on laboratory results obtained during the participants’ visit to UMMC for biochemical screen- ing. As assessments for PIM use were done retrospectively, some assumptions were made when medical or medication history were incomplete for specific assessments. These assump- tions are detailed in S1 File. Potential drug-drug interactions (PDDIs) were assessed using the Thompson Microme- dex1 2.0 interaction database (Truven Health Analytics Inc., Greenwood Village, CO, USA). Only interactions listed as major or contraindicated with clinically relevant and potentially serious consequences were included in the present study. PDDIs were assessed using this inter- action database as it included potential interactions with dietary supplements which were not captured in the updated Beers Criteria [29]. Falls were defined based on self-reported responses to the question “Have you fallen in the past 12 months?” Functional disability was assessed using the Modified Katz Index of Independence in Activ- ities of Daily Living (ADL) [30] and Lawton Instrumental Activities of Daily Living (IADL) [31]. For ADL, participants were classified as dependent if they reported needing help with one or more daily living activities: walking, bathing, personal grooming, dressing, eating, get- ting from bed to chair and using the toilet. For IADL, participants who reported low function in one or more of the following activities were classified as dependent: using telephone, shop- ping, preparing food, doing housework, travelling, taking medicine and managing money. Healthcare utilisation was assessed based on the participants’ reported number of visits to any of the following health facilities in the past 12 months: public hospital or clinic, private hospital or clinic and traditional or alternative medical health practitioners. Quality of life (QoL) was assessed using 12 items which originated from the CASP-19 (Con- trol, Autonomy, Self-realisation and Pleasure) questionnaire [32]. CASP-12 as previously described [33] is based on 12 Likert-like scale items which assessed four theoretically derived dimensions of QoL in older adults: control, autonomy, self-realisation and pleasure. Each item was numerically coded so that the most positive response was scored as 3 while the most nega- tive response as 0. This outcome was analysed as a continuous variable (score range of 0–36), with lower scores associated with poorer QoL. Chronic health problems were assessed based on self-reported diseases by participants and verified by the medications taken. Payment method for healthcare services was assessed based on participants reported mode of payment when they utilized any healthcare facility in the preceding 12 months. Study definitions and outcome measures Polypharmacy was defined as the concurrent use of five or more medications [28]. These included prescribed, non-prescribed and over-the counter (OTC) medications including die- tary supplements such as vitamins, minerals, herbals and other botanicals. Combined formula- tions (multiple active ingredients in a preparation) were considered as a single medication. This definition for polypharmacy is among the most widely used in the literature, is objective and allows comparability across different studies. Potential inappropriate medications (PIMs) use was defined according to the American Geriatrics Society 2015 Updated Beers Criteria [29]. This guideline lists medications to be avoided in older adults in all settings except in hospice and palliative care, with the aim to reduce the risk of adverse drug events in older individuals. The updated Beers Criteria was 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Characteristics of participants The median (interquartile range, IQR) age of the participants was 69 (63–74) years, ranging from 55 to 97 years. The participants were almost evenly distributed among the three main ethnic groups in Malaysia that is, Indian (35.6%), Chinese (33.3%) and Malay (30.5%). The median (IQR) number of comorbidities were 3 (2–4) and the maximum reported was 11. A summary of the participants’ characteristics is shown in Table 1. The three most common chronic health conditions reported were cardiovascular disorders (1002 participants; 79.8%), endocrine disorders (472; 37.6%), bone and joint disorders (359; 28.6%). The three most com- monly used medication classes were those that act on the cardiovascular system (1030; 82.0%), alimentary tract and metabolism (860; 68.5%), blood and blood forming organs (371; 29.5%). A complete list of medication classes used are detailed in S1 Table. Polypharmacy among community-dwelling older adults produced a p<0.250 in the univariate analysis were included in the final multivariate model. For the analysis of health outcomes associated with polypharmacy, models were adjusted for the number of comorbidities, age, ethnicity and gender. All statistical analyses were performed using the Statistical Package for Social Science (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA) and Stata Statistical Software: Release 14 (StataCorp LP, College Station, TX). Statistical significance was defined as p<0.050. Results A total of 1504 participants aged 55 years and above were recruited into the MELoR study from October 2013 through June 2015. 55% of eligible and contactable participants declined to participate either due to time constraints or inability to attend the research clinic. There was no difference in terms of gender and ethnicity (p>0.05 for all on chi-square analysis) among those who declined to participate compared to those included in the study. Of the 1504 recruited, 248 individuals did not receive any medications and hence, only 1256 participants were included in the present study. Ethics and sample size calculation The study protocol was approved by the Medical Ethics Committee of University Malaya Med- ical Centre (MEC 943.6) and all participants provided informed written consent. Based on pre- vious reported rates of polypharmacy in Malaysia in a hospital setting (46%), we estimated a minimum sample size of 400 would be required for this study using the binomial exact method with a 95% level of confidence and a 90% power. Statistical analysis Graphical tests were used to assess the normality of the data distribution. Parameters which were missing in 1% of participants or less were considered as negligible [34]. The number of medications taken as a count variable was used as a surrogate measure of polypharmacy in the analysis of risk factors and health outcomes associated with polypharmacy. Multivariate Pois- son regression analysis was performed to identify risk factors associated with polypharmacy. Ordinal logistic regression was used to assess the health outcomes associated with polyphar- macy specifically falls, functional disability, PIMs and PDDIs while the association of poly- pharmacy with healthcare utilisation and QoL were assessed using multivariate linear regression. In the analysis of risk factors associated with polypharmacy, only variables that 4 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Prevalence of polypharmacy and supplement use A total of 5804 medications (prescribed or non-prescribed medications and dietary supple- ments) were used, with a median of 4 (2–6) medications per person and a maximum of 20. The prevalence of polypharmacy was 45.9% with 576 individuals using at least five medica- tions. Among those with polypharmacy exposure, 499 (86.6%) received 5 to 9 medications, 65 (11.3%) received 10 to 14 medications while 12 (2.0%) received 15 or more. The percentage of participants using 5 medications or more increased with age (Table 1). Polypharmacy was also more prevalent among Indian participants compared to the Malay and Chinese. In terms of chronic health conditions, more than 70.0% of the participants who reported gastrointestinal, renal and psychiatric disorders were also taking 5 medications or more. In addition, 76.0% of the participants with PDDIs were classified in the polypharmacy cohort and also 67.8% of those with PIMs. A majority of the participants (54.4%) who reported 5 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Polypharmacy among community-dwelling older adults Table 1. Characteristics of all participants included in the study (n = 1256) as well as those with polypharmacy (n = 576) and dietary supplement users (n = 715). Characteristics Total, n(column %) or median (IQR) Polypharmacy, n(row %) or median (IQR) Dietary supplement users, n(row %) or median (IQR) Total 1256 (100.0) 576 (45.9) 715 (56.9) Age (n = 1256) 69 (63–74) 71 (66–76) 70 (64–75) 55–59 124 (9.9) 39 (31.5) 76 (61.3) 60–64 243 (19.4) 79 (32.5) 126 (51.9) 65–69 279 (22.2) 122(43.7) 160 (57.3) 70–74 300 (23.9) 151 (50.3) 178 (59.3) 75–79 202 (16.1) 118 (58.4) 119 (58.9) 80+ 108 (8.6) 67 (62.0) 65 (60.2) Gender (n = 1256) Male 532 (42.3) 259 (48.7) 269 (50.6) Female 724 (57.7) 317 (43.8) 446 (61.6) Ethnic (n = 1255) Malay 383 (30.5) 137 (35.8) 133 (34.7) Chinese 418 (33.3) 170 (40.7) 294 (70.3) Indian 447 (35.6) 265 (59.3) 284 (63.5) Others 7 (0.6) 4 (50.0) 4 (50.0) Highest education level (n = 1252) Primary or No Formal 326 (26.0) 133 (40.8) 121 (37.1) Secondary 538 (43.0) 232 (43.1) 317 (58.9) Tertiary 388 (31.0) 210 (54.1) 275 (70.9) Employment status (n = 1255) No 1021 (81.4) 483 (47.3) 572 (56.0) Yes 234 (18.6) 93 (39.7) 142 (60.7) Payment method for healthcare service in the last 12 months† (n = 1145) Used free service 56 (4.9) 32 (57.1) 34 (60.7) Self/Out of pocket 591 (51.6) 252 (42.6) 332 (56.2) Employer 292 (25.5) 156 (52.7) 156 (53.4) Relatives 127 (11.1) 64 (50.4) 67 (52.8) Welfare 112 (9.8) 62 (55.4) 67 (59.8) Insurance 13 (1.1) 4 (30.8) 10 (76.9) Non-governmental/Religious organizations 4 (0.4) 3 (75.0) 4 (100) Chronic health problems† (n = 1256) Cardiovascular disorders 1002 (79.8) 607 (50.6) 519 (51.8) Endocrine disorders 472 (37.6) 296 (62.7) 229 (48. Polypharmacy among community-dwelling older adults Table 1. (Continued) Characteristics Total, n(column %) or median (IQR) Polypharmacy, n(row %) or median (IQR) Dietary supplement users, n(row %) or median (IQR) Katz ADL 44 (3.5) 25 (56.8) 27 (61.4) Lawton IADL 388 (30.9) 210 (54.1) 192 (49.5) PIMs use (n = 1256) 400 (31.8) 271 (67.8) 177 (44.3) PDDIs (n = 1256) 281 (22.4) 215 (76.5) 141 (50.2) Smoking status (n = 1236) Never 985 (79.7) 446 (45.3) 584 (59.3) Current smoker 96 (7.8) 36 (37.5) 40 (41.7) Ex-smoker 155 (12.5) 87 (56.1) 78 (50.3) Alcohol consumption (n = 1250) Never 889 (71.1) 388 (43.6) 467 (52.5) Yes 280 (22.4) 147 (52.5) 204 (72.9) Used to 81 (6.5) 37 (45.7) 40 (49.4) More than 1 comorbidity 979 (78.0) 521 (53.2) 636 (65.0) Using dietary supplements 715 (56.9) 401 (56.1) - Quality of life (QoL) (n = 124), CASP 12 score 28 (24–31) 28 (24–31) 28 (25–31) Number of visits to any healthcare facility in the last 12 months† (n = 1202) 4 (2–6) 4 (2–7) 3 (1–6) Number of comorbidities (n = 1256) 3 (2–4) 3 (2–5) 3 (1–4) Number of prescribed and non-prescribed drugs (n = 1256) 3 (1–5) 5 (3–6) 2 (1–4) Number of dietary supplements (n = 1256) 1 (0–2) 2 (0–3) 2 (1–3) Total number of medications (n = 1256) 4 (2–6) 6 (5–8) 5 (3–7) IQR: interquartile range; ADL: activities of daily living; IADL: instrumental activities of daily living; PIMs: potential inappropriate medications; PDDIs: potential drug-drug interactions †More than one choice can be chosen. d i 10 1371/j l 0173466 001 IQR: interquartile range; ADL: activities of daily living; IADL: instrumental activities of daily living; PIMs: potential inappropriate medications; PDDIs: potential drug-drug interactions †More than one choice can be chosen. having functional disability, assessed using either Katz ADL or Lawton IADL, and almost half of those who reported having falls, were categorised in the polypharmacy group. More than half of the participants (56.9%) reported the use of at least one dietary supple- ment, with the median (IQR) of 1 (0–2) in the total cohort and 2 (0–3) in the polypharmacy group. Dietary supplements made up a total of 1782 or 30.7% of the total medications used (details in S2 Table) and were utilised by 401 participants (69.6%) who had polypharmacy exposure. When analysed by ethnic background, the proportion of Chinese who reported the use of dietary supplements was almost double that of the Malays (Table 1). The chronic condi- tions associated with the highest usage of dietary supplements were oncologic disorders, fol- lowed by bone and joint disorders, and gastrointestinal disorders. Among participants with PDDIs and PIMs, 50.2% and 44.3% respectively, reported the use of supplements. 5) Bone and joint disorders 359 (28.6) 211 (58.8) 246 (68.5) Urologic disorders 321 (25.6) 170 (53.0) 182 (56.7) Ophthalmic disorders 237 (18.9) 130 (54.9) 134 (56.5) Respiratory disorders 104 (8.3) 58 (55.8) 53 (51.0) Oncologic disorders 84 (6.7) 47 (56.0) 72 (85.7) Gastrointestinal disorders 64 (5.1) 45 (70.3) 43 (67.2) Renal disorders 39 (3.2) 30 (76.9) 21 (53.8) Neurologic disorders 22 (1.8) 14 (63.6) 12 (54.5) Falls in the last 12 months (n = 1256) 294 (23.4) 146 (49.7) 151 (51.4) Functional disability (Continued) nts included in the study (n = 1256) as well as those with polypharmacy (n = 576) and dietary supplement (Continued) PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 6 / 18 vities of daily living; IADL: instrumental activities of daily living; PIMs: potential inappropriate medications; PDDIs: Polypharmacy among community-dwelling older adults variables were constant. Similarly, when the other variables were held constant in the model, male participants had an incidence rate ratio (for the number of medications taken) of 1.09 times that of female participants (an increase of 9%). As participants increased the number of dietary supplements taken, they had a 19% increase in the total number of medications they were taking (IRR 1.19; 95% CI: 1.15, 1.23, p<0.001). Similarly, as the number of comorbidities increased, there was also an increased use of medications (IRR 1.14; 95% CI: 1.11, 1.17, p<0.001). Specifi- cally, participants who had cardiovascular disorders (IRR 1.23; 95% CI: 1.14, 1.33, p<0.001), endocrine disorders (IRR 1.47; 95% CI: 1.31, 1.64, p<0.001) and gastrointestinal disorders (IRR 1.13; 95% CI: 1.03, 1.24, p = 0.010) had a higher incidence rate ratio for the number of medica- tions taken compared to those without these disorders. However, those with urologic disorders were associated with a lower number of medication use. Health outcomes associated with polypharmacy Multivariate logistic analysis found that PIMs (OR 1.27; 95% CI: 1.20, 1.34, p<0.001) and PDDIs (OR 1.34; 95% CI: 1.26, 1.42, p<0.001) were significantly associated with the increase in number of medications used after adjusting for age, gender, ethnicity and the number of comorbidities but not whether falls occurred in the past 12 months or functional impairment existed (Table 3). Multivariate linear regression models found a significant association between the increase in number of medications used and healthcare utilisation (β = 0.44; 95% CI: 0.30, 0.59, p <0.001) but not the quality of life scores. In a similar analysis performed for prescription/non-prescription drugs and dietary supple- ments separately, it was found that increased supplement use remained significantly associated with PDDIs but quality of life scores also improved (data not shown). The health impact asso- ciated with prescription/non-prescription polypharmacy were similar to that found when the total medication used was analysed. Risk factors associated with polypharmacy Risk factors associated with increasing medication use as a surrogate of polypharmacy are summarised in Table 2. Multivariate Poisson regression analysis identified an increasing num- ber of medications use as age increased compared to those aged 55–64 (p<0.001) when adjusted for the other variables in the model. Malays (incidence rate ratio, IRR 0.89; 95% confi- dence interval, CI: 0.30, 0.94,) and Chinese (IRR 0.83; 95% CI: 0.78, 0.88) had a lower inci- dence rate ratio of medication use when compared to Indians (p<0.001) whilst all the other 7 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Discussion This study assessed the prevalence, risk factors and health outcomes associated with polyphar- macy, including the contributory role of dietary supplement use among older individuals tak- ing chronic medications in the community setting in Malaysia. The study found a high prevalence of polypharmacy (46%) with dietary supplements constituting a third of the total number of medications used in older individuals. Risk factors independently associated with increased medication use were advanced age, being male, increasing number of comorbidities specifically cardiovascular, endocrine, and gastrointestinal disorders, and dietary supplement use. As for ethnic groups, Chinese and Malays were associated with lower risk of polyphar- macy compared to Indians as well as individuals diagnosed with urologic disorders. The health outcomes associated with increased medication use were potential inappropriate medication use (PIMs), potential drug-drug interactions (PDDIs) and increased healthcare utilisation while increased medication use was not found to be associated with falls, functional disability and quality of life in this cohort. Increased use of dietary supplement was independently asso- ciated with increased PDDIs and improved quality of life scores after adjusting for age, gender, ethnicity and number of comorbidities. The prevalence of polypharmacy in older individuals varied between 39% and 89% in the Asian regions, depending on the definition of polypharmacy used, the age range of the partici- pants and the research settings [28, 35–39]. Prior studies on polypharmacy among the Malay- sian elderly people, reported rates between 39% and 55% in the hospital setting [17, 19] but currently, no data is available for community dwelling individuals. Studies assessing polyphar- macy in the community are logistically harder to conduct and reports from other countries 8 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Polypharmacy among community-dwelling older adults Table 2. Risk factors associated with polypharmacy among urban community-dwelling older adults. Variables Univariate Poisson regression Multivariate Poisson regression Crude IRR (95%CI) p-value Adjusted IRR (95%CI) p-value Number of dietary supplements 1.13 (1.11–1.15) <0.001 1.19 (1.15–1.23) <0.001 Number of comorbidities 1.15 (1.13–1.16) <0.001 1.14 (1.11–1.17) <0.001 Ethnic <0.001 <0.001 Indian (reference) 1.00 1.00 Malay 0.73 (0.67–0.79) 0.89 (0.30–0.94) Chinese 0.77 (0.71–0.83) 0.83 (0.78–0.88) Age <0.001 <0.001 55–59 (reference) 1.00 1.00 60–64 1.05 (0.93–1.21) 1.05 (0.95–1.15) 65–69 1.27 (1.11–1.44) 1.11 (1.01–1.22) 70–74 1.47 (1.29–1.67) 1.23 (1.12–1.34) 75–79 1.55 (1.36–1.77) 1.28 (1.15–1.41) 80+ 1.63 (1.40–1.90) 1.29 (1.13–1.46) Chronic health problems† Cardiovascular disorders 1.42 (1.28–1.58) <0.001 1.23 (1.14–1.33) <0.001 Endocrine disorders 1.48 (1.38–1.58) <0.001 1.47 (1.31–1.64) <0.001 Bone and joint disorders 1.26 (1.17–1.35) <0.001 _ Urologic disorders 1.12 (1.03–1.21) 0.006 0.91 (0.86–0.97) 0.002 Ophthalmic disorders 1.15 (1.06–1.25) 0.001 – Respiratory disorders 1.13 (1.00–1.28) 0.051 _ Oncologic disorders 1.12 (1.03–1.32) 0.014 _ Gastrointestinal disorders 1.48 (1.29–1.68) <0.001 1.13 (1.03–1.24) 0.010 Renal disorders 1.59 (1.36–1.85) <0.001 – Neurologic disorders 1.31 (0.97–1.77) 0.077 – Psychiatric disorders 1.08 (0.71–1.64) 0.714 – Smoke cigarettes 0.002 No (reference) 1.00 – _ Current smoker 0.86 (0.76–0.97) 0.013 _ Ex-smoker 1.125 (1.01–1.23) 0.031 _ Payment method for healthcare service in last 12 months† Used Free Service 1.14 1.01–1.28) 0.044 – Self/Out of pocket 0.90 (0.84–0.97) 0.004 – Employer 1.14 (1.05–1.23) 0.001 – Relative 1.09(0.98–1.23) 0.123 – Welfare 1.14 (1.02–1.28) 0.024 _ Insurance 0.73 (0.55–0.97) 0.028 _ Non-governmental/ religious organization 3.59 (0.37–34.56 0.270 _ Alcohol consumption history 0.01 No (reference) 1.00 – Yes 1.10 (1.02–1.19) 0.013 - Used to 1.17 (1.01–1.35) 0.037 Employment† 0.88 (0.81–0.97) 0.009 – Highest education level <0.001 Primary or below 1.00 _ _ Secondary 1.06(0.98–1.15) 0.170 _ Tertiary 1.22 (1.12–1.33) <0.001 _ (Continued) PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 9 / 18 Notably, 1 in 3 older individuals who were taking chronic medications in the community setting used five or more medications concurrently, with 1 in 2 reporting dietary supplement use. Additionally, more than 30% of the cohort experienced PIMs similar to a prior study among elderly nursing home residents in Malaysia [42] while one fifth were exposed to clinically significant PDDIs. These data highlight high rates of medication related issues among the community-dwelling older individuals in Malaysia, which currently does not have a national programme for medication review in the elderly population, unlike many other countries [43–45]. Given that Malaysia will experience a sizable increase in their older population over the next decade, the introduc- tion of such a programme is timely and the findings from this study would provide baseline data to monitor the changes over time. Table 3. Health outcomes associated with polypharmacy among urban-community dwelling older adults. Health outcomes associated with polypharmacy Categorical variables Crude OR (95%CI) p-value †Adjusted OR (95%CI) p-value PIMs* 1.31 (1.25–1.37) <0.001 1.27 (1.20–1.34) <0.001 PDDIs* 1.38 (1.31–1.46) <0.001 1.34 (1.26–1.42) <0.001 Fall in last 12 months 1.04 (0.99–1.09) 0.060 – – Katz ADL 1.04 (0.94–1.14) 0.473 – – Lawton IADL 1.09 (1.05–1.14) <0.001 – – Continuous variables Crude β (95%CI) p-value †Adjusted β (95%CI) p-value Healthcare utilisation 0.46 (0.32–0.60) <0.001 0.44 (0.30–0.59) <0.001 CASP 12 QoL -0.11 (-2.08 -,0.01) 0.040 - - OR: odd ratio; CI: confidence interval; PIMs: potential inappropriate medications; PDDIs: potential drug-drug interactions; ADL: activities of daily living; IADL: instrumental activities of daily living †Adjusted for age, gender, number of comorbidities and ethnicity Healthcare utilisation unadjusted model:, R2 = 0.003; adjusted model: R2 = 0.038 CASP 12 QoL unadjusted model:, R2 = 0.003; β: regression coefficient; CI: confidence interval; R2: coefficient of determination; CASP 12 QoL: control, autonomy, self realisation and pleasure measure of quality of life Table 3. Health outcomes associated with polypharmacy among urban-community dwelling older adults. doi:10.1371/journal.pone.0173466.t003 Polypharmacy among community-dwelling older adults Table 2. (Continued) Variables Univariate Poisson regression Multivariate Poisson regression Crude IRR (95%CI) p-value Adjusted IRR (95%CI) p-value Gender Male 1.06 (0.99–1.14) 0.085 1.09 (1.03–1.14) 0.001 IRR:incidence rate ratio; CI: confidence interval †”No” is the reference group Multicollinearity Overdispersion and interaction terms were checked and not found. Interactions were between the number of supplements and the number of comorbidities and the presence of endocrine disorders and number of comorbidities Area under the receiver operating characteristic (ROC) curve (79.9%) were applied to check the goodness of qfit doi:10.1371/journal.pone.0173466.t002 often utilized data from health insurance claims [40] or telephone interviews [41] to estimate the rates of polypharmacy in the community. These approaches may be inaccurate due to underreporting and the omission of out of pocket purchases of non-prescription drugs includ- ing dietary supplements. In the present study, medication use was reported by the study partic- ipants and verified against the actual medications taken and prescriptions during home visits which provided a more accurate estimate of the total medications taken. Notably, 1 in 3 older individuals who were taking chronic medications in the community setting used five or more medications concurrently, with 1 in 2 reporting dietary supplement use. Additionally, more than 30% of the cohort experienced PIMs similar to a prior study among elderly nursing home residents in Malaysia [42] while one fifth were exposed to clinically significant PDDIs. These data highlight high rates of medication related issues among the community-dwelling older individuals in Malaysia, which currently does not have a national programme for medication review in the elderly population, unlike many other countries [43–45]. Given that Malaysia will experience a sizable increase in their older population over the next decade, the introduc- tion of such a programme is timely and the findings from this study would provide baseline data to monitor the changes over time. often utilized data from health insurance claims [40] or telephone interviews [41] to estimate the rates of polypharmacy in the community. These approaches may be inaccurate due to underreporting and the omission of out of pocket purchases of non-prescription drugs includ- ing dietary supplements. In the present study, medication use was reported by the study partic- ipants and verified against the actual medications taken and prescriptions during home visits which provided a more accurate estimate of the total medications taken. Polypharmacy among community-dwelling older adults Among the risk factors associated with polypharmacy, advanced age and increasing num- ber of comorbidities were associated with increased medication use, similar to that reported in other studies [17, 36, 46, 47]. Specifically, the diagnoses with cardiovascular, endocrine or gas- trointestinal disorders were associated with a significant increase in medication use compared to other disease conditions as previously described [17, 38, 46, 48]. Prior studies in the general population in Malaysia which assessed medication related issues in individuals diagnosed with cardiovascular and endocrine disorders had similarly highlighted a profile of multiple concur- rent medication use in the management of these conditions [49, 50]. In hypertension and dia- betes, which are among the most common cardiovascular and endocrine disorders in older people, numerous studies had found that the overzealous prescribing of medications to reach specific treatment targets may have only marginal benefits compared to the harms associated with hypoglycemia and hypotension in this age group. These may lead to falls, fractures and head injuries which could further impact physical function and quality of life [51–54]. There- fore, many guidelines including those in Malaysia recommend a less intensified approach for blood glucose and blood pressure management among the elderly patients [55–58] though this may not be widely practised [59]. Additionally, Malaysia does not have a national health insur- ance scheme and thus patients are not managed by a single medical practitioner but are free to visit any medical establishment to seek treatment. This system encourages multi-clinic visits and over-prescription [60] which invariably leads to polypharmacy. The lack of separation between prescribing and dispensing roles between doctors and pharmacists in Malaysia fur- ther complicates this situation. This study found ethnic groups, Malay and Chinese were less likely to be associated with polypharmacy compared to the Indians when all other covariates were held constant. The rea- sons for this are not entirely clear but polypharmacy disparities among different ethnicities have been previously reported [47, 61] and may be associated with different underlying socio- economic status [47, 62]. The present study did not have detailed information on the socio- economic status of the study participants but similar to a prior study [62], the univariate analy- sis showed that individuals paying from health insurance or paying out of pocket for health services were less likely to experience polypharmacy compared to individuals receiving free or subsidised healthcare services. Health outcomes associated with polypharmacy Categorical variables Crude OR (95%CI) p-value †Adjusted OR (95%CI) p-value PIMs* 1.31 (1.25–1.37) <0.001 1.27 (1.20–1.34) <0.001 PDDIs* 1.38 (1.31–1.46) <0.001 1.34 (1.26–1.42) <0.001 Fall in last 12 months 1.04 (0.99–1.09) 0.060 – – Katz ADL 1.04 (0.94–1.14) 0.473 – – Lawton IADL 1.09 (1.05–1.14) <0.001 – – Continuous variables Crude β (95%CI) p-value †Adjusted β (95%CI) p-value Healthcare utilisation 0.46 (0.32–0.60) <0.001 0.44 (0.30–0.59) <0.001 CASP 12 QoL -0.11 (-2.08 -,0.01) 0.040 - - Table 3. Health outcomes associated with polypharmacy among urban-community dwelling older adults. OR: odd ratio; CI: confidence interval; PIMs: potential inappropriate medications; PDDIs: potential drug-drug interactions; ADL: activities of daily living; IADL: instrumental activities of daily living j β: regression coefficient; CI: confidence interval; R2: coefficient of determination; CASP 12 QoL: control, autonomy, self realisation and pleasure measure of quality of life 10 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Polypharmacy among community-dwelling older adults negative impact on quality of life [71], depending on the disease setting. In the present study, an increase in supplement use in the elderly was associated with better quality of life scores. It is important to note that the CASP-12 tool used to measure QoL in this study only encom- passed the psychosocial constructs of quality of life including domains of control, autonomy, pleasure and self-realization but not physical aspects. However, no association was found between an increase in supplement use and measures of physical functioning including ADL and IADL. Therefore, the association between supplement use and improved quality of life should be interpreted with caution as other factors including socio-economic status which could also impact QoL were not assessed and adjusted in the present model. j Thus far, few studies have assessed health outcomes associated with polypharmacy in the community dwelling elderly (reviewed in [8]). In the multivariate analysis, it was found that increasing medication use (prescribed, non-prescribed and supplement use) was associated with PDDIs and PIMS after adjusting for age, sex, ethnicity and the number of comorbidities, as previously described [9, 72, 73]. In the present study, only drug interactions which were considered clinically significant and could potentially lead to serious consequences were assessed while PIMS identified the use of medications that had a high propensity to cause adverse events in the elderly population. Both of these outcomes have significant morbidity and mortality implications in the elderly population and are frequent causes of preventable medication related hospitalisations [54, 74, 75]. In addition, it was found that increasing medi- cation use was independently associated with increased healthcare visits to any healthcare facility in the prior 12 months though the reasons for these visits were not explored in this study. The use of multiple drugs is sometimes unavoidable in individuals with multiple comor- bidities. However, this needs to be weighed against the potential harms of increasing treatment burden in the elderly population. The findings in the present study highlight an urgent need to introduce medication reconciliation programmes among community-dwelling elderly popula- tion and increase educational initiatives among healthcare practitioners with regards to pre- scribing issues in older individuals. Approaches which have been shown to reduce adverse drug events and hospitalisation associated with polypharmacy should be considered [76–79]. PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 Future studies should explore the interaction between ethnicity and socio-economic status on polypharmacy and medication related issues in Malaysia. In the multivariate analysis, an increase in the use of dietary supplements was associated with almost double the incident risk ratio of polypharmacy (15%) compared to the increase in the number of comorbidities (8%). This highlights the important contributory role of supple- ment use to polypharmacy in the present study setting. A market survey on the profile of regu- lar supplement users in Malaysia found older individuals often considered taking dietary supplements as ‘nutritional insurance’ because they were concerned about their health follow- ing the diagnosis of chronic conditions [63]. More than two-thirds of the participants who reported using dietary supplement in the present study, had two or more comorbidities. Sup- plement use was also highest among elderly individuals diagnosed with oncologic disorders, as previously reported [64–66] though scientific evidence on the safety and efficacy of supple- ments in cancer has so far been inconclusive (reviewed in [67]). In the present study, a signifi- cant association between supplement use and potential drug-drug interactions was found and this remained significant after adjusting for age, gender, ethnicity and multiple comorbidities. Thus, the high use of supplements among elderly people is a major concern, particularly given that a majority of patients in our setting have previously reported not to disclose information on their supplement intake to their healthcare practitioners [64, 65]. Association between supplement use and quality of life scores has not been consistent in the literature. Some studies reported a positive association [68–70] while others reported a PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 11 / 18 Polypharmacy among community-dwelling older adults often omitted in studies which assessed polypharmacy. The sample size was large and represen- tative of the urban community setting in Malaysia. Conclusions A number of significant health related issues specifically, PIMS, PDDIs and increased health- care utilisation were found to be associated with polypharmacy among older urban commu- nity dwelling individuals who were taking chronic medications. Dietary supplement use was also high among older individuals and was significantly associated with PDDIs. Individuals diagnosed with cardiovascular, endocrine and gastrointestinal disorders had a high propensity to receive multiple medications which contributed significantly to polypharmacy. The present study adds to the growing evidence that a significant proportion of older adults are exposed to multiple medication related issues. More stringent guidelines with regards to the appropriate- ness and therapeutic need of dietary supplements among older adults is also crucial, given its contribution to polypharmacy in the present study. Healthcare providers should be vigilant of such usage to prevent any interactions with prescribed medications. Pharmacists, especially those in the community setting should provide targeted medication reviews for older individu- als despite the lack of a national programme to reduce the potential morbidity associated with polypharmacy in this age group. S2 File. Supplementary data 3: Relevant sections of the questionnaire used for the study (English and Malay versions). (PDF) (English and Malay versions). (PDF) S3 File. Supplementary data 4: Minimal dataset for the study. (XLS) S1 Table. Supplementary Table 1: Prevalence of medication classes used according to the Anatomical Therapeutic Chemical (ATC) Classification System (ATC first level) among 1256 urban-community dwelling older adults. (PDF) S2 Table. Supplementary Table 2. Details of the products taken by the 715 dietary supple- ments users. (PDF) Supporting information S1 File. Supplementary data 1: Assumptions made during assessments of potential inap- propriate medications (PIMs) use according to the American Geriatrics Society 2015 Updated Beers Criteria. (PDF) Updated Beers Criteria. (PDF) S2 File. Supplementary data 3: Relevant sections of the questionnaire used for the study (English and Malay versions). (PDF) S3 File. Supplementary data 4: Minimal dataset for the study. (XLS) S1 Table. Supplementary Table 1: Prevalence of medication classes used according to the Anatomical Therapeutic Chemical (ATC) Classification System (ATC first level) among 1256 urban-community dwelling older adults. (PDF) S2 Table. Supplementary Table 2. Details of the products taken by the 715 dietary supple- ments users. (PDF) Acknowledgments We would like to acknowledge the contribution of the staff and participants of the MELoR study. This work was supported by the University of Malaya (UM) High Impact Research (HIR) Grant from the Department of Higher Education, Ministry of Education, Malaysia [UM.C/625/1/HIR/MOHE/ASH/02] and the University Malaya Research Grant from UM [UMRG RP029-14HTM]. Polypharmacy was not associated with falls, physical function (measured by ADL and IADL) nor quality of life scores in this study though these associations were significant in the univari- ate model and a model which did not include comorbidities as a covariate. This implies that the presence of multiple comorbidities has a stronger modulating effect on these outcomes. Additionally, recent studies including those in the present study setting found that the con- sumption of falls-risk increasing drugs, rather than polypharmacy per say were more impor- tant in predicting the risk of falls in the elderly population [18, 80]. There were a number of limitations in the present study. Firstly, participants’ adherence to the medications in their possession was not assessed and this could have affected the other vari- ables. Secondly, there were some intrinsic limitations to the use of the 2015 Beers criteria to assess PIMS as many of the medications listed in Beers were not available in the present study setting. The Beers criteria was used instead of STOPP/START, the other commonly used tool to measure PIMS in the elderly population. This was because the participants’ medical notes were not available and hence, assessing the appropriateness of medications prescribed as required by the latter tool could not be conducted. The present study could not exclude the possibility that safer alternatives to the inappropriate medications listed in Beers had not already been tried in the past or that it was the participants’ choice to continue using these medications despite the inherent risks. The strengths of the present study include the study approach which involved home visits that allowed a realistic estimate of the number of medications taken by the partici- pants. In addition, the use of dietary supplements could be documented in the cohort as this is PLOS ONE | DOI:10.1371/journal.pone.0173466 March 8, 2017 12 / 18 References 1. United Nations Department of Economic and Social Affairs. World Population Aging 2015; 2015. Report No.: ST/ESA/SER.A/390. 2. Tey NP, Siraj SB, Kamaruzzaman SB, Chin AV, Tan MP, Sinnappan GS, et al. Aging in Multi-ethnic Malaysia. The Gerontologist. 2015 Nov 9. 3. Marengoni A, Angleman S, Melis R, Mangialasche F, Karp A, Garmen A, et al. Aging with multimorbid- ity: a systematic review of the literature. Ageing Res Rev. 2011 Sep; 10(4):430–9. doi: 10.1016/j.arr. 2011.03.003 PMID: 21402176 4. Boyd CM, Darer J, Boult C, Fried LP, Boult L, Wu AW. 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Mitochondrial One-Carbon Flux has a Growth- Independent Role in Promoting Breast Cancer Metastasis Nicole Kiweler  Luxembourg Institute of Health Catherine Delbrouck  Luxembourg Institute of Health Laura Neises  Luxembourg Institute of Health Vitaly Pozdeev  University of Luxembourg Leticia Soriano-Baguet  Luxembourg Institute of Health https://orcid.org/0000-0002-4822-624X Feng Xiang  Luxembourg Institute of Health Mohaned Benzarti  Luxembourg Institute of Health Lara Haase  University of Luxembourg Maryse Schmoetten  University of Luxembourg Christian Jaeger  University of Luxembourg Muhammad Noman  Luxembourg Institute of Health Alexei Vazquez  German Aerospace Center (DLR) https://orcid.org/0000-0003-2764-3244 Bassam Janji Mitochondrial One-Carbon Flux has a G Independent Role in Promoting Breast Metastasis Nicole Kiweler  Luxembourg Institute of Health Catherine Delbrouck  Luxembourg Institute of Health Laura Neises  Luxembourg Institute of Health Vitaly Pozdeev  University of Luxembourg Leticia Soriano-Baguet  Luxembourg Institute of Health https://orcid.org/0000-0002-4822-624X Feng Xiang  Luxembourg Institute of Health Mohaned Benzarti  Luxembourg Institute of Health Lara Haase  University of Luxembourg Maryse Schmoetten  University of Luxembourg Christian Jaeger  University of Luxembourg Muhammad Noman  Luxembourg Institute of Health Alexei Vazquez  German Aerospace Center (DLR) https://orcid.org/0000-0003-2764-3244 Bassam Janji  Luxembourg Institute of Health Gunnar Dittmar  Luxembourg Institute of Health https://orcid.org/0000-0003-3647-8623 Dirk Brenner  Luxembourg Institute of Health https://orcid.org/0000-0001-8979-1045 Elisabeth Letellier Nicole Kiweler Laura Neises  Luxembourg Institute of Health Vitaly Pozdeev  University of Luxembourg Vitaly Pozdeev  University of Luxembourg University of Luxembourg Johannes Meiser  (  Johannes.Meiser@lih.lu ) Luxembourg Institute of Health Article Keywords: One-Carbon Metabolism, Formate Over§ow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate, Methotrexate Posted Date: July 12th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-639444/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nature Communications on May 16th, University of Luxembourg Johannes Meiser  (  Johannes.Meiser@lih.lu ) Luxembourg Institute of Health Article Keywords: One-Carbon Metabolism, Formate Over§ow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate, Methotrexate Posted Date: July 12th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-639444/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nature Communications on May 16th, 2022. See the published version at https://doi.org/10.1038/s41467-022-30363-y. Mitochondrial One-Carbon Flux has a Growth-Independent Role in Promoting Breast Cancer 1 Metastasis 2 Nicole Kiweler1, Catherine Delbrouck1,2, Laura Neises1, Vitaly I. Pozdeev3, Leticia Soriano-Baguet2,4,5, Feng 3 Xian6, Mohaned Benzarti1,2, Lara Haase1,2,5, Maryse Schmoetten3, Christian Jaeger5, Muhammad Zaeem 4 Noman1, Alexei Vazquez7, Bassam Janji1, Gunnar Dittmar2,6, Dirk Brenner4,5,8, Elisabeth Letellier3, Johannes 5 Meiser1* 6 7 1Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg 8 2Faculty of Science, Technology and Medicine, University of Luxembourg, 2 avenue de Université, Esch-sur-Alzette, 9 Luxembourg 10 3Faculty of Science, Technology and Medicine, Department of Life Sciences and Medicine, Molecular Disease 11 Mechanisms Group, University of Luxembourg, Esch-sur-Alzette, Luxembourg 12 4Experimental & Molecular Immunology, Department of Infection and Immunity, Luxembourg Institute of Health, 13 Luxembourg, Luxembourg 14 5Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg 15 6Proteomics of Cellular Signaling, Quantitative Biology Unit, Luxembourg Institute of Health, Luxembourg, Luxembourg 16 7Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom 17 8Odense Research Center for Anaphylaxis, Department of Dermatology and Allergy Center, Odense University Hospital, 18 University of Southern Denmark, Odense, Denmark 19 20 *Correspondence: 21 Email: johannes.meiser@lih.lu 22 Twitter: @JohannesMeiser 23 Mitochondrial One-Carbon Flux has a Growth-Independent Role in Promoting Breast Cancer 1 Metastasis 2 24 25 Graphical Abstract 26 27 28 One-Carbon Metabolism, Formate Overflow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate, 48 Methotrexate 49 Abstract 29 In summary, we identified mitochondrial serine catabolism as a targetable, growth- 44 independent metabolic vulnerability to limit metastatic progression. 45 46 Keywords 47 One-Carbon Metabolism, Formate Overflow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate, 48 Methotrexate 49 50 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients 30 with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer 31 cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify 32 metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients 30 with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer 31 cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify 32 metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33 Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did 34 not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35 Prolonged treatment even selected for more motile cancer subpopulations. We found that cytosolic inhibition 36 of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly 37 active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate 38 overflow are increased, suggesting that mitochondria provide a protective environment that allows serine 39 catabolism to support cellular motility during nucleotide synthesis inhibition. 40 Enhanced motility of growth-arrested cells was reduced by inhibition of PHGDH-dependent de novo serine 41 synthesis and genetic silencing of mitochondrial one-carbon cycle. In vivo targeting of mitochondrial one- 42 carbon cycle and formate overflow strongly and significantly reduced lung metastasis formation in an orthotopic 43 breast cancer model. In summary, we identified mitochondrial serine catabolism as a targetable, growth- 44 independent metabolic vulnerability to limit metastatic progression. 45 46 Keywords 47 One-Carbon Metabolism, Formate Overflow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate, 48 Methotrexate 49 50 Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did 34 not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35 Prolonged treatment even selected for more motile cancer subpopulations. Abstract 29 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients 30 with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer 31 cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify 32 metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33 Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did 34 not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35 Prolonged treatment even selected for more motile cancer subpopulations. We found that cytosolic inhibition 36 of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly 37 active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate 38 overflow are increased, suggesting that mitochondria provide a protective environment that allows serine 39 catabolism to support cellular motility during nucleotide synthesis inhibition. 40 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients 30 with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer 31 cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify 32 metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33 Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did 34 not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35 Prolonged treatment even selected for more motile cancer subpopulations. We found that cytosolic inhibition 36 of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly 37 active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate 38 overflow are increased, suggesting that mitochondria provide a protective environment that allows serine 39 catabolism to support cellular motility during nucleotide synthesis inhibition. 40 Enhanced motility of growth-arrested cells was reduced by inhibition of PHGDH-dependent de novo serine 41 synthesis and genetic silencing of mitochondrial one-carbon cycle. In vivo targeting of mitochondrial one- 42 carbon cycle and formate overflow strongly and significantly reduced lung metastasis formation in an orthotopic 43 breast cancer model. Introduction 51 84 However, these recent works focused mainly on the relevance of serine de novo synthesis via 85 phosphoglycerate dehydrogenase (PHGDH) itself instead of addressing the subsequent catabolism of serine 86 via SOC for metastatic progression. Mechanistic studies on the role of SOC in context of metastasis were 87 mainly focused on the proliferative capacities essential for metastatic outgrowth [11, 12, 14] rather than 88 analyzing metabolic requirements that support the growth-independent metastatic processes of dissemination. 89 In the absence of such growth demands and under certain metabolic stress conditions, we have previously 90 However, changes in cancer cell metabolism during stages of reduced biomass production, for example upon 61 chemotherapy or when cancer cells transition along the metastatic cascade, are far less described [3]. 62 Targeting metabolic cues to not only reduce cancer growth but also to metabolically interfere with invasive and 63 migratory properties of cancer cells represents a desirable therapeutic approach to ultimately prevent 64 metastatic progression of the initial disease. We have previously shown that a reduction of biomass demand 65 can result in increased formate release from cancer cells to their surrounding microenvironment [4]. The 66 mechanistic basis for this formate overflow phenomenon particularly in growth inhibited cancer cells, is the 67 rate of serine catabolism through serine one-carbon cycle (SOC). 68 Within this metabolic pathway, which spans between cytosol and mitochondrion [5], extracellular and de novo 69 synthesized serine is catabolized to support all three essential pillars of metabolism: biomass production, redox 70 equivalent and bioenergy generation [6]. Especially metastasizing cancer cells need to selectively and 71 dynamically adopt their cellular metabolism to fulfil these three major metabolic constraints along every step 72 of the metastatic cascade [3, 7]. Previous studies have shown that metabolic rewiring during the growth- 73 independent process of tumor cell escape and dissemination is dominated by bioenergetic and redox demands 74 rather than biosynthetic needs [3, 7, 8]. Importantly, serine plasticity allows to readjust the metabolic outputs 75 of serine catabolism through the various routes of SOC towards either of the three metabolic hallmarks [6]. 76 For example, upon high growth rates, serine, glycine and formate production from SOC is required to support 77 the synthesis of anabolic building blocks such as proteins, lipids, heme, glutathione, and nucleotides. Abstract 29 We found that cytosolic inhibition 36 of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly 37 active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate 38 overflow are increased, suggesting that mitochondria provide a protective environment that allows serine 39 catabolism to support cellular motility during nucleotide synthesis inhibition. 40 Enhanced motility of growth-arrested cells was reduced by inhibition of PHGDH-dependent de novo serine 41 synthesis and genetic silencing of mitochondrial one-carbon cycle. In vivo targeting of mitochondrial one- 42 carbon cycle and formate overflow strongly and significantly reduced lung metastasis formation in an orthotopic 43 breast cancer model. In summary, we identified mitochondrial serine catabolism as a targetable, growth- 44 independent metabolic vulnerability to limit metastatic progression. 45 50 Introduction 51 Many primary tumors can be targeted by specific treatment schemes. Thus survival rates of patients harboring 52 primary tumors steadily increases. However, in case of tumor relapse and metastatic disease, effective 53 therapies are mostly lacking with the consequence that secondary tumors account for the majority of cancer 54 deaths [1]. 55 Classical chemotherapeutic approaches to counteract cancer growth aim to target biomass production by 56 interfering with the synthesis of proteins, lipids, and nucleotides and thereby directly interfere with central 57 pathways of cancer cell metabolism. Consequently, it is well studied that beneficial metabolic reprogramming 58 is fundamental for cancer cells to maximize the synthesis of essential lipids, proteins and nucleotides in order 59 to drive the excessive growth rates observed within the primary tumor and secondary metastasis [2, 3]. 60 However, changes in cancer cell metabolism during stages of reduced biomass production, for example upon 61 chemotherapy or when cancer cells transition along the metastatic cascade, are far less described [3]. 62 Targeting metabolic cues to not only reduce cancer growth but also to metabolically interfere with invasive and 63 migratory properties of cancer cells represents a desirable therapeutic approach to ultimately prevent 64 metastatic progression of the initial disease. We have previously shown that a reduction of biomass demand 65 can result in increased formate release from cancer cells to their surrounding microenvironment [4]. The 66 mechanistic basis for this formate overflow phenomenon particularly in growth inhibited cancer cells, is the 67 rate of serine catabolism through serine one-carbon cycle (SOC). 68 Many primary tumors can be targeted by specific treatment schemes. Thus survival rates of patients harboring 52 primary tumors steadily increases. However, in case of tumor relapse and metastatic disease, effective 53 therapies are mostly lacking with the consequence that secondary tumors account for the majority of cancer 54 deaths [1]. 55 Classical chemotherapeutic approaches to counteract cancer growth aim to target biomass production by 56 interfering with the synthesis of proteins, lipids, and nucleotides and thereby directly interfere with central 57 pathways of cancer cell metabolism. Consequently, it is well studied that beneficial metabolic reprogramming 58 is fundamental for cancer cells to maximize the synthesis of essential lipids, proteins and nucleotides in order 59 to drive the excessive growth rates observed within the primary tumor and secondary metastasis [2, 3]. Introduction 51 60 However, changes in cancer cell metabolism during stages of reduced biomass production, for example upon 61 chemotherapy or when cancer cells transition along the metastatic cascade, are far less described [3]. 62 Targeting metabolic cues to not only reduce cancer growth but also to metabolically interfere with invasive and 63 migratory properties of cancer cells represents a desirable therapeutic approach to ultimately prevent 64 metastatic progression of the initial disease. We have previously shown that a reduction of biomass demand 65 can result in increased formate release from cancer cells to their surrounding microenvironment [4]. The 66 mechanistic basis for this formate overflow phenomenon particularly in growth inhibited cancer cells, is the 67 rate of serine catabolism through serine one-carbon cycle (SOC). 68 Within this metabolic pathway, which spans between cytosol and mitochondrion [5], extracellular and de novo 69 synthesized serine is catabolized to support all three essential pillars of metabolism: biomass production, redox 70 equivalent and bioenergy generation [6]. Especially metastasizing cancer cells need to selectively and 71 dynamically adopt their cellular metabolism to fulfil these three major metabolic constraints along every step 72 of the metastatic cascade [3, 7]. Previous studies have shown that metabolic rewiring during the growth- 73 independent process of tumor cell escape and dissemination is dominated by bioenergetic and redox demands 74 rather than biosynthetic needs [3, 7, 8]. Importantly, serine plasticity allows to readjust the metabolic outputs 75 of serine catabolism through the various routes of SOC towards either of the three metabolic hallmarks [6]. 76 For example, upon high growth rates, serine, glycine and formate production from SOC is required to support 77 the synthesis of anabolic building blocks such as proteins, lipids, heme, glutathione, and nucleotides. In 78 contrast, upon energy limitation, most of serine derived carbon is excreted in the form of formate or CO2 and 79 SOC is maximized towards energy production in form of NADH and ATP [4]. Alternatively, upon oxidative 80 stress, the demand for glycine to support GSH synthesis is prioritized [9]. We have previously shown that the 81 rate of serine catabolism to formate is significantly increased in adenomas of the small intestine and breast 82 tumors compared to normal tissue and, that increased formate levels can increase the invasiveness of cancer 83 cells [10]. In addition, other labs also reported a role of serine metabolism in the context of metastasis [11-13]. Introduction 51 Given this pro-migratory effect of nucleotide synthesis inhibition 129 in growth-arrested cancer cells, we generated MTX-resistant MDA-MB468 cells by long-term cultivation in 130 demonstrated that cells increase their rate of formate overflow via SOC [4, 10], which further manifests that 91 regulation of SOC is a key metabolic switch especially in, but not limited to, the growth-independent stages of 92 metastatic outgrowth. 93 Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells 106 To assess the relative importance of different metabolic pathways for cancer cell migration, we employed a 107 panel of metabolic perturbations targeting glycolysis, mitochondrial oxidative phosphorylation (OxPhos) as well 108 as lipid, protein, and nucleotide synthesis (Figure 1A). All metabolic interventions effectively inhibited the 109 growth and cell cycle progression of MDA-MB468 breast cancer cells and the chosen drug concentrations had 110 none to mild toxic effects after 48 h treatment (Figure 1B-C, S1A). Inhibition of glycolysis, OxPhos, lipid, and 111 protein synthesis significantly reduced migration of MDA-MB468 cells (Figure 1D). Surprisingly and in contrast 112 to all other growth-inhibiting conditions, inhibition of nucleotide synthesis with various drugs did not diminish 113 migration of MDA-MB468 cells (Figure 1E). Calculation of the area under curve (AUC) allowed us to 114 quantitatively compare wound closure upon the diverse metabolic perturbations over time (Figure 1D, E) and 115 confirmed that wound closure was significantly reduced upon rotenone (Rot), galactose (Gal), simvastatin 116 (SIM) and sirolimus (rapamycin, SIR) treatment. However, treatment with the antifolates methotrexate (MTX) 117 and pemetrexed (PEM), as well as with hydroxyurea (HU) and clofarabine (CLO) did not affect or did even 118 significantly increase wound closure in scratch assays in comparison to fully proliferative, untreated control 119 cells (Figure 1D, E). This divergent effect of nucleotide synthesis inhibition from all other metabolic 120 perturbations was further realized, when directly correlating our data on MDA-MB468 cell migration with cell 121 growth in response to treatment (Figure 1F, G). While cell migration correlates with reduced cell growth for all 122 other metabolic perturbations, nucleotide synthesis inhibition does not reduce cell migration in correlation to 123 growth inhibition (Figure 1F, G). This finding was verified in LN229 glioblastoma and 4T1 breast cancer cells 124 (Figure S1B-D). To further confirm this finding, we assessed migration of MDA-MB468 cells in a trans-well 125 assay. Introduction 51 In 78 contrast, upon energy limitation, most of serine derived carbon is excreted in the form of formate or CO2 and 79 SOC is maximized towards energy production in form of NADH and ATP [4]. Alternatively, upon oxidative 80 stress, the demand for glycine to support GSH synthesis is prioritized [9]. We have previously shown that the 81 rate of serine catabolism to formate is significantly increased in adenomas of the small intestine and breast 82 tumors compared to normal tissue and, that increased formate levels can increase the invasiveness of cancer 83 cells [10]. In addition, other labs also reported a role of serine metabolism in the context of metastasis [11-13]. 84 However, these recent works focused mainly on the relevance of serine de novo synthesis via 85 phosphoglycerate dehydrogenase (PHGDH) itself instead of addressing the subsequent catabolism of serine 86 via SOC for metastatic progression. Mechanistic studies on the role of SOC in context of metastasis were 87 mainly focused on the proliferative capacities essential for metastatic outgrowth [11, 12, 14] rather than 88 analyzing metabolic requirements that support the growth-independent metastatic processes of dissemination. 89 In the absence of such growth demands and under certain metabolic stress conditions, we have previously 90 demonstrated that cells increase their rate of formate overflow via SOC [4, 10], which further manifests that 91 regulation of SOC is a key metabolic switch especially in, but not limited to, the growth-independent stages of 92 metastatic outgrowth. 93 In the present study, our goal was to identify metabolic processes that are specifically important to support 94 cancer cell motility in the growth independent process of metastatic dissemination. We have discovered, that 95 antifolates, while having strong growth arresting properties do not decrease the cellular motility potential. Using 96 MTX as a tool compound to separate metabolic processes of growth from motility processes, we have identified 97 SOC as a central metabolic pathway that supports cellular motility potential. Specifically, the mitochondrial 98 SOC is required to support the observed motility, while the cytosolic part, which is essential for proliferation, is 99 dispensable for the cellular motility potential. Finally, we provide evidence that formate overflow via 100 mitochondrial SOC is a targetable growth-independent vulnerability to limit breast cancer metastasis. This 101 identified metabolic vulnerability might have clinical implications for metastatic progression in context of 102 growth-inhibiting chemotherapies. Introduction 51 103 104 Results 105 Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells 106 To assess the relative importance of different metabolic pathways for cancer cell migration, we employed a 107 panel of metabolic perturbations targeting glycolysis, mitochondrial oxidative phosphorylation (OxPhos) as well 108 as lipid, protein, and nucleotide synthesis (Figure 1A). All metabolic interventions effectively inhibited the 109 growth and cell cycle progression of MDA-MB468 breast cancer cells and the chosen drug concentrations had 110 none to mild toxic effects after 48 h treatment (Figure 1B-C, S1A). Inhibition of glycolysis, OxPhos, lipid, and 111 protein synthesis significantly reduced migration of MDA-MB468 cells (Figure 1D). Surprisingly and in contrast 112 to all other growth-inhibiting conditions, inhibition of nucleotide synthesis with various drugs did not diminish 113 migration of MDA-MB468 cells (Figure 1E). Calculation of the area under curve (AUC) allowed us to 114 quantitatively compare wound closure upon the diverse metabolic perturbations over time (Figure 1D, E) and 115 confirmed that wound closure was significantly reduced upon rotenone (Rot), galactose (Gal), simvastatin 116 (SIM) and sirolimus (rapamycin, SIR) treatment. However, treatment with the antifolates methotrexate (MTX) 117 and pemetrexed (PEM), as well as with hydroxyurea (HU) and clofarabine (CLO) did not affect or did even 118 significantly increase wound closure in scratch assays in comparison to fully proliferative, untreated control 119 cells (Figure 1D, E). This divergent effect of nucleotide synthesis inhibition from all other metabolic 120 perturbations was further realized, when directly correlating our data on MDA-MB468 cell migration with cell 121 growth in response to treatment (Figure 1F, G). While cell migration correlates with reduced cell growth for all 122 other metabolic perturbations, nucleotide synthesis inhibition does not reduce cell migration in correlation to 123 growth inhibition (Figure 1F, G). This finding was verified in LN229 glioblastoma and 4T1 breast cancer cells 124 (Figure S1B-D). To further confirm this finding, we assessed migration of MDA-MB468 cells in a trans-well 125 assay. Similar to our previous findings, there was no observable reduction of cell migration in response to MTX 126 or PEM (Figure 1H). Furthermore, using ECM-collagen coating in trans-well assay, we found that nucleotide 127 synthesis inhibition had no impact on cancer cell invasion (Figure 1H). These findings were also confirmed 128 using LN229 and 4T1 cells (Figure S1E, F). MTX treatment triggers a ROS-driven EMT phenotype MTX treatment triggers a ROS driven EMT phenotype 142 One of the most established and extensively described nucleotide synthesis-inhibiting clinical drugs is the 143 dihydrofolate reductase inhibitor MTX. MTX impedes the regeneration of tetrahydrofolate (THF), which is a 144 central enzymatic cofactor during purine and pyrimidine synthesis [15]. As MTX induced the strongest growth 145 repression without impacting cell death (Figure 1B, C) and had no effect on cell migration (Figure 1E), we 146 selected it as a tool compound to identify metabolic processes that are essential to support cell migration and 147 metastasis independent of cell proliferation. We tested multiple MTX concentrations and chose a concentration 148 of 50 nM, which is not only significantly growth- and cell cycle-arresting in MDA-MB468 cells (Figure 1B, S1A, 149 S2A), but has also been shown to be within the therapeutic window [16-19]. We observed that 48 h MTX 150 treatment of MDA-MB468 cells resulted in altered cellular morphology with individual cells visibly increasing in 151 cell size (Figure 2A). Following treatment, single cells exhibited an outstretched cell shape with protruding 152 edges, indicative of a remodeling of the cellular cytoskeleton (Figure 2A). MTX-resistant MDA-MB468 cells 153 showed comparable morphologic alterations (Figure S2B). We found that the observed cell cycle and 154 proliferation arrest upon MTX treatment was reversible, as MDA-MB468 cells promptly regained growth 155 properties (Figure S2C) and re-established proliferation by resuming cell cycle progression (Figure S2D) upon 156 MTX removal. Using [U-13C]serine tracer and metabolic flux analysis, we confirmed that 50 nM MTX was 157 sufficient to significantly diminish the flux of labelled serine carbon units via methylenetetrahydrofolate to ATP 158 (Figure 2B). Of note, nucleotide synthesis was partially restored in MTX-resistant MDA-MB468 cells (Figure 159 S2E). Using stable isotope labeling by amino acids in cell culture (SILAC) and subsequent quantitative 160 proteomics analysis, we confirmed the efficiency of the selected MTX dose by determining expression changes 161 of mechanistic targets of MTX such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and 162 thymidine kinase (TK) which were indeed regulated by treatment as expected (Figure 2C, S2F). Interestingly, 163 we also found superoxide dismutase (SOD) to be one of the most significantly upregulated enzymes in 164 response to treatment (Figure 2C, S2F), which corresponds to prior reports that depict MTX as a potent 165 inducer of oxidative stress [20-22]. Introduction 51 Similar to our previous findings, there was no observable reduction of cell migration in response to MTX 126 or PEM (Figure 1H). Furthermore, using ECM-collagen coating in trans-well assay, we found that nucleotide 127 synthesis inhibition had no impact on cancer cell invasion (Figure 1H). These findings were also confirmed 128 using LN229 and 4T1 cells (Figure S1E, F). Given this pro-migratory effect of nucleotide synthesis inhibition 129 in growth-arrested cancer cells, we generated MTX-resistant MDA-MB468 cells by long-term cultivation in 130 50 nM MTX for 2 months. The resulting MTX-resistant MDA-MB468 cells proliferate in the presence of 131 increasing concentrations of MTX and show enhanced migratory capacity when compared to the parental 132 MDA-MB468 cell line (Figure 1I, J). 133 In conclusion, the perturbation of any major anabolic route results in growth repression, its impact on cell 134 migration however highly depends on the targeted metabolic pathway, with nucleotide synthesis being an 135 ineffective target to abrogate cell motility. Considering that cell proliferation contributes to the experimental 136 outcome in scratch and trans-well assays, cell migration must in fact be increased upon nucleotide synthesis 137 inhibition to compensate for the reduced growth rate. Different means of metabolic growth repression thus 138 differentially impact cell motility and do not necessarily result in decreased cell migration. Furthermore, 139 prolonged nucleotide synthesis inhibition in drug-resistant cells acts as a selection pressure for more motile 140 cancer subpopulations. 141 50 nM MTX for 2 months. The resulting MTX-resistant MDA-MB468 cells proliferate in the presence of 131 increasing concentrations of MTX and show enhanced migratory capacity when compared to the parental 132 MDA-MB468 cell line (Figure 1I, J). 133 In conclusion, the perturbation of any major anabolic route results in growth repression, its impact on cell 134 migration however highly depends on the targeted metabolic pathway, with nucleotide synthesis being an 135 ineffective target to abrogate cell motility. Considering that cell proliferation contributes to the experimental 136 outcome in scratch and trans-well assays, cell migration must in fact be increased upon nucleotide synthesis 137 inhibition to compensate for the reduced growth rate. Different means of metabolic growth repression thus 138 differentially impact cell motility and do not necessarily result in decreased cell migration. Furthermore, 139 prolonged nucleotide synthesis inhibition in drug-resistant cells acts as a selection pressure for more motile 140 cancer subpopulations. 141 MTX treatment triggers a ROS-driven EMT phenotype We validated these prior findings by measuring a moderate but significant 166 increase of mitochondrial and cytosolic reactive oxidant species (ROS) in MDA-MB468 cells in response to 167 MTX (Figure 2D, E). Moreover, the mesenchymal marker protein vimentin (VIM) and transforming growth 168 factor-beta2 (TGFB2) were found to be highly upregulated in the global proteomics approach following MTX 169 treatment (Figure 2C, S2F). This finding corresponds to earlier reports, which highlight the potential of 170 oxidative stress as a driver of cancer cell migration and the transformation of cancer cells via epithelial- 171 mesenchymal transition (EMT) [6, 23-28]. We thus analyzed expression levels of the major regulators of EMT 172 - the transcription factors ZEB1, ZEB2, SNAIL, and SLUG - in MDA-MB468 cells and found that ZEB1 and 173 ZEB2 expression as well as VIM expression was upregulated in a time-dependent manner in response to MTX 174 treatment (Figure 2F). Similar upregulation of ZEB1, ZEB2, and VIM expression was observed in the MTX- 175 resistant MDA-MB468 cell line compared to the parental MDA-MB468 cell line (Figure 2G). Similarly to mRNA 176 expression, there was an upregulation of VIM at the protein level following MTX-treatment as well as in the 177 MTX-resistant MDA-MB468 cell line (Figure 2H, I). MTX-treatment also induced the expression of the 178 collagenase MMP9 (Figure 2J), an enzyme which is involved in the degradation of the extracellular matrix 179 during cancer cell migration [29]. To investigate the relevance of ROS as a potential trigger for the observed 180 EMT phenotype in response to MTX treatment, we treated cell with the antioxidant N-acetylcysteine (NAC) 181 which reduced ROS levels in MTX-treated MDA-MB468 cells (Figure 2K). While NAC alone already reduced 182 the cell migration, combined treatment with NAC and MTX resulted in a significant reduction of MTX-induced 183 cell migration (Figure 2L). Correspondingly, NAC treatment also prevented the EMT phenotype induction in 184 MTX-treated MDA-MB468 cells with the exception of ZEB1 (Figure 2M). These findings indicate that ROS 185 induction upon MTX promotes EMT and sustains migration. In conclusion, the moderate increase in cellular 186 ROS levels after MTX acts as a pro-migratory stimulus that subsequently results in the establishment and 187 long-term selection of phenotypically altered cancer cell subpopulations. MTX treatment triggers a ROS-driven EMT phenotype However, research in the past has 188 raised awareness, that antioxidant treatment is not a promising cancer treatment strategy with mixed, and not 189 always beneficial, outcomes in clinical trials [30, 31]. Therefore, our aim was to further investigate the metabolic 190 mechanisms that support enhanced cell migration in the presence of the nucleotide synthesis and growth- 191 inhibiting tool compound MTX. 192 MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis Having established that growth arrested MDA-MB468 cells maintain full migratory and invasive properties 194 under MTX treatment, we wanted to define metabolic mechanisms that maintain this pro-invasive EMT 195 phenotype. To this end, we focused on central carbon metabolism using [U-13C]glutamine and [U-13C]glucose 196 to monitor glycolytic activity and glucose- or glutamine-derived carbon oxidation through the TCA cycle (Figure 197 3A, D). Absolute uptake rates of glutamine and glutamate were sustained in response to MTX treatment 198 (Figure 3B), while the relative flux of [U-13C]glutamine throughout the TCA-cycle was significantly increased 199 in response to MTX (Figure 3C). Absolute quantification of uptake and release rates of glucose and lactate 200 also revealed sustained high glycolytic rates upon MTX treatment (Figure 3E). This came as a surprise, as 201 lactate release rates were previously shown to generally correlate with cell growth rates [32]. Here however, 202 MTX-treated and growth-arrested cells maintained their glycolytic rate and associated lactate release rates at 203 constant high levels. This indicates a constant generation of glycolysis-derived ATP even in the presence of 204 reduced energetic demand for anabolic reactions under growth-arresting conditions. Consequently, [U- 205 13C]glucose distribution within the TCA-cycle was comparable in untreated and MTX-treated MDA-MB468 cells 206 (Figure 3F) as well as in 4T1 and LN229 cells (Figure S3A, B). It has previously been shown that MTX inhibits 207 OCR in HCT116 cells [33]. We could replicate this finding and observed a ~ 30 % reduction in OCR in HCT116 208 cells upon MTX (Figure S3C). However, OCR in MDA-MB468, LN229, and 4T1 cells was sustained at control 209 level in response to MTX (Figure 3G), which indicates cell line specific effects of MTX on OCR. Overall, these 210 findings indicate that, despite decreased growth rates, central carbon metabolism and mitochondrial 211 respiration are sustained in response to MTX treatment, with an additional increase in oxidative glutamine 212 metabolism. Furthermore, we observed that MTX treatment significantly increased the relative flux of glucose 213 to serine via the serine de novo synthesis pathway despite decreased anabolic demands for nucleotide 214 synthesis (Figure 3H, S3D). The serine de novo synthesis pathway is catalyzed by three enzymes - PHGDH, 215 PSPH, and PSAT1, whose protein levels remained unchanged in response to MTX treatment in MDA-MB468, 216 MDA-MB231, 4T1 and LN229 cells (Figure S3E-H). In agreement with Diehl et al. Mitochondria protect SOC-dependent serine catabolism in the presence of MTX Mitochondria protect SOC-dependent serine catabolism in the presence of MTX 226 Given our finding of increased [U-13C]glucose-derived formate (Figure 3J), we hypothesized that the influence 227 of MTX-mediated DHFR inhibition on mitochondrial SOC is, in contrast to cytosolic SOC, limited (Figure 4A). 228 To test this hypothesis, we employed [U-13C]serine tracing to further characterize serine flux through SOC. We 229 observed that despite inducing growth-arrest, MTX did not alter the rates of serine consumption and glycine 230 release in MDA-MB468 or LN229 cells (Figure 4B, S4A). This is a remarkable observation for two reasons: 231 (i) since the major route of serine catabolism is via SOC [4], one would have expected that MTX-mediated 232 inhibition of DHFR would also cause a decrease in cellular serine consumption; (ii) since cell dry mass 233 composition is constituted by around 60 % of proteins [35], growth arresting conditions upon MTX should result 234 in decreased consumption rates of proteinogenic amino acids. In fact, a general trend for decreased 235 consumption of other essential amino acids was observed (Figure S4B) in response to MTX. Most strikingly, 236 the total release rate of formate as well as of labeled M+1 formate derived from [U13C]serine into the culture 237 medium was significantly increased (Figure 4C) in MDA-MB468 cells and sustained in LN229 cells (Figure 238 S4C). We could confirm this observation of sustained serine and glycine consumption and release (CORE) 239 rates and increased formate overflow via SOC in the presence of MTX in Plasmax medium [36], which is a 240 culture medium closer to the physiological state of human physiology (Figure S4D). SOC follows a cycle in 241 which serine can be resynthesized in the cytoplasm and mitochondrion from glycine via serine 242 hydroxymethyltransferase 1 and 2 (SHMT1, SHMT2) (Figure 4A). As the mitochondrial SHMT2 reaction is 243 highly reversible, the serine labeling pattern from [U13C]serine is composed of a mix of fully labeled M+3 244 isotopologues as well as M+1 and M+2 isotopologues, which represent the different recombination events with 245 labeled and unlabeled glycine [4, 5, 37]. Consequently, M+1 and M+2 serine isotopologues are expected to 246 be absent upon complete SOC inhibition. Intriguingly, DHFR inhibition with MTX neither completely abrogated 247 serine M+1 and M+2 isotopologues upon [U-13C]serine nor did it affect the serine to glycine ratio in MDA- 248 MB468 or LN229 cells (Figure 4D, E, S4E, F). MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis 228 To test this hypothesis, we employed [U-13C]serine tracing to further characterize serine flux through SOC. We 229 observed that despite inducing growth-arrest, MTX did not alter the rates of serine consumption and glycine 230 release in MDA-MB468 or LN229 cells (Figure 4B, S4A). This is a remarkable observation for two reasons: 231 (i) since the major route of serine catabolism is via SOC [4], one would have expected that MTX-mediated 232 inhibition of DHFR would also cause a decrease in cellular serine consumption; (ii) since cell dry mass 233 composition is constituted by around 60 % of proteins [35], growth arresting conditions upon MTX should result 234 in decreased consumption rates of proteinogenic amino acids. In fact, a general trend for decreased 235 consumption of other essential amino acids was observed (Figure S4B) in response to MTX. Most strikingly, 236 the total release rate of formate as well as of labeled M+1 formate derived from [U13C]serine into the culture 237 medium was significantly increased (Figure 4C) in MDA-MB468 cells and sustained in LN229 cells (Figure 238 S4C). We could confirm this observation of sustained serine and glycine consumption and release (CORE) 239 rates and increased formate overflow via SOC in the presence of MTX in Plasmax medium [36], which is a 240 culture medium closer to the physiological state of human physiology (Figure S4D). SOC follows a cycle in 241 which serine can be resynthesized in the cytoplasm and mitochondrion from glycine via serine 242 hydroxymethyltransferase 1 and 2 (SHMT1, SHMT2) (Figure 4A). As the mitochondrial SHMT2 reaction is 243 highly reversible, the serine labeling pattern from [U13C]serine is composed of a mix of fully labeled M+3 244 isotopologues as well as M+1 and M+2 isotopologues, which represent the different recombination events with 245 labeled and unlabeled glycine [4, 5, 37]. Consequently, M+1 and M+2 serine isotopologues are expected to 246 be absent upon complete SOC inhibition. Intriguingly, DHFR inhibition with MTX neither completely abrogated 247 serine M+1 and M+2 isotopologues upon [U-13C]serine nor did it affect the serine to glycine ratio in MDA- 248 MB468 or LN229 cells (Figure 4D, E, S4E, F). This observation of diminished, but not abolished serine M+1 249 and M+2 isotopologues indicates a sole inhibition of cytosolic but not mitochondrial SOC under MTX treatment. 250 MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis [34] who showed that 217 changes in NAD+/NADH ratio can increase PHGDH activity and in consequence serine de novo synthesis 218 rates, we observed an increased NAD+/NADH ratio upon MTX (Figure 3I). Congruently, yet somewhat 219 surprisingly, MTX treatment did also increase the proportion of labeled, extracellular formate derived from [U- 220 13C]glucose through serine and the mitochondrial SOC (Figure 3J). This may indicate that the mitochondrial 221 part of the SOC is not as inhibited by MTX treatment as its cytosolic part. In summary, we demonstrate that, 222 despite decreased metabolic demand for biomass production, MTX-treated cells sustain high metabolic rates 223 comparable to fully proliferating cells. Additionally, treated cells exhibit increased rates of serine synthesis and 224 formate excretion. 225 findings indicate that, despite decreased growth rates, central carbon metabolism and mitochondrial 211 respiration are sustained in response to MTX treatment, with an additional increase in oxidative glutamine 212 metabolism. Furthermore, we observed that MTX treatment significantly increased the relative flux of glucose 213 to serine via the serine de novo synthesis pathway despite decreased anabolic demands for nucleotide 214 synthesis (Figure 3H, S3D). The serine de novo synthesis pathway is catalyzed by three enzymes - PHGDH, 215 PSPH, and PSAT1, whose protein levels remained unchanged in response to MTX treatment in MDA-MB468, 216 MDA-MB231, 4T1 and LN229 cells (Figure S3E-H). In agreement with Diehl et al. [34] who showed that 217 changes in NAD+/NADH ratio can increase PHGDH activity and in consequence serine de novo synthesis 218 rates, we observed an increased NAD+/NADH ratio upon MTX (Figure 3I). Congruently, yet somewhat 219 surprisingly, MTX treatment did also increase the proportion of labeled, extracellular formate derived from [U- 220 13C]glucose through serine and the mitochondrial SOC (Figure 3J). This may indicate that the mitochondrial 221 part of the SOC is not as inhibited by MTX treatment as its cytosolic part. In summary, we demonstrate that, 222 despite decreased metabolic demand for biomass production, MTX-treated cells sustain high metabolic rates 223 comparable to fully proliferating cells. Additionally, treated cells exhibit increased rates of serine synthesis and 224 formate excretion. 225 Mitochondria protect SOC-dependent serine catabolism in the presence of MTX 226 Given our finding of increased [U-13C]glucose-derived formate (Figure 3J), we hypothesized that the influence 227 of MTX-mediated DHFR inhibition on mitochondrial SOC is, in contrast to cytosolic SOC, limited (Figure 4A). argeting of serine de novo synthesis decreases MTX-dependent cancer cell migration Targeting of serine de novo synthesis decreases MTX-dependent cancer cell migration 264 Having confirmed increased serine synthesis from glucose and subsequent formate overflow through 265 mitochondrial SOC in response to MTX treatment, we sought to determine whether interfering with this 266 metabolic pathway is a means to counteract enhanced cell migration upon MTX. To identify a suitable inhibitor 267 of serine de novo synthesis, we tested multiple available allosteric and one competitive inhibitor of PHGDH. 268 We found that the competitive inhibitor BI-4916 (BI) had superior efficiency when compared to the allosteric 269 inhibitors WQ-2101 (WQ), NCT-502 (NCT), and CBR-5884 (CBR) as can be observed in the isotopic labelling 270 pattern of serine from [U-13C]glucose tracer (Figure 5A). Isotopic labelling of released formate from [U- 271 13C]glucose tracer was also profoundly diminished after BI treatment alone or in combination with MTX (Figure 272 5B). Additionally, routine screened for metabolic side effects revealed that treatment with the allosteric 273 inhibitors WQ, NCT, and CBR alone did result in a significant reduction of mitochondrial OCR (Figure 5C), 274 which was not observed with the competitive inhibitor BI (Figure 5C). WQ and NCT also negatively affected 275 TCA cycle activity as characterized by a reduction of M+2 isotopologue abundance of prominent TCA 276 metabolites following [U-13C]glucose tracing (Figure 5D). In contrast, BI had no adverse effect on TCA cycle 277 activity or proliferation rate and thus emerged as the preferred PHGDH inhibitor in all subsequent experiments 278 (Figure 5D, S5A-D). Combined treatment with the specific PHGDH inhibitor BI significantly reduced MTX- 279 mediated cell migration (Figure 5E), which further indicated that mitochondrial SOC provides a metabolic 280 foundation to support cancer cell motility upon cytosolic DHFR inhibition. This inhibitory effect on MTX- 281 mediated cell motility was further enhanced upon combined PHGDH inhibition and serine and glycine (S/G) 282 starvation (Figure 5F). While serine and glycine starvation alone resulted only in moderate inhibitory effects 283 on migration of MTX-treated cells, combined treatment with PHGDH inhibition fully blunted cell migration, 284 indicating that serine is not only essential to support biomass production but also to support the metabolic 285 program required for migration (Figure 5F). Mitochondria protect SOC-dependent serine catabolism in the presence of MTX Overall, these results indicate that mitochondria provide a protected cellular 261 environment that permits serine catabolism with formate overflow via SOC even in the presence of the 262 antifolate MTX. 263 Mitochondria protect SOC-dependent serine catabolism in the presence of MTX This observation of diminished, but not abolished serine M+1 249 and M+2 isotopologues indicates a sole inhibition of cytosolic but not mitochondrial SOC under MTX treatment. 250 To comparatively demonstrate how sole or dual inhibition of cytosolic and mitochondrial SOC affects serine 251 MID, we analyzed a panel of CRISPR generated knockouts in HAP1 cells in which either the cytosolic 252 (SHMT1), the mitochondrial (MFT, SHMT2), or both compartments (FPGS, MFT+SHMT1) of SOC were 253 abrogated [38]. In concordance with our previous findings and resulting conclusion that MTX solely abolishes 254 cytosolic SOC, cytosolic SHMT1 KO did not eliminate intermediary M+1 and M+2 serine isotoplogues from [U- 255 13C]serine, whereas mitochondrial or combined cytosolic and mitochondrial inhibition of SOC upon MFT KO, 256 SHMT2 KO, FPGS KO and MFT+SHMT1 KO resulted in a complete loss of M+1 and M+2 serine isotopologues 257 (Figure 4F). SHMT1 KO reduced serine M+1 and M+2 isotopologue abundance comparable to MTX treatment 258 (Figure 4F). In mutants harboring a full inhibition of SOC or mitochondrial SOC, we additionally observed an 259 increase of the serine to glycine ratio, whereas MTX treatment and SHMT1 KO slightly reduced serine to 260 glycine ratio (Figure 4G). Overall, these results indicate that mitochondria provide a protected cellular 261 environment that permits serine catabolism with formate overflow via SOC even in the presence of the 262 antifolate MTX. 263 To comparatively demonstrate how sole or dual inhibition of cytosolic and mitochondrial SOC affects serine 251 MID, we analyzed a panel of CRISPR generated knockouts in HAP1 cells in which either the cytosolic 252 (SHMT1), the mitochondrial (MFT, SHMT2), or both compartments (FPGS, MFT+SHMT1) of SOC were 253 abrogated [38]. In concordance with our previous findings and resulting conclusion that MTX solely abolishes 254 cytosolic SOC, cytosolic SHMT1 KO did not eliminate intermediary M+1 and M+2 serine isotoplogues from [U- 255 13C]serine, whereas mitochondrial or combined cytosolic and mitochondrial inhibition of SOC upon MFT KO, 256 SHMT2 KO, FPGS KO and MFT+SHMT1 KO resulted in a complete loss of M+1 and M+2 serine isotopologues 257 (Figure 4F). SHMT1 KO reduced serine M+1 and M+2 isotopologue abundance comparable to MTX treatment 258 (Figure 4F). In mutants harboring a full inhibition of SOC or mitochondrial SOC, we additionally observed an 259 increase of the serine to glycine ratio, whereas MTX treatment and SHMT1 KO slightly reduced serine to 260 glycine ratio (Figure 4G). Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility 302 Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility 302 Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility 302 To further reinforce the role of mitochondrial SOC in sustaining cell migration under nucleotide synthesis 303 inhibition, we employed MDA-MB468 cells with a shRNA mediated knockdown (KD) of MTHFD2, a key enzyme 304 of the mitochondrial SOC [39]. This KD cell line exhibited significantly reduced MTHFD2 expression (Figure 305 S6A) and displayed a significant reduction of formate overflow even in the presence of MTX (Figure 6A), 306 indicating effective abrogation of MTHFD2 activity. MTHFD2 KD did not significantly alter cell proliferation and 307 both cell lines were comparatively sensitive to the growth-arresting effects of MTX (Figure 6B). We confirmed 308 that MTX treatment itself did not upregulate protein levels of MTHFD2 or any of the other SOC enzymes 309 SHMT1, SHMT2, or MTHFD1L (Figure S6B-E). In line with our prediction, MTHFD2 KD markedly reduced 310 MTX-mediated cell motility (Figure 6C), confirming that mitochondrial SOC activity is a major metabolic 311 determinant of cell migration in growth-arrested cells under MTX treatment. Of note, cell death rates upon MTX 312 were not affected by MTHFD2 KD, which further confirmed that the observed reduction upon MTX and 313 MTHFD2 KD was not related to enhanced cell death or further reduced growth rates, but solely based on an 314 inherent reduction of migratory capacity upon loss of mitochondrial SOC activity (Figure 6B, 6D). In summary, 315 we uncovered that mitochondrial SOC acts as a metabolic facilitator of cell migration under stress conditions 316 such as chemotherapeutic nucleotide-synthesis inhibition. Based on these observations, we speculate that 317 different growth inhibiting and stress inducing conditions, such as antifolate therapy or energy limitation [4], 318 can trigger increased rates of formate overflow and concomitant accumulation of formate within the tumor 319 microenvironment. Such increased concentrations of formate might act in an autocrine and/or paracrine 320 manner to trigger a pro-invasive phenotype, which represents the first stage to enter the metastasis process. 321 Conclusively, targeting formate overflow emerges as a conceivable intervention to limit metastasis formation. 322 G ti t ti f it h d i l SOC d t t i f ti i i 323 argeting of serine de novo synthesis decreases MTX-dependent cancer cell migration Furthermore, while neither BI treatment alone nor serine and 286 glycine starvation alone were sufficient to prevent the induction of the previously observed EMT-phenotype in 287 response to MTX (Figure 2G, 5G), combined inhibition and starvation did minimize ZEB1, ZEB2, and VIM 288 upregulation (Figure 5G). This indicated that the efficient restriction of both, extracellular and de novo- 289 synthesized serine to inhibit mitochondrial SOC, is effective to prevent the pro-migratory, phenotypic change 290 upon MTX. Notably, serine and glycine starvation did not further increase cell death after combined MTX and 291 BI treatment (Figure 5H). Since MTX-treated cells were already in full growth arrest, the additional perturbation 292 with S/G starvation and/or PHGDH inhibition reveals that serine catabolism fulfills an important function to 293 support cell motility. This function is independent of serine’s anabolic function to support biosynthesis 294 processes. (Figure S5E). Using the MTX-resistant MDA-MB468 cell line, we found that BI treatment was also 295 sufficient to reduce the migratory capacity of such selected, pro-migratory, and potentially metastatic cancer 296 subpopulations (Figure 5I). In summary, depletion of de novo-synthesized serine using a specific PHGDH 297 inhibitor uncovered that the mitochondrial SOC is essential to sustain maximal cancer cell migration under 298 growth-arresting stimuli such as nucleotide synthesis inhibition by MTX. This implies that the mitochondrial 299 SOC represents a metabolic vulnerability that can be targeted to limit cell migration during certain 300 chemotherapeutic interventions (Figure 5J). 301 Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Having previously reported that formate overflow via SOC is a characteristic of 360 invasive cancer cells [10], and knowing that MTX has a direct inhibiting effect on SOC and proliferation, 361 studying this metabolic pathway emerged as a conceivable starting point for such further mechanistic 362 investigations. Within SOC, the non-essential amino acid serine is catabolized in the mitochondrion to glycine 363 and formyl-tetrahydrofolate (formyl-THF), which are required building blocks for cytosolic purine and thymidine 364 (a pyrimidine) synthesis. During thymidine synthesis, the co-factor THF is oxidized to dihydrofolate (DHF), 365 which subsequently requires regeneration to keep the folate in its bioactive form. This reduction of DHF to THF 366 is dependent on the enzyme DHF reductase (DHFR), which is a target of MTX. Hence, upon MTX treatment 367 mammary fat pads of immunocompetent BALB/c mice and monitored primary tumor growth over 6 weeks 331 (Figure 7A). In concordance with in vitro data, primary tumor growth in vivo and tumor weight at the endpoint 332 were not affected by Mthfd1l knockdown (Figure 7B, C). After 6 weeks, mice were sacrificed and lung tissue 333 was stained with H&E staining. Staining shows that micrometastases formation was visibly reduced in 334 response to MTHFD1L KD (Figure 7D). In addition, the number of macroscopic lung metastases were counted 335 and we found primary tumors derived from Mthfd1l-depleted 4T1 cells had significantly less pulmonary 336 metastases compared to 4T1 cells expressing MTHFD1L (Figure 7E). While all mice that were injected with 337 MTHFD1L-expressing 4T1 cells, exhibited lung metastases over 50% of mice injected with Mthfd1l knockdown 338 cells did not show metastatic lesions (Figure 7E). These in vivo findings further strengthen the key relevance 339 of mitochondrial SOC and specifically mitochondrial formate generation as a cornerstone that supports cancer 340 cell migration independent of primary tumor growth and cancer cell proliferation rates. 341 342 In this study, we uncovered a growth-independent function for serine and its mitochondrial catabolism that 344 drives cancer cell dissemination. Importantly, by using MTX as a tool compound to study migration 345 independent of proliferation, we could show that mitochondria sustain an autarkic SOC that is sufficient to 346 support the cells migratory capacity. This finding implies the dismal possibility that chemotherapeutic 347 approaches with nucleotide synthesis inhibiting drugs, which are undertaken to reduce primary cancer growth, 348 might not sufficiently suppress the motility capacities of remaining cancer cells. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo The fact, that in our hands, 349 breast cancer cells even showed a transitioning phenotype and were selected over time to be drug resistant, 350 while at the same time showing enhanced migratory potential, could warrant further work to comparatively 351 investigate metastatic disease progression after such therapies. Of note, aside from its use in cancer therapy, 352 the antifolate MTX represents the “anchor drug” for chronic treatment of autoimmune diseases such as 353 rheumatoid arthritis. Here, intracellular erythrocyte concentration of MTX and mean MTX plasma concentration 354 after chronic, low-dose MTX were previously reported to be in a comparable, even slightly higher, nM range 355 to our chosen drug concentration of 50 nM [40, 41]. This implies an additional, potential significance of our 356 findings in cancer patients that undergo chronic MTX therapy for an arthritic comorbidity and might warrant 357 further clinical retrospective and prospective investigations beyond the scope of this study. Our aim here was 358 however to comprehensively analyze the metabolic pathways that foster such sustained cell migration under 359 growth-arresting conditions. Having previously reported that formate overflow via SOC is a characteristic of 360 invasive cancer cells [10], and knowing that MTX has a direct inhibiting effect on SOC and proliferation, 361 studying this metabolic pathway emerged as a conceivable starting point for such further mechanistic 362 investigations. Within SOC, the non-essential amino acid serine is catabolized in the mitochondrion to glycine 363 and formyl-tetrahydrofolate (formyl-THF), which are required building blocks for cytosolic purine and thymidine 364 (a pyrimidine) synthesis. During thymidine synthesis, the co-factor THF is oxidized to dihydrofolate (DHF), 365 which subsequently requires regeneration to keep the folate in its bioactive form. This reduction of DHF to THF 366 is dependent on the enzyme DHF reductase (DHFR), which is a target of MTX. Hence, upon MTX treatment 367 mammary fat pads of immunocompetent BALB/c mice and monitored primary tumor growth over 6 weeks 331 (Figure 7A). In concordance with in vitro data, primary tumor growth in vivo and tumor weight at the endpoint 332 were not affected by Mthfd1l knockdown (Figure 7B, C). After 6 weeks, mice were sacrificed and lung tissue 333 was stained with H&E staining. Staining shows that micrometastases formation was visibly reduced in 334 response to MTHFD1L KD (Figure 7D). Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo In addition, the number of macroscopic lung metastases were counted 335 and we found primary tumors derived from Mthfd1l-depleted 4T1 cells had significantly less pulmonary 336 metastases compared to 4T1 cells expressing MTHFD1L (Figure 7E). While all mice that were injected with 337 MTHFD1L-expressing 4T1 cells, exhibited lung metastases over 50% of mice injected with Mthfd1l knockdown 338 cells did not show metastatic lesions (Figure 7E). These in vivo findings further strengthen the key relevance 339 of mitochondrial SOC and specifically mitochondrial formate generation as a cornerstone that supports cancer 340 cell migration independent of primary tumor growth and cancer cell proliferation rates. 341 342 Discussion 343 In this study, we uncovered a growth-independent function for serine and its mitochondrial catabolism that 344 drives cancer cell dissemination. Importantly, by using MTX as a tool compound to study migration 345 independent of proliferation, we could show that mitochondria sustain an autarkic SOC that is sufficient to 346 support the cells migratory capacity. This finding implies the dismal possibility that chemotherapeutic 347 approaches with nucleotide synthesis inhibiting drugs, which are undertaken to reduce primary cancer growth, 348 might not sufficiently suppress the motility capacities of remaining cancer cells. The fact, that in our hands, 349 breast cancer cells even showed a transitioning phenotype and were selected over time to be drug resistant, 350 while at the same time showing enhanced migratory potential, could warrant further work to comparatively 351 investigate metastatic disease progression after such therapies. Of note, aside from its use in cancer therapy, 352 the antifolate MTX represents the “anchor drug” for chronic treatment of autoimmune diseases such as 353 rheumatoid arthritis. Here, intracellular erythrocyte concentration of MTX and mean MTX plasma concentration 354 after chronic, low-dose MTX were previously reported to be in a comparable, even slightly higher, nM range 355 to our chosen drug concentration of 50 nM [40, 41]. This implies an additional, potential significance of our 356 findings in cancer patients that undergo chronic MTX therapy for an arthritic comorbidity and might warrant 357 further clinical retrospective and prospective investigations beyond the scope of this study. Our aim here was 358 however to comprehensively analyze the metabolic pathways that foster such sustained cell migration under 359 growth-arresting conditions. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo We sought to confirm our hypothesis on the central role of mitochondrial SOC as a facilitator of cancer cell 324 migration and ultimately metastasis formation in vivo. To specifically inhibit formate overflow, we generated a 325 stable knockdown of Mthfd1l in 4T1 breast cancer cells. The engineered cells exhibited a functional knockdown 326 of the enzyme as seen from the prominent reduction in formate release rates when compared to non-targeting 327 shRNA transfected 4T1 cells (Figure S7A). In line with our previous reports [4, 10], knockdown of Mthfd1l had 328 no effect on cell proliferation in vitro (Figure S7B) and reduced invasion (Figure S7C). To test if formate 329 overflow suppression can reduce metastasis formation in vivo, we injected both murine cell lines into the 330 mammary fat pads of immunocompetent BALB/c mice and monitored primary tumor growth over 6 weeks 331 (Figure 7A). In concordance with in vitro data, primary tumor growth in vivo and tumor weight at the endpoint 332 were not affected by Mthfd1l knockdown (Figure 7B, C). After 6 weeks, mice were sacrificed and lung tissue 333 was stained with H&E staining. Staining shows that micrometastases formation was visibly reduced in 334 response to MTHFD1L KD (Figure 7D). In addition, the number of macroscopic lung metastases were counted 335 and we found primary tumors derived from Mthfd1l-depleted 4T1 cells had significantly less pulmonary 336 metastases compared to 4T1 cells expressing MTHFD1L (Figure 7E). While all mice that were injected with 337 MTHFD1L-expressing 4T1 cells, exhibited lung metastases over 50% of mice injected with Mthfd1l knockdown 338 cells did not show metastatic lesions (Figure 7E). These in vivo findings further strengthen the key relevance 339 of mitochondrial SOC and specifically mitochondrial formate generation as a cornerstone that supports cancer 340 cell migration independent of primary tumor growth and cancer cell proliferation rates. 341 342 Discussion 343 In this study, we uncovered a growth-independent function for serine and its mitochondrial catabolism that 344 drives cancer cell dissemination. Importantly, by using MTX as a tool compound to study migration 345 independent of proliferation, we could show that mitochondria sustain an autarkic SOC that is sufficient to 346 support the cells migratory capacity. This finding implies the dismal possibility that chemotherapeutic 347 approaches with nucleotide synthesis inhibiting drugs, which are undertaken to reduce primary cancer growth, 348 might not sufficiently suppress the motility capacities of remaining cancer cells. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo The fact, that in our hands, 349 breast cancer cells even showed a transitioning phenotype and were selected over time to be drug resistant, 350 while at the same time showing enhanced migratory potential, could warrant further work to comparatively 351 investigate metastatic disease progression after such therapies. Of note, aside from its use in cancer therapy, 352 the antifolate MTX represents the “anchor drug” for chronic treatment of autoimmune diseases such as 353 rheumatoid arthritis. Here, intracellular erythrocyte concentration of MTX and mean MTX plasma concentration 354 after chronic, low-dose MTX were previously reported to be in a comparable, even slightly higher, nM range 355 to our chosen drug concentration of 50 nM [40, 41]. This implies an additional, potential significance of our 356 findings in cancer patients that undergo chronic MTX therapy for an arthritic comorbidity and might warrant 357 further clinical retrospective and prospective investigations beyond the scope of this study. Our aim here was 358 however to comprehensively analyze the metabolic pathways that foster such sustained cell migration under 359 growth-arresting conditions. Having previously reported that formate overflow via SOC is a characteristic of 360 invasive cancer cells [10], and knowing that MTX has a direct inhibiting effect on SOC and proliferation, 361 studying this metabolic pathway emerged as a conceivable starting point for such further mechanistic 362 investigations. Within SOC, the non-essential amino acid serine is catabolized in the mitochondrion to glycine 363 and formyl-tetrahydrofolate (formyl-THF), which are required building blocks for cytosolic purine and thymidine 364 (a pyrimidine) synthesis. During thymidine synthesis, the co-factor THF is oxidized to dihydrofolate (DHF), 365 which subsequently requires regeneration to keep the folate in its bioactive form. This reduction of DHF to THF 366 is dependent on the enzyme DHF reductase (DHFR), which is a target of MTX. Hence, upon MTX treatment 367 the folate co-factor accumulates in its inactive form DHF, which prevents nucleotide synthesis and cell growth 368 through obstruction of SOC activity [5] [42]. In agreement, our tool compound MTX effectively abolishes SOC- 369 dependent nucleotide synthesis in the cytosol. However, using [U13C]serine- and [U13C]glucose-assisted 370 metabolic flux analyses, we found that mitochondrial one-carbon cycle was preserved under antifolate-induced 371 nucleotide synthesis inhibition. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Such compartmentalization of SOC is on the one hand enabled by the lack of 372 oxidation of THF to DHF in the mitochondrial compartment, but is also based on the chemical modification of 373 folate species and their transport activities across the mitochondrial membrane. For maximal biologic activity, 374 folate species need to be polyglutamated by folylpolyglutamate synthase (FPGS) and the resulting 375 polyglutamate species were shown to be poorly transported across the mitochondrial membrane [43, 44]. 376 Consequently, polyglutamated folate species can be chemically trapped within the mitochondria to sustain an 377 autarkic mitochondrial SOC especially upon growth arrest and subsequent unaltered mitochondrial content. 378 Furthermore, there have been implications that the mitochondrial folate transporter is specific for reduced 379 folates with the possibility of limiting the transport of MTX and folic acid itself across the mitochondrial 380 membrane [45].Taken together, such compartmentalization of SOC and autarkic function of mitochondrial 381 SOC emerges as a selective advantage upon perturbation of cytosolic SOC and indicates an additional 382 metabolic functionality of the mitochondrial SOC pathway during growth-arresting conditions. Specifically, 383 plasticity of serine catabolism through this sustained mitochondrial pathway could offer a metabolic reserve in 384 the form of ATP and redox equivalent generation that might allow growth-arrested cancer cells to undergo 385 epithelial-mesenchymal transition and to enhance their migratory capacity in order to escape from sites of 386 nutrient deprivation or chemotherapeutic targeting. In concert with this notion, we identified mitochondrial SOC 387 as a targetable metabolic vulnerability by using genetic and pharmacologic inhibition of mitochondrial one- 388 carbon cycle activity to reduce migration in vitro and metastasis formation in vivo. These findings are in line 389 with previous reports indicating that serine de novo synthesis rates and serine uptake rates are associated 390 with disease progression and even metastatic dissemination [11-13, 46]. However to our knowledge, this is 391 the first report to show that it is indeed the enhanced activity of mitochondrial serine catabolism to formate via 392 SOC which translates these enhanced serine synthesis rates or serine uptake rates to a pro-migratory signal 393 downstream. Based on these findings, we hypothesize that serine plasticity and the alternative usage of serine 394 catabolism in the mitochondria can selectively promote formate overflow in response to extrinsic or intrinsic 395 stress stimuli such as growth inhibition or nutrient deprivation. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Consequently, the resulting increase in local 396 formate concentrations in the TME could subsequently serve as a local stress signal in an auto- or paracrine 397 manner to promote the formation of invasive cancer cells. These cells are, as a result, more prone and capable 398 in escaping a non-beneficial primary TME, which eventually advances dissemination and metastasis formation. 399 Thus, restricting mitochondrial SOC, which is at the root of this stress signaling cascade, emerges as a 400 promising, preventive strategy to hinder metastatic tumor progression early on. 401 402 We are grateful to Saverio Tardito (Cancer Research UK Beatson Institute, Glasgow, UK) for providing 404 Plasmax medium to us. We are also thankful to Clément Thomas (LIH, Luxembourg) for providing 4T1 cells, 405 Lewis Cantley (Weill Cornell Medical College) for providing MDA-MB468 cells and to Patel’s laboratory 406 (Cambridge, UK) for providing HAP1 cells [38]. 407 Plasmax medium to us. We are also thankful to Clément Thomas (LIH, Luxembourg) for providing 4T1 cells, 405 Lewis Cantley (Weill Cornell Medical College) for providing MDA-MB468 cells and to Patel’s laboratory 406 (Cambridge, UK) for providing HAP1 cells [38]. 407 We would like to thank: the LCSB Metabolomics Platform, especially Xiangyi Dong and Floriane Vanhalle, for 408 providing technical and analytical support; the National Cytometry Platform (Quantitative Biology Unit, LIH) 409 and especially Thomas Cerutti for support with flow cytometric analyses; Magretta Adiamah for her diligent 410 We would like to thank: the LCSB Metabolomics Platform, especially Xiangyi Dong and Floriane Vanhalle, for 408 providing technical and analytical support; the National Cytometry Platform (Quantitative Biology Unit, LIH) 409 and especially Thomas Cerutti for support with flow cytometric analyses; Magretta Adiamah for her diligent 410 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411 All graphical figures were produced with BioRender.com. 412 J.M. is supported by the FNR-ATTRACT program (A18/BM/11809970), INTER-BMBF grant (18/13399110) 413 and i2Tron (PRIDE19/14254520). N.K. is supported by the LIH Career Launchpad program (Legs Baertz). 414 D.B. is supported by the FNR-ATTRACT program (A14/BM/7632103), the FNR-CORE (C18/BM/12691266), 415 i2Tron (PRIDE19/14254520) and the FNR-RIKEN (TregBar/11228353) grant. D.B. and L.S.B. are funded by 416 the FNR-PRIDE (PRIDE/11012546/NEXTIMMUNE) scheme. E.L. is supported by the FNR-CORE program 417 (C16/BM/11282028 and C20/BM/14591557), by a Proof of Concept FNR grant (PoC/18/12554295), a 418 PRIDE17/11823097 and by i2Tron (PRIDE19/14254520). G.D. and F.X. are supported by FNR-CORE 419 (C17/BM/11642138). B.J. and M.Z.N. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo are supported by Luxembourg National Research Fund 420 (C18/BM/12670304/COMBATIC) and FNRS Televie grant (7.4579.20). 421 422 Author Contributions 423 Conceptualization, J.M., N.K.; Methodology, J.M., N.K., C.J., M.Z.N., B.J., E.L., D.B., G.D., V.I.P.; Software, 424 A.V., F.X.; Validation, N.K., J.M., L.N., C.D.; Formal Analysis, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S., 425 C.J.; Investigation, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S., E.L.; Resources, C.J., A.V., J.M., E.L.; 426 Data Curation, N.K., J.M., F.X., A.V.; Writing – Original Draft, N.K., J.M.; Writing – Review & Editing, All 427 Authors; Visualization, N.K., J.M., L.S.B., F.X., V.I.P.; Supervision, J.M., D.B., A.V., E.L., G.D.; Project 428 Administration, J.M., Funding Acquisition, J.M., D.B., E.L., A.V., G.D.. 429 430 Materials & Methods 431 Chemicals 432 Methotrexate, rotenone, PKUMDL-WQ-2101, galactose, hydroxyurea, NAC, and fluorouracil were purchased 433 from Sigma Aldrich. Clofarabine, pemetrexed disodium hydrate, sirolismus were purchased from Bio Connect. 434 Simvastatin was purchased from Sanbio. CBR-5884 and BI-4916 were purchased from MedChemExpress. 435 NCT-502 was purchased from ApeXBio. 436 Cell Culture 437 All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) without phenol red, glucose, and 438 glutamine (Thermo Fisher Scientific) and supplemented with 2 mM glutamine, 17 mM glucose, and 10 % fetal 439 bovine serum (FBS) at 37°C and 5 % CO2. For galactose treatment, supplemented glucose was replaced by 440 17 mM galactose at the beginning of the experiment. MDA-MB468 and LN229 were obtained from ATCC. 441 HCT-116 was obtained from the group of E. Letellier (LSRU, University of Luxembourg), and 4T1 from C. 442 Thomas (LIH, Luxembourg). HAP1 cells were obtained from Patel’s laboratory [38] and cultured in IMDM 443 medium supplemented with 10 % FBS. MTX-resistant MDA-MB468 cells were generated through prolonged 444 culturing of MDA-MB468 cells with 50 nM MTX for 2 months until cells started to regrowth under MTX. All cell 445 lines were routinely tested for mycoplasma contamination. 446 Metabolic Characterization 447 For metabolic characterization of cell lines, previously established protocols for absolute quantification of 448 exchange fluxes and intracellular fluxes of one-carbon metabolism were applied [4]. 449 Stable Isotope Tracing and Metabolite Extraction 450 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411 All graphical figures were produced with BioRender.com. 412 J.M. is supported by the FNR-ATTRACT program (A18/BM/11809970), INTER-BMBF grant (18/13399110) 413 and i2Tron (PRIDE19/14254520). N.K. is supported by the LIH Career Launchpad program (Legs Baertz). Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 414 D.B. is supported by the FNR-ATTRACT program (A14/BM/7632103), the FNR-CORE (C18/BM/12691266), 415 i2Tron (PRIDE19/14254520) and the FNR-RIKEN (TregBar/11228353) grant. D.B. and L.S.B. are funded by 416 the FNR-PRIDE (PRIDE/11012546/NEXTIMMUNE) scheme. E.L. is supported by the FNR-CORE program 417 (C16/BM/11282028 and C20/BM/14591557), by a Proof of Concept FNR grant (PoC/18/12554295), a 418 PRIDE17/11823097 and by i2Tron (PRIDE19/14254520). G.D. and F.X. are supported by FNR-CORE 419 (C17/BM/11642138). B.J. and M.Z.N. are supported by Luxembourg National Research Fund 420 (C18/BM/12670304/COMBATIC) and FNRS Televie grant (7.4579.20). 421 422 Author Contributions 423 Conceptualization, J.M., N.K.; Methodology, J.M., N.K., C.J., M.Z.N., B.J., E.L., D.B., G.D., V.I.P.; Software, 424 A.V., F.X.; Validation, N.K., J.M., L.N., C.D.; Formal Analysis, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S., 425 C.J.; Investigation, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S., E.L.; Resources, C.J., A.V., J.M., E.L.; 426 Data Curation, N.K., J.M., F.X., A.V.; Writing – Original Draft, N.K., J.M.; Writing – Review & Editing, All 427 Authors; Visualization, N.K., J.M., L.S.B., F.X., V.I.P.; Supervision, J.M., D.B., A.V., E.L., G.D.; Project 428 Administration, J.M., Funding Acquisition, J.M., D.B., E.L., A.V., G.D.. 429 430 Materials & Methods 431 Chemicals 432 Methotrexate, rotenone, PKUMDL-WQ-2101, galactose, hydroxyurea, NAC, and fluorouracil were purchased 433 from Sigma Aldrich. Clofarabine, pemetrexed disodium hydrate, sirolismus were purchased from Bio Connect. 434 Simvastatin was purchased from Sanbio. CBR-5884 and BI-4916 were purchased from MedChemExpress. 435 NCT-502 was purchased from ApeXBio. 436 Cell Culture 437 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411 All graphical figures were produced with BioRender.com. 412 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411 All graphical figures were produced with BioRender.com. 412 Methotrexate, rotenone, PKUMDL-WQ-2101, galactose, hydroxyurea, NAC, and fluorouracil were purchased 433 from Sigma Aldrich. Clofarabine, pemetrexed disodium hydrate, sirolismus were purchased from Bio Connect. 434 Simvastatin was purchased from Sanbio. CBR-5884 and BI-4916 were purchased from MedChemExpress. 435 NCT-502 was purchased from ApeXBio. 436 Stable isotope tracing experiments with [U-13C]-glucose tracer or [U-13C]-glutamine tracer (Cambridge Isotope 451 Laboratories, CLM-1396) were performed in DMEM supplemented with 2 mM glutamine, 17 mM glucose 452 tracer, and 10 % FBS. Stable isotope tracing experiments with [U-13C]-serine tracer (Cambridge Isotope 453 Laboratories) were performed in MEM (Thermo Fisher Scientific) supplemented with 400 μM glycine, 2 mM 454 glutamine, 17 mM glucose, 400 μM serine tracer, and 10 % FBS. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo GC- 476 MS analysis was performed using an Agilent 7890A GC coupled to an Agilent 5975C inert XL Mass Selective 477 Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in 478 split mode (20:1) at 270 °C. The gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) 479 DB-5MS capillary column (Agilent J&W GC Column, 122-5532G). Helium was used as carrier gas with a 480 constant flow rate of 1.4 ml/min. GC oven temperature was held at 80 °C for 1 min and increased to 130 °C at 481 10 °C/min followed by a post run time of 4 min at 280 °C. Total run time was 15 min. Transfer line temperature 482 was set to 280 °C. Mass selective detector (MSD) was operating under electron ionization at 70 eV. MS source 483 was held at 230 °C and the quadrupole at 150 °C. For precise quantification, measurements were performed 484 in selected ion monitoring mode. Target ions (m/z) and dwell times are shown in Table S2. GC-MS 485 chromatograms were processed using Agilent MassHunter Quantitative Analysis for GC-MS, Version B.08.00. 486 Final determination of release rates was performed as described in [4]. 487 Determination of MIDs of Intracellular TCA Cycle Metabolites following Stable Isotope Tracing: 488 Polar metabolites were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c = 20 mg/ml) in pyridine 489 under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MTBSTFA w/ 1% TBDMCS. GC- 490 y Formate derivatization in the culture medium was performed using MCF derivatization as described in [4]. GC- 476 MS analysis was performed using an Agilent 7890A GC coupled to an Agilent 5975C inert XL Mass Selective 477 Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in 478 split mode (20:1) at 270 °C. The gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) 479 DB-5MS capillary column (Agilent J&W GC Column, 122-5532G). Helium was used as carrier gas with a 480 constant flow rate of 1.4 ml/min. GC oven temperature was held at 80 °C for 1 min and increased to 130 °C at 481 10 °C/min followed by a post run time of 4 min at 280 °C. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo To allow for adaptation, cells were cultivated 455 in MEM for at least one passage prior to [U-13C]-serine tracer experiments. 150,000 to 200,000 cells were 456 seeded in 12-well plates in triplicates for each experimental condition. Identical triplicate wells were seeded to 457 allow for cell count and cell volume determination (to calculate the packed cell volume (PCV)) at the start and 458 end of each tracing experiment. The day after seeding, growth medium was replaced by tracer medium and 459 cells were cultured for 24 h. In parallel, 3 wells per condition were counted to assess starting PCV. After 24 h, 460 triplicate wells were counted to assess PCV at the end of the experiment and one set of triplicates was used 461 for subsequent metabolite extraction. Medium of these triplicates were collected and analyzed for exchange 462 rates. To determine the basal medium composition for the subsequent calculation of exchange rates, identical 463 medium was incubated in triplicates in empty 12 wells throughout the experiment and analyzed in parallel to 464 the medium samples. Collected medium samples were centrifuged at 300 g for 5 min. Supernatant was 465 collected and stored at -20°C until extraction of metabolites. Formate extraction, derivatization, and 466 quantification as well as metabolite extraction for LC-MS analysis was performed as described in [4]. For 467 metabolite extraction for GC-MS analysis after [U-13C]-glucose tracing and [U-13C]-glutamine tracing, cells 468 were washed with cold 0.9 % NaCl solution. 400 μl ice-cold MeOH/H2OMQ [(ratio, 1:1) containing the internal 469 standards pentanedioc-d6 acid and [U-13C]-ribitol at a final concentration of 1 μg/ml and Tridecanoid-d25 acid 470 at a final concentration of 5 μg/ml] was added to each well. Plates were incubated for 5 min at 4°C on a rocking 471 shaker. Supernatant was collected, mixed with 200 μl CHCl3, and centrifuged for 5 min at 13,000 g at 4°C. 472 Upper polar phase was collected and stored at -20°C for subsequent MS analysis of polar metabolites. 473 GC-MS Measurements 474 Stable isotope tracing experiments with [U-13C]-glucose tracer or [U-13C]-glutamine tracer (Cambridge Isotope 451 Laboratories, CLM-1396) were performed in DMEM supplemented with 2 mM glutamine, 17 mM glucose 452 tracer, and 10 % FBS. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Stable isotope tracing experiments with [U-13C]-serine tracer (Cambridge Isotope 453 Laboratories) were performed in MEM (Thermo Fisher Scientific) supplemented with 400 μM glycine, 2 mM 454 glutamine, 17 mM glucose, 400 μM serine tracer, and 10 % FBS. To allow for adaptation, cells were cultivated 455 in MEM for at least one passage prior to [U-13C]-serine tracer experiments. 150,000 to 200,000 cells were 456 seeded in 12-well plates in triplicates for each experimental condition. Identical triplicate wells were seeded to 457 allow for cell count and cell volume determination (to calculate the packed cell volume (PCV)) at the start and 458 end of each tracing experiment. The day after seeding, growth medium was replaced by tracer medium and 459 cells were cultured for 24 h. In parallel, 3 wells per condition were counted to assess starting PCV. After 24 h, 460 triplicate wells were counted to assess PCV at the end of the experiment and one set of triplicates was used 461 for subsequent metabolite extraction. Medium of these triplicates were collected and analyzed for exchange 462 rates. To determine the basal medium composition for the subsequent calculation of exchange rates, identical 463 medium was incubated in triplicates in empty 12 wells throughout the experiment and analyzed in parallel to 464 the medium samples. Collected medium samples were centrifuged at 300 g for 5 min. Supernatant was 465 collected and stored at -20°C until extraction of metabolites. Formate extraction, derivatization, and 466 quantification as well as metabolite extraction for LC-MS analysis was performed as described in [4]. For 467 metabolite extraction for GC-MS analysis after [U-13C]-glucose tracing and [U-13C]-glutamine tracing, cells 468 were washed with cold 0.9 % NaCl solution. 400 μl ice-cold MeOH/H2OMQ [(ratio, 1:1) containing the internal 469 standards pentanedioc-d6 acid and [U-13C]-ribitol at a final concentration of 1 μg/ml and Tridecanoid-d25 acid 470 at a final concentration of 5 μg/ml] was added to each well. Plates were incubated for 5 min at 4°C on a rocking 471 shaker. Supernatant was collected, mixed with 200 μl CHCl3, and centrifuged for 5 min at 13,000 g at 4°C. 472 Upper polar phase was collected and stored at -20°C for subsequent MS analysis of polar metabolites. 473 GC-MS Measurements 474 Analysis of Formate Release Rates: 475 Formate derivatization in the culture medium was performed using MCF derivatization as described in [4]. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Total run time was 15 min. Transfer line temperature 482 was set to 280 °C. Mass selective detector (MSD) was operating under electron ionization at 70 eV. MS source 483 was held at 230 °C and the quadrupole at 150 °C. For precise quantification, measurements were performed 484 in selected ion monitoring mode. Target ions (m/z) and dwell times are shown in Table S2. GC-MS 485 chromatograms were processed using Agilent MassHunter Quantitative Analysis for GC-MS, Version B.08.00. 486 Final determination of release rates was performed as described in [4]. 487 Determination of MIDs of Intracellular TCA Cycle Metabolites following Stable Isotope Tracing: 488 Polar metabolites were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c = 20 mg/ml) in pyridine 489 under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MTBSTFA w/ 1% TBDMCS. GC- 490 MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977A Mass Selective Detector 491 (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in splitless 492 mode at 270 °C. Gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) ZB-35MS capillary 493 column with 5 m guard column (Phenomenex). Helium was used as carrier gas with a constant flow rate of 1.2 494 ml/min. GC oven temperature was held at 100 °C for 2 min and increased to 300 °C at 10 °C/min and held for 495 4 min. Total run time was 26 min. Transfer line temperature was set to 280 °C. Mass selective detector (MSD) 496 was operating under electron ionization at 70 eV. MS source was held at 230 °C and the quadrupole at 150 497 °C. For precise quantification of the MID, measurements were performed in selected ion monitoring mode. 498 Target ions (m/z) and dwell times are shown in Table S3. 499 The MetaboliteDetector software package (Version 3.220180913) was used for mass spectrometric data post 500 processing, quantification, MID calculations, correction of natural isotope abundance, and determinations of 501 fractional carbon contributions [47]. 502 Analysis of Medium Exchange Rates: 503 Polar metabolites of the culture medium were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c = 504 20 mg/ml) in pyridine under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MSTFA. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 505 GC-MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977A Mass Selective 506 Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in 507 splitless mode at 270 °C. Gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) ZB-35MS 508 capillary column with 5 m guard column (Phenomenex). Helium was used as carrier gas with a constant flow 509 rate of 1.2 ml/min. GC oven temperature program: 90° C for 1 min, 9° C/min to 270° C, 25° C/min to 320° C 510 and held for 7 min. Total run time was 30 min. Transfer line temperature was set to 280° C. MSD was operated 511 under electron ionization at 70 eV. MS source was held at 230° C and the quadrupole at 150° C. Full scan 512 mass spectra were acquired between m/z 70 and 700. 513 The MetaboliteDetector software package (Version 3.220180913) was used for quantification. Briefly, peak 514 areas of all isotopologues of defined quantification ions were summed up and divided by the peak area of the 515 internal standard for normalization. In addition, a calibration curve was prepared to calculate absolute 516 concentrations. Absolute uptake and release rates were calculated as described in [4] 517 LC-MS Measurements 518 Untargeted LC-MS analysis was carried out as previously described in [4]. Targeted analysis of GSH/GSSG 519 was performed as described in [48]. 520 Nucleotide and NAD/NADH Analysis 521 The following analytical conditions are based on a protocol from [4]. Metabolite analyses were performed using 522 a Thermo Vanquish Flex Quaternary LC coupled to a Thermo Q Exactive HF mass spectrometer. 523 Chromatography was carried out with a SeQuant ZIC-pHILIC 5µm polymer (150 x 2.1 mm) column connected 524 to the corresponding SeQuant ZIC-pHILIC Guard (20 x 2.1 mm) pre-column. Column temperature was 525 maintained at 45 °C. The flow rate was set to 0.2 mL/min and the mobile phases consisted of 20 mmol/L 526 ammonium carbonate in water, pH 9.2 (Eluent A) and Acetonitrile (Eluent B). The gradient was: 0 min, 80% B; 527 The MetaboliteDetector software package (Version 3.220180913) was used for mass spectrometric data post 500 processing, quantification, MID calculations, correction of natural isotope abundance, and determinations of 501 fractional carbon contributions [47]. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 536 follows: sheath gas flow rate, 25; aux gas flow rate, 15; sweep gas flow rate, 0; spray voltage, 4.5 kV (+) / 3.5 531 kV (–); capillary temperature, 325 °C; S-lense RF level, 50; aux gas heater temperature, 50 °C. The Orbitrap 532 mass analyzer was operated at a resolving power of 30,000 in full-scan mode (scan range: m/z 75…1000; 533 automatic gain control target: 1e6; maximum injection time: 250 ms). Data were acquired with Thermo Xcalibur 534 software (Version 4.3.73.11) and analyzed with TraceFinder (Version 4.1). Subsequent data analysis for 535 normalization and natural isotope subtraction were performed using in house scripts as in [4]. 536 Western Blot 537 Total cell lysates were prepared by 30 min incubation of cell pellets on ice in cell lysis buffer (150 mM NaCl, 538 1 mM EDTA, 50 mM Tris-HCl, 1% NP-40). Lysis efficiency was maximized by sonification. Lysis solution was 539 centrifuged at 13,000 g for 15 min at 4°C and supernatant was collected and stored at -80°C. Protein 540 concentration was determined by Bradford assay. 15 – 25 μg of total protein were loaded on NuPAGE 4-12% 541 Bis-Tris gels (Life Technologies) or RunBlue 4-12% Bis-Tris gels (Westburg) using 4x NuPage LDS Sample 542 buffer (Thermo Fisher Scientific) supplemented with 10 mM DTT (Sigma Aldrich) and blotted on nitrocellulose 543 membrane according to standard protocols. Membranes were stained with REVERT staining solution (LI-COR) 544 and analyzed for total protein abundance. Subsequently, membranes were blocked with Odyssey TBS 545 blocking buffer (LI-COR) or 5% milk-powder in TBST and incubated with the indicated primary antibodies over 546 night at 4°C. Incubation with secondary antibody occurred for 2 h at RT. Detection was performed with the 547 Odyssey CLx Infrared Imaging System (LI-COR). ImageStudioLite Software Vers.5.2 (LI-COR) was used for 548 image analysis. Antibodies used for Western blot analysis in this study: MTHFD1L (16113-1-AP) from 549 Proteintech; vimentin (3390), β-actin (3700), and MTHFD2 (41377) from Cell Signaling Technology; PHGDH 550 (HPA021241), PSAT1 (HPA042924), PSPH (HPA020376), SHMT1 (HPA023314), and SHMT2 (HPA020549) 551 from Sigma Aldrich; IRDye 680RD Goat Anti-Mouse IgG (H+L) and IRDye 800CW Donkey Anti-Rabbit IgG 552 (H+L) from LI-COR. 553 Total cell lysates were prepared by 30 min incubation of cell pellets on ice in cell lysis buffer (150 mM NaCl, 538 1 mM EDTA, 50 mM Tris-HCl, 1% NP-40). Lysis efficiency was maximized by sonification. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 502 Polar metabolites of the culture medium were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c = 504 20 mg/ml) in pyridine under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MSTFA. 505 GC-MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977A Mass Selective 506 Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in 507 splitless mode at 270 °C. Gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) ZB-35MS 508 capillary column with 5 m guard column (Phenomenex). Helium was used as carrier gas with a constant flow 509 rate of 1.2 ml/min. GC oven temperature program: 90° C for 1 min, 9° C/min to 270° C, 25° C/min to 320° C 510 and held for 7 min. Total run time was 30 min. Transfer line temperature was set to 280° C. MSD was operated 511 under electron ionization at 70 eV. MS source was held at 230° C and the quadrupole at 150° C. Full scan 512 mass spectra were acquired between m/z 70 and 700. 513 The MetaboliteDetector software package (Version 3.220180913) was used for quantification. Briefly, peak 514 areas of all isotopologues of defined quantification ions were summed up and divided by the peak area of the 515 internal standard for normalization. In addition, a calibration curve was prepared to calculate absolute 516 concentrations. Absolute uptake and release rates were calculated as described in [4] 517 Untargeted LC-MS analysis was carried out as previously described in [4]. Targeted analysis of GSH/GSSG was performed as described in [48]. follows: sheath gas flow rate, 25; aux gas flow rate, 15; sweep gas flow rate, 0; spray voltage, 4.5 kV (+) / 3.5 531 kV (–); capillary temperature, 325 °C; S-lense RF level, 50; aux gas heater temperature, 50 °C. The Orbitrap 532 mass analyzer was operated at a resolving power of 30,000 in full-scan mode (scan range: m/z 75…1000; 533 automatic gain control target: 1e6; maximum injection time: 250 ms). Data were acquired with Thermo Xcalibur 534 software (Version 4.3.73.11) and analyzed with TraceFinder (Version 4.1). Subsequent data analysis for 535 normalization and natural isotope subtraction were performed using in house scripts as in [4]. Seahorse Measurements 560 The day prior to measuring OCR, 40,000 cells were seeded on poly-L-lysine coated plates. Cells were treated 561 as indicated. XF96 Extracellular Flux Analyzer (Seahorse Bioscience) was used to measure basal OCR 562 following manufacturer’s instructions. Basal OCR was normalized to the protein concentration in the wells 563 following the protocol described in [49] using Bradford assay. 564 Poly-L-lysine Coating 554 Poly-L-lysine (P1274) was purchased from Sigma-Aldrich and reconstituted at 500 μg/ml in H2OMQ. Wells were 555 coated with PLL prior to Seahorse measurement and migration analysis by Scratch assay. To this end, PLL 556 solution was diluted 1:20 in H2OMQ and added to the plates at least 1 h prior to seeding. Following incubation 557 with PLL and before cells were seeded, plates were washed twice with H2OMQ and allowed to dry under the 558 hood. 559 Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Lysis solution was 539 centrifuged at 13,000 g for 15 min at 4°C and supernatant was collected and stored at -80°C. Protein 540 concentration was determined by Bradford assay. 15 – 25 μg of total protein were loaded on NuPAGE 4-12% 541 Bis-Tris gels (Life Technologies) or RunBlue 4-12% Bis-Tris gels (Westburg) using 4x NuPage LDS Sample 542 buffer (Thermo Fisher Scientific) supplemented with 10 mM DTT (Sigma Aldrich) and blotted on nitrocellulose 543 membrane according to standard protocols. Membranes were stained with REVERT staining solution (LI-COR) 544 and analyzed for total protein abundance. Subsequently, membranes were blocked with Odyssey TBS 545 blocking buffer (LI-COR) or 5% milk-powder in TBST and incubated with the indicated primary antibodies over 546 night at 4°C. Incubation with secondary antibody occurred for 2 h at RT. Detection was performed with the 547 Odyssey CLx Infrared Imaging System (LI-COR). ImageStudioLite Software Vers.5.2 (LI-COR) was used for 548 image analysis. Antibodies used for Western blot analysis in this study: MTHFD1L (16113-1-AP) from 549 Proteintech; vimentin (3390), β-actin (3700), and MTHFD2 (41377) from Cell Signaling Technology; PHGDH 550 (HPA021241), PSAT1 (HPA042924), PSPH (HPA020376), SHMT1 (HPA023314), and SHMT2 (HPA020549) 551 from Sigma Aldrich; IRDye 680RD Goat Anti-Mouse IgG (H+L) and IRDye 800CW Donkey Anti-Rabbit IgG 552 (H+L) from LI-COR. 553 Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death Cell 591 proliferation was either determined as measurement of cell density (confluence) or and through quantification 592 of red objects by using the IncuCyte ® NucLight Rapid Red Reagent (Essen Bioscience). Staining was 593 performed according to manufacturer’s instructions. Viable cell number was determined by trypan blue staining 594 and automatic counting using a Countess™ Cell Counting Chamber Slide (Thermo Fisher). 595 Migration Assay (Wound Closure) 596 Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen 597 Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells 598 per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker Tool 599 (Essen Bioscience) and indicated individual treatment conditions were applied simultaneously. Measurement 600 for individual treatment conditions was performed in biological replicates (n = 4 – 8) and repeated as 601 independent experiments as stated for the respective experiment (see figure legends). 602 Invasion and Migration Assay (Transwell Assay) 603 Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018 604 [10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. 605 Treatment was carried out for 18 h to 24 h. 606 RNA Extraction cDNA Synthesis and qPCR Analysis 607 Subsequent centrifugation yielded a pellet that was resuspended in 100 μl PBS and fixed with ice-cold 80% 571 EtOH. Fixed cells were stored at -20°C for at least 1 h and maximum 5 days prior to measurement. Cells were 572 centrifuged, pellet was incubated for 1 h in 200 μl RNAse A in PBS (30 μg/ml) at RT. Immediately prior to 573 measurement, 98 μl propidium iodide (PI) in PBS (50 μg/ml) was added. Flow cytometric analysis was 574 performed using BD FACSCanto and BD FACSDiva software. Analysis was performed in FlowJo. 575 C ll D h A l i 6 A fresh pellet following centrifugation was resuspended in 50 μl AnnexinV-FITC staining solution (5% 577 AnnexinV-FITC in AnnexinV binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA 578 in ddH2O)) and incubated for 15 min on ice in the dark. Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death 200,000 cells were seeded in 2 ml DMEM and treated the subsequent day as indicated. After incubation, 566 medium was collected and cells were washed with PBS. PBS fraction was collected and cells were detached 567 with trypsin. Trypsination was stopped by DMEM addition and this fraction as well as a subsequent PBS 568 washing fraction were collected. The combined solutions were centrifuged and washed with PBS. 569 Cell Cycle Distribution 570 Subsequent centrifugation yielded a pellet that was resuspended in 100 μl PBS and fixed with ice-cold 80% 571 EtOH. Fixed cells were stored at -20°C for at least 1 h and maximum 5 days prior to measurement. Cells were 572 centrifuged, pellet was incubated for 1 h in 200 μl RNAse A in PBS (30 μg/ml) at RT. Immediately prior to 573 measurement, 98 μl propidium iodide (PI) in PBS (50 μg/ml) was added. Flow cytometric analysis was 574 performed using BD FACSCanto and BD FACSDiva software. Analysis was performed in FlowJo. 575 Cell Death Analysis 576 A fresh pellet following centrifugation was resuspended in 50 μl AnnexinV-FITC staining solution (5% 577 AnnexinV-FITC in AnnexinV binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA 578 in ddH2O)) and incubated for 15 min on ice in the dark. 450 μl PI-staining solution (1.1 μg/ml PI in AnnexinV 579 binding buffer) was added immediately prior to measurement using BD FACSCanto and BD FACSDiva 580 software (for wild-type cells) or NovoCyte Quanteon (for GFP-positive transfected cells). Analysis was 581 performed in FlowJo. 582 Flow Cytometric Analysis of ROS Levels 583 250,000 MDA-MB468 cells were seeded in 2 ml medium in 6 well plates and treated as indicated. Following 584 incubation, adherent cells were detached with trypsin, centrifuged at 350 g for 5 min and washed with warm 585 DMEM. Live cells were stained in 100 μl DMEM supplemented with 1:2,000 DAPI and 1:500 DCFDA for 30 min 586 at 37°C. Following incubation, samples were centrifuged at 350 g for 5 min and washed with PBS. Following 587 centrifugation at 350 g for 5 min, cells were resuspended in 100 μl PBS and measured using the BD 588 LSRFortessaTM system and BD FACSDiva software. Data analysis was performed using FlowJo software. 589 Cell Proliferation 590 Cell proliferation was determined using the IncuCyte® Live-Cell Analysis system (Essen Bioscience). Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death 605 Treatment was carried out for 18 h to 24 h. 606 RNA Extraction, cDNA Synthesis, and qPCR Analysis 607 Total RNA was extracted directly from the cell culture dishes using RNeasy Mini Kit (Qiagen, 74104). For 608 qPCR, RNA was converted to cDNA using High-capacity cDNA Reverse Transcription Kit (ThermoFisher, 609 4368814). qPCR was performed from 20 ng cDNA per samples using Fast SYBR™ Green Master Mix 610 Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen 597 Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells 598 per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker Tool 599 (Essen Bioscience) and indicated individual treatment conditions were applied simultaneously. Measurement 600 for individual treatment conditions was performed in biological replicates (n = 4 – 8) and repeated as 601 independent experiments as stated for the respective experiment (see figure legends). 602 Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018 604 [10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. 605 Treatment was carried out for 18 h to 24 h. 606 Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018 604 [10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. 605 Treatment was carried out for 18 h to 24 h. 606 Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death 450 μl PI-staining solution (1.1 μg/ml PI in AnnexinV 579 binding buffer) was added immediately prior to measurement using BD FACSCanto and BD FACSDiva 580 software (for wild-type cells) or NovoCyte Quanteon (for GFP-positive transfected cells). Analysis was 581 performed in FlowJo. 582 software (for wild type cells) or NovoCyte Quanteon (for GFP positive transfected cells). Analysis was 581 performed in FlowJo. 582 Flow Cytometric Analysis of ROS Levels 583 250,000 MDA-MB468 cells were seeded in 2 ml medium in 6 well plates and treated as indicated. Following 584 incubation, adherent cells were detached with trypsin, centrifuged at 350 g for 5 min and washed with warm 585 DMEM. Live cells were stained in 100 μl DMEM supplemented with 1:2,000 DAPI and 1:500 DCFDA for 30 min 586 at 37°C. Following incubation, samples were centrifuged at 350 g for 5 min and washed with PBS. Following 587 centrifugation at 350 g for 5 min, cells were resuspended in 100 μl PBS and measured using the BD 588 LSRFortessaTM system and BD FACSDiva software. Data analysis was performed using FlowJo software. 589 Cell Proliferation 590 Cell proliferation was determined using the IncuCyte® Live-Cell Analysis system (Essen Bioscience). Cell 591 proliferation was either determined as measurement of cell density (confluence) or and through quantification 592 of red objects by using the IncuCyte ® NucLight Rapid Red Reagent (Essen Bioscience). Staining was 593 performed according to manufacturer’s instructions. Viable cell number was determined by trypan blue staining 594 and automatic counting using a Countess™ Cell Counting Chamber Slide (Thermo Fisher). 595 Migration Assay (Wound Closure) 596 Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen 597 Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells 598 per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker Tool 599 (Essen Bioscience) and indicated individual treatment conditions were applied simultaneously. Measurement 600 for individual treatment conditions was performed in biological replicates (n = 4 – 8) and repeated as 601 independent experiments as stated for the respective experiment (see figure legends). 602 Invasion and Migration Assay (Transwell Assay) 603 Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018 604 [10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. RNA Extraction, cDNA Synthesis, and qPCR Analysis Total RNA was extracted directly from the cell culture dishes using RNeasy Mini Kit (Qiagen, 74104). For 608 qPCR, RNA was converted to cDNA using High-capacity cDNA Reverse Transcription Kit (ThermoFisher, 609 4368814). qPCR was performed from 20 ng cDNA per samples using Fast SYBR™ Green Master Mix 610 Total RNA was extracted directly from the cell culture dishes using RNeasy Mini Kit (Qiagen, 74104). For 608 qPCR, RNA was converted to cDNA using High-capacity cDNA Reverse Transcription Kit (ThermoFisher, 609 4368814). qPCR was performed from 20 ng cDNA per samples using Fast SYBR™ Green Master Mix 610 (ThermoFisher, 4385612). All samples were analyzed in technical duplicates or triplicates. qPCR using Fast 611 SYBR Green was conducted at 95°C for 20 s and then 40 cycles of 95°C for 1 s and 60°C for 20 s. Specificity 612 of the reaction was verified by melt curve analysis. The relative quantification of each mRNA was performed 613 using the comparative Ct method. Experiments were performed using the QuantStudio 5 Real-Time PCR 614 System (Applied Biosciences, ThermoFisher Scientific) and data processing was performed using 615 QuantStudio Design&Analysis v1.5.1 software (Applied Biosciences, ThermoFisher Scientific). All expression 616 data was normalized to two housekeeping controls (GAPDH and CycloA). All human primers used in this study 617 are listed in the table below: 618 Gene Name Sequence Fwd (5’ – 3’) Sequence Rev (5’ – 3’) CDH1 AGTGCCAACTGGACCATTCA TCTTTGACCACCGCTCTCCT CycloA CAGACAAGGTCCCAAAGACA CCATTATGGCGTGTGAAGTC GAPDH CATGAGAAGTATGACAACAGCCT AGTCCTTCCACGATACCAAAGT MMP9 CGCGCTGGGCTTAGATCATT GGTTCAGGGCGAGGACCATA SLUG CTACAGCGAACTGGACACACA TGGAATGGAGCAGCGGTAGT SNAIL CGAAAGGCCTTCAACTGCAAA TGACATCTGAGTGGGTCTGGA VIM CCTTGAACGCAAAGTGGAATC GACATGCTGTTCCTGAATCTGAG ZEB1 AAGAACTGCTGGGAGGATGACA CCTCTTCAGGTGCCTCAGGAAAA ZEB2 GCAAGAGGCGCAAACAAGC GGGTTGGCAATACCGTCATCC 619 shRNA mediated Gene Silencing 620 Gene knockdown with shRNA was performed in MDA-MB468 and 4T1 cells using lentiviral particles 621 expressing two different shRNAs targeting the gene of interest and one non-silencing control vector (pGIPZ- 622 shSCR: TTACTCTCGCCCAAGCGAG (Code: RHS4346). For silencing of the MTHFD2 gene in MDA-MB468 623 cells, pGIPZ shMTHFD2-2: ATTGCATTTCTATTGGCCT (V2LHS_90968) purchased from Horizon Discovery 624 was used. For silencing of the Mthfd1l gene in 4T1 cells, pGIPZ shMTHFD1L-2: TAGATTTCAATTTCATCTG 625 (Code: V2LHS_96542) purchased from Horizon Discovery was used. All plasmids used for generation of 626 lentivirus were first amplified from glycerol stocks, isolated (Machery&Nagel plasmid isolation kit), and used 627 for transfection of HEK293T cells. RNA Extraction, cDNA Synthesis, and qPCR Analysis Survey scans of peptide precursors from 375 682 For H&E staining, snap-frozen lungs were cut in 15 μm sections using Leica Cryostat (Leica Biosystems) and 654 embedded in Tissue-Tek® O.C.T (Sakura). 5 sections in a distance of 150 μm to each other were stained per 655 lung. Staining was performed according to established protocols. Briefly, selected sections were dehydrated 656 with MeOH and stained with Gill 2 hematoxylin. Sections were neutralized with successive washes of tap 657 water, hard water (10g MgSO4 and 0.7g NaHCO3 per L), and distilled water. Subsequently, sections were 658 stained with Eosin-solution and dehydrated through successive washes with 80%, 95%, 100% EtOH, and Xylol 659 prior to mounting. 660 SILAC (Stable Isotope Labeling with Amino acids in Cell culture) strategy was used for the proteomic analysis. 662 In general, MDA-MB468 cells were cultivated in DMEM-F12 SILAC medium supplemented either with Lys0 663 and Arg0 (light channel) or Lys8 and Arg10 (heavy channel). After 6 passages, labeling efficiency of heavy 664 channel was checked using LC-MS/MS. Cells in light channel were treated with 50 nM MTX for 24h and 72h, 665 while heavy channel was used as control. Cell pellets of biological duplicates were collected after the 666 treatment. Proteins were extracted in lysis buffer (50mM ammonium bicarbonate, 6M Urea, 2M Thio-urea, pH 667 8) following a 30 min incubation at 4 °C in the presence of protease inhibitors (cOmplete™ EDTA-free Protease 668 Inhibitor Cocktail, Roche). Following centrifugation at 16,000 g for 10 min, supernatants were taken for protein 669 quantification. Samples from light channel (50 μg protein) were mixed with control heavy channel (50 μg 670 protein) for protein reduction (5 mM DTT, 1 h incubation at 37°C) and alkylation (10 mM IAA, 45min in dark at 671 room temperature). Protein digestion was performed with Lys-C (FUJIFILM Wako, 125-05061) at 1:30 ratio 672 (enzyme/protein substances) for 4 h at 37 °C, then samples were diluted 4 times with 50 mM ammonium 673 bicarbonate and digested overnight with 1 μg of trypsin at 37 °C. Digestion was terminated through addition of 674 formic acid (1% final concentration). Digested peptides were cleaned up with reverse phase Sep-Pak C18 1 675 cc Vac Cartridge (Waters, WAT054955) and eluted with 1 mL 50% ACN. Eluted peptides were dried by 676 Speedvac (Thermo Fisher Scientific) and re-suspended in 0.1% formic acid. Peptide concentration was 677 measured with Nanodrop. RNA Extraction, cDNA Synthesis, and qPCR Analysis 662 In general, MDA-MB468 cells were cultivated in DMEM-F12 SILAC medium supplemented either with Lys0 663 and Arg0 (light channel) or Lys8 and Arg10 (heavy channel). After 6 passages, labeling efficiency of heavy 664 channel was checked using LC-MS/MS. Cells in light channel were treated with 50 nM MTX for 24h and 72h, 665 while heavy channel was used as control. Cell pellets of biological duplicates were collected after the 666 treatment. Proteins were extracted in lysis buffer (50mM ammonium bicarbonate, 6M Urea, 2M Thio-urea, pH 667 8) following a 30 min incubation at 4 °C in the presence of protease inhibitors (cOmplete™ EDTA-free Protease 668 Inhibitor Cocktail, Roche). Following centrifugation at 16,000 g for 10 min, supernatants were taken for protein 669 housed in a specific pathogen free (SPF) facility at a relative humidity of 40–70%, at 22 °C, and in 12 h 643 dark/light cycles. 644 housed in a specific pathogen free (SPF) facility at a relative humidity of 40–70%, at 22 °C, and in 12 h 643 dark/light cycles. 644 Syngeneic 4T1 mammary carcinoma cells were orthotopically implanted into the left mammary fat pads (1 645 injection/mouse) of immune competent female Balb/c mice according to established protocols [50]. Briefly, 646 each injection contained 2000 cells in a mixture of 25 μL PBS and 25 μL matrigel. 9 mice per group were 647 injected with either 4T1 SCR or 4T1 MTHFD1L KD cells. Primary tumor growth was monitored between day 7 648 and day 35 of the experiment. Weight was monitored over time and no weight loss was observed. Experiment 649 was terminated after 6 weeks and lung and liver were prepared for examination of metastatic outgrowth. No 650 metastases were found in the liver. Macroscopic lung metastases were blindly counted under a microscope 651 and microscopic lung metastases were visualized by H&E staining. 652 H&E staining 653 For H&E staining, snap-frozen lungs were cut in 15 μm sections using Leica Cryostat (Leica Biosystems) and 654 embedded in Tissue-Tek® O.C.T (Sakura). 5 sections in a distance of 150 μm to each other were stained per 655 lung. Staining was performed according to established protocols. Briefly, selected sections were dehydrated 656 with MeOH and stained with Gill 2 hematoxylin. Sections were neutralized with successive washes of tap 657 water, hard water (10g MgSO4 and 0.7g NaHCO3 per L), and distilled water. RNA Extraction, cDNA Synthesis, and qPCR Analysis Subsequently, sections were 658 stained with Eosin-solution and dehydrated through successive washes with 80%, 95%, 100% EtOH, and Xylol 659 prior to mounting. 660 Proteomics 661 SILAC (Stable Isotope Labeling with Amino acids in Cell culture) strategy was used for the proteomic analysis. 662 In general, MDA-MB468 cells were cultivated in DMEM-F12 SILAC medium supplemented either with Lys0 663 and Arg0 (light channel) or Lys8 and Arg10 (heavy channel). After 6 passages, labeling efficiency of heavy 664 channel was checked using LC-MS/MS. Cells in light channel were treated with 50 nM MTX for 24h and 72h, 665 while heavy channel was used as control. Cell pellets of biological duplicates were collected after the 666 treatment. Proteins were extracted in lysis buffer (50mM ammonium bicarbonate, 6M Urea, 2M Thio-urea, pH 667 8) following a 30 min incubation at 4 °C in the presence of protease inhibitors (cOmplete™ EDTA-free Protease 668 Inhibitor Cocktail, Roche). Following centrifugation at 16,000 g for 10 min, supernatants were taken for protein 669 quantification. Samples from light channel (50 μg protein) were mixed with control heavy channel (50 μg 670 protein) for protein reduction (5 mM DTT, 1 h incubation at 37°C) and alkylation (10 mM IAA, 45min in dark at 671 room temperature). Protein digestion was performed with Lys-C (FUJIFILM Wako, 125-05061) at 1:30 ratio 672 (enzyme/protein substances) for 4 h at 37 °C, then samples were diluted 4 times with 50 mM ammonium 673 bicarbonate and digested overnight with 1 μg of trypsin at 37 °C. Digestion was terminated through addition of 674 formic acid (1% final concentration). Digested peptides were cleaned up with reverse phase Sep-Pak C18 1 675 cc Vac Cartridge (Waters, WAT054955) and eluted with 1 mL 50% ACN. Eluted peptides were dried by 676 Speedvac (Thermo Fisher Scientific) and re-suspended in 0.1% formic acid. Peptide concentration was 677 measured with Nanodrop. Peptides were measured by LC-MS/MS on Q-Exactive HF mass spectrometer 678 (Thermo Fisher) connected to a Dionex Ultimate 3000 (Thermo Fisher). 500 ng of peptides were loaded onto 679 a trap column (Acclaim PepMap 75 μm x 2 cm, C18, 3 μm) and separated on a 25 cm Acclaim pepmap RSLC 680 column (75 μm x 25 cm, C18, 2 μm) using a 150 min gradient (2% to 90% acetonitrile) with a flow rate of 0.3 681 μL/min. MS data were acquired in data dependent mode (DDA). RNA Extraction, cDNA Synthesis, and qPCR Analysis Lentivirus was produced for 24 h in HEK293T cells co-transfected with the 628 viral core packaging construct pCMVR8.74, the VSV-G envelope protein vector pMD2.G and the respective 629 pGIPZ-shRNA-target plasmid using lipofectamine 3000 (Invitrogen #L3000008) as a transfection reagent. 630 Filtered virus supernatant was used to transduce MDA-MB468 and 4T1 cells in DMEM supplemented with 2% 631 FBS. After incubation at 37°C for 24 h, the virus was removed and cells were cultured in DMEM supplemented 632 with 10 % FBS for 48 h. 96 h post transduction cells were cultured in selection media containing 2 µg/mL of 633 puromycin (Sigma #P8833) for five passages to obtain stably transduced GFP-positive cells. 634 housed in a specific pathogen free (SPF) facility at a relative humidity of 40–70%, at 22 °C, and in 12 h 643 dark/light cycles. 644 Syngeneic 4T1 mammary carcinoma cells were orthotopically implanted into the left mammary fat pads (1 645 injection/mouse) of immune competent female Balb/c mice according to established protocols [50]. Briefly, 646 each injection contained 2000 cells in a mixture of 25 μL PBS and 25 μL matrigel. 9 mice per group were 647 injected with either 4T1 SCR or 4T1 MTHFD1L KD cells. Primary tumor growth was monitored between day 7 648 and day 35 of the experiment. Weight was monitored over time and no weight loss was observed. Experiment 649 was terminated after 6 weeks and lung and liver were prepared for examination of metastatic outgrowth. No 650 metastases were found in the liver. Macroscopic lung metastases were blindly counted under a microscope 651 and microscopic lung metastases were visualized by H&E staining. 652 H&E staining 653 For H&E staining, snap-frozen lungs were cut in 15 μm sections using Leica Cryostat (Leica Biosystems) and 654 embedded in Tissue-Tek® O.C.T (Sakura). 5 sections in a distance of 150 μm to each other were stained per 655 lung. Staining was performed according to established protocols. Briefly, selected sections were dehydrated 656 with MeOH and stained with Gill 2 hematoxylin. Sections were neutralized with successive washes of tap 657 water, hard water (10g MgSO4 and 0.7g NaHCO3 per L), and distilled water. Subsequently, sections were 658 stained with Eosin-solution and dehydrated through successive washes with 80%, 95%, 100% EtOH, and Xylol 659 prior to mounting. 660 Proteomics 661 SILAC (Stable Isotope Labeling with Amino acids in Cell culture) strategy was used for the proteomic analysis. RNA Extraction, cDNA Synthesis, and qPCR Analysis 716 6. Benzarti, M., et al., Metabolic Potential of Cancer Cells in Context of the Metastatic Cascade. Cells, 717 2020. 9(9). 718 7. Elia, I., G. Doglioni, and S.M. Fendt, Metabolic Hallmarks of Metastasis Formation. Trends Cell Biol, 719 2018. 28(8): p. 673-684. 720 8. Faubert, B., A. Solmonson, and R.J. DeBerardinis, Metabolic reprogramming and cancer 721 progression. Science, 2020. 368(6487). 722 9. LeBoeuf, S.E., et al., Activation of Oxidative Stress Response in Cancer Generates a Druggable 723 Dependency on Exogenous Non-essential Amino Acids. Cell Metab, 2020. 31(2): p. 339-350 e4. 724 10. Meiser, J., et al., Increased formate overflow is a hallmark of oxidative cancer. Nat Commun, 2018. 725 9(1): p. 1368. 726 11. Pollari, S., et al., Enhanced serine production by bone metastatic breast cancer cells stimulates 727 osteoclastogenesis. Breast Cancer Res Treat, 2011. 125(2): p. 421-30. 728 12. Rossi, M., et al., Heterogeneity in PHGDH protein expression potentiates cancer cell dissemination 729 and metastasis. bioRxiv, 2021: p. 2021.01.24.427949. 730 Unpaired t-test with Welch’s correction was applied for pairwise comparison (two-sided) using GraphPad 699 Software Vers.8. For normalization, data points of one experiment were either normalized to the untreated 700 control or divided by the global mean of this individual experiment. We define one n as one independent 701 biological experiment (consisting of several wells, e.g. triplicate wells for all stable isotope tracing experiments). 702 The mean of one experiment was considered as one n. The mean values of several independent experiments 703 (as indicated in figure legends) were plotted and used for statistical analysis as indicated. 704 2. DeBerardinis, R.J. and N.S. Chandel, Fundamentals of cancer metabolism. Sci Adv, 2 e1600200. Bergers, G. and S.M. Fendt, The metabolism of cancer cells during metastasis. Nat Rev Cancer 2021. 4. Meiser, J., et al., Serine one-carbon catabolism with formate overflow. Sci Adv, 2016. 2(10): p. 713 e1601273. 714 4. Meiser, J., et al., Serine one-carbon catabolism with formate overflow. Sci Adv, 2016. 2(10): p. 713 e1601273. 714 5. Tibbetts, A.S. and D.R. Appling, Compartmentalization of Mammalian folate-mediated one-carbon 715 metabolism. Annu Rev Nutr, 2010. 30: p. 57-81. 716 5. Tibbetts, A.S. and D.R. Appling, Compartmentalization of Mammalian folate-mediated one-carbon 715 metabolism. Annu Rev Nutr, 2010. 30: p. 57-81. 716 6. Benzarti, M., et al., Metabolic Potential of Cancer Cells in Context of the Metastatic Cascade. Cells, 717 2020. 9(9). 718 6. RNA Extraction, cDNA Synthesis, and qPCR Analysis Peptides were measured by LC-MS/MS on Q-Exactive HF mass spectrometer 678 (Thermo Fisher) connected to a Dionex Ultimate 3000 (Thermo Fisher). 500 ng of peptides were loaded onto 679 a trap column (Acclaim PepMap 75 μm x 2 cm, C18, 3 μm) and separated on a 25 cm Acclaim pepmap RSLC 680 column (75 μm x 25 cm, C18, 2 μm) using a 150 min gradient (2% to 90% acetonitrile) with a flow rate of 0.3 681 μL/min. MS data were acquired in data dependent mode (DDA). Survey scans of peptide precursors from 375 682 to 1500 m/z were performed at 70,000 resolution with a 3×106 ion count target and the top 12 abundant peaks 683 from survey scan were selected for fragmentation. Tandem MS was performed by isolation at 1.4 m/z with the 684 quadrupole, HCD fragmentation with a normalized collision energy of 28. The MS2 ion count target was set to 685 1×105 and the max injection time was 45 ms. Only precursors with a charge state of 2–7 were sampled for 686 MS2. The dynamic exclusion duration was set to 20 s with a 10 ppm mass tolerance around the selected 687 precursor and its isotopes. Each sample was analyzed twice as technical replicates. All raw data was analyzed 688 with MaxQuant (version 1.6.7.0) and searched with Andromeda against the Homo sapiens database from 689 Uniprot. The minimal peptide length was set to 7 amino acids and the maximum of 3 missed cleavages were 690 allowed. The search included variable modifications of methionine oxidation and N-terminal acetylation, 691 deamidation (N and Q) and fixed modification of carbamidomethyl cysteine. The “Match between run” was 692 checked within 1 min retention time window. Mass tolerance for peptide precursor and fragments were set as 693 10 ppm and 20 ppm, respectively. The FDR was set to 0.01 for peptide and protein identifications. SILAC 694 based protein quantification (MaxQuant built-in) was used for quantitative evaluation of identified protein. 695 ProTIGY (https://github.com/broadinstitute/protigy), an R based tool, was used for differential analysis of 696 MaxQuant output. 697 to 1500 m/z were performed at 70,000 resolution with a 3×106 ion count target and the top 12 abundant peaks 683 from survey scan were selected for fragmentation. Tandem MS was performed by isolation at 1.4 m/z with the 684 quadrupole, HCD fragmentation with a normalized collision energy of 28. RNA Extraction, cDNA Synthesis, and qPCR Analysis The MS2 ion count target was set to 685 1×105 and the max injection time was 45 ms. Only precursors with a charge state of 2–7 were sampled for 686 MS2. The dynamic exclusion duration was set to 20 s with a 10 ppm mass tolerance around the selected 687 precursor and its isotopes. Each sample was analyzed twice as technical replicates. All raw data was analyzed 688 with MaxQuant (version 1.6.7.0) and searched with Andromeda against the Homo sapiens database from 689 Uniprot. The minimal peptide length was set to 7 amino acids and the maximum of 3 missed cleavages were 690 allowed. The search included variable modifications of methionine oxidation and N-terminal acetylation, 691 deamidation (N and Q) and fixed modification of carbamidomethyl cysteine. The “Match between run” was 692 checked within 1 min retention time window. Mass tolerance for peptide precursor and fragments were set as 693 10 ppm and 20 ppm, respectively. The FDR was set to 0.01 for peptide and protein identifications. SILAC 694 based protein quantification (MaxQuant built-in) was used for quantitative evaluation of identified protein. 695 ProTIGY (https://github.com/broadinstitute/protigy), an R based tool, was used for differential analysis of 696 MaxQuant output. 697 Statistics 698 Unpaired t-test with Welch’s correction was applied for pairwise comparison (two-sided) using GraphPad 699 Software Vers.8. For normalization, data points of one experiment were either normalized to the untreated 700 control or divided by the global mean of this individual experiment. We define one n as one independent 701 biological experiment (consisting of several wells, e.g. triplicate wells for all stable isotope tracing experiments). 702 The mean of one experiment was considered as one n. The mean values of several independent experiments 703 (as indicated in figure legends) were plotted and used for statistical analysis as indicated. 704 705 References 706 1. Siegel, R.L., K.D. Miller, and A. 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Burgos-Barragan, G., et al., Mammals divert endogenous genotoxic formaldehyde into o 785 metabolism. Nature, 2017. 548(7669): p. 549-554. 786 39. Zheng, Y., et al., Mitochondrial One-Carbon Pathway Supports Cytosolic Folate Integrity 787 Cells. Cell, 2018. 175(6): p. 1546-1560 e17. 788 40. den Boer, E., et al., Determinants of erythrocyte methotrexate polyglutamate levels in rh 789 arthritis. J Rheumatol, 2014. 41(11): p. 2167-78. 790 Samanta, D., et al., PHGDH Expression Is Required for Mitochondrial Redox Homeostasis, Brea Cancer Stem Cell Maintenance, and Lung Metastasis. Cancer Res, 2016. 76(15): p. 4430-42. g ( ) p 14. Ngo, B., et al., Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to 733 PHGDH Inhibition. Cancer Discov, 2020. 10(9): p. 1352-1373. 734 , ( ) p Hryniuk, W., Mechanism of Action of Methotrexate. Proceedings of the American Association fo Cancer Research, 1974. 15(Mar): p. 136-136. ( ) p 16. 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Cardona-Munoz, The Role of Oxidative Stress in 746 Physiopathology and Pharmacological Treatment with Pro- and Antioxidant Properties in Chronic 747 Diseases. Oxid Med Cell Longev, 2020. 2020: p. 2082145. 748 21. Phillips, D.C., K.J. Woollard, and H.R. Griffiths, The anti-inflammatory actions of methotrexate are 749 critically dependent upon the production of reactive oxygen species. Br J Pharmacol, 2003. 138(3): 750 p. 501-11. 751 p 22. Babiak, R.M., et al., Methotrexate: pentose cycle and oxidative stress. RNA Extraction, cDNA Synthesis, and qPCR Analysis Cell Biochem Funct, 1998. 752 16(4): p. 283-93. 753 23. Nguyen, H.L., et al., Oxidative stress and prostate cancer progression are elicited by membrane- 754 type 1 matrix metalloproteinase. Mol Cancer Res, 2011. 9(10): p. 1305-18. 755 24. Rhyu, D.Y., et al., Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein 756 kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. 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RNA Extraction, cDNA Synthesis, and qPCR Analysis Benzarti, M., et al., Metabolic Potential of Cancer Cells in Context of the Metastatic Cascade. Cells, 717 2020. 9(9). 718 7. Elia, I., G. Doglioni, and S.M. Fendt, Metabolic Hallmarks of Metastasis Formation. Trends Cell Biol, 719 2018. 28(8): p. 673-684. 720 7. Elia, I., G. Doglioni, and S.M. Fendt, Metabolic Hallmarks of Metastasis Formation. Trends Cell Biol, 719 2018. 28(8): p. 673-684. 720 8. Faubert, B., A. Solmonson, and R.J. DeBerardinis, Metabolic reprogramming 721 progression. Science, 2020. 368(6487). 722 Faubert, B., A. Solmonson, and R.J. DeBerardinis, Metabolic reprogramming and cancer rogression. Science, 2020. 368(6487). 9. LeBoeuf, S.E., et al., Activation of Oxidative Stress Response in Cancer Generates a Druggable 723 Dependency on Exogenous Non-essential Amino Acids. Cell Metab, 2020. 31(2): p. 339-350 e4. 724 10. Meiser, J., et al., Increased formate overflow is a hallmark of oxidative cancer. 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Tajan, M., et al., Serine synthesis pathway inhibition cooperates with dietary serine and glycine 803 limitation for cancer therapy. Nat Commun, 2021. 12(1): p. 366. 804 47. Hiller, K., et al., MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted 805 GC/MS based metabolome analysis. Anal Chem, 2009. 81(9): p. 3429-39. 806 48. Mak, T.W., et al., Glutathione Primes T Cell Metabolism for Inflammation. Immunity, 2017. 46(6): p. 807 1089-1090. 808 49. Dranka, B.P., et al., Assessing bioenergetic function in response to oxidative stress by metabolic 809 profiling. Free Radic Biol Med, 2011. 51(9): p. 1621-35. 810 50. Kocaturk, B. and H.H. Versteeg, Orthotopic injection of breast cancer cells into the mammary fat pad 811 of mice to study tumor growth. J Vis Exp, 2015(96). 812 813 814 41. Zhu, T., et al., Investigation of Potential Drug-Drug Interactions between Peficitinib (ASP015K) and 791 Methotrexate in Patients with Rheumatoid Arthritis. 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Effect of 797 folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and 798 folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism. J Biol Chem, 799 1993. 268(29): p. 21665-73. 800 44. Lowe, K.E., et al., Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of 797 folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and 798 folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism. J Biol Chem, 799 1993. 268(29): p. 21665-73. 800 ( ) p Horne, D.W., R.S. Holloway, and H.M. Said, Uptake of 5-formyltetrahydrofolate in isolated rat liv mitochondria is carrier-mediated. RNA Extraction, cDNA Synthesis, and qPCR Analysis Morscher, R.J., et al., Mitochondrial translation requires folate-dependent tRN 775 2018. 554(7690): p. 128-132. 776 Morscher, R.J., et al., Mitochondrial translation requires folate-dependent tRNA methylation. Na 2018. 554(7690): p. 128-132. 34. Diehl, F.F., et al., Cellular redox state constrains serine synthesis and nucleotide production to 777 impact cell proliferation. Nat Metab, 2019. 1(9): p. 861-867. 778 p p , ( ) p 35. Palm, W. and C.B. Thompson, Nutrient acquisition strategies of mammalian cells. Nature, 2017. 779 546(7657): p. 234-242. 780 36. Vande Voorde, J., et al., Improving the metabolic fidelity of cancer models with a physiological cell 781 culture medium. Sci Adv, 2019. 5(1): p. eaau7314. 782 37. Ducker, G.S., et al., Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the 783 Mitochondrial Folate Pathway. Cell Metab, 2016. 24(4): p. 640-641. 784 y ( ) p 38. Burgos-Barragan, G., et al., Mammals divert endogenous genotoxic formalde 785 metabolism. Nature, 2017. 548(7669): p. 549-554. 786 y ( ) p Burgos-Barragan, G., et al., Mammals divert endogenous genotoxic formaldehyde into one-carb metabolism. Nature, 2017. 548(7669): p. 549-554. , ( ) p Zheng, Y., et al., Mitochondrial One-Carbon Pathway Supports Cytosolic Folate Integrity in Can Cells. Cell, 2018. 175(6): p. 1546-1560 e17. ( ) p 39. Zheng, Y., et al., Mitochondrial One-Carbon Pathway Supports Cytosolic Fol 787 Cells. Cell, 2018. 175(6): p. 1546-1560 e17. 788 40. den Boer, E., et al., Determinants of erythrocyte methotrexate polyglutamate levels in rheumatoid 789 arthritis. J Rheumatol, 2014. 41(11): p. 2167-78. 790 40. den Boer, E., et al., Determinants of erythrocyte methotrexate polyglutamate levels in rheumatoid 789 arthritis. J Rheumatol, 2014. 41(11): p. 2167-78. 790 41. Zhu, T., et al., Investigation of Potential Drug-Drug Interactions between Peficitinib (ASP015K) and 791 Methotrexate in Patients with Rheumatoid Arthritis. Clin Drug Investig, 2020. 40(9): p. 827-838. 792 42. Garcia-Martinez, L.F. and D.R. Appling, Characterization of the folate-dependent mitochondrial 793 oxidation of carbon 3 of serine. Biochemistry, 1993. 32(17): p. 4671-6. 794 43. Lawrence, S.A., et al., Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase 795 maintain the subcellular compartmentalization of folates. J Biol Chem, 2014. 289(42): p. 29386-96. 796 44. Lowe, K.E., et al., Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of 797 folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and 798 folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism. J Biol Chem, 799 1993. 268(29): p. 21665-73. 800 45. Horne, D.W., R.S. g p 50. Kocaturk, B. and H.H. Versteeg, Orthotopic injection of breast cancer cells into the mammary fat pad 811 of mice to study tumor growth. J Vis Exp, 2015(96). 812 813 RNA Extraction, cDNA Synthesis, and qPCR Analysis J Nutr, 1992. 122(11): p. 2204-9. 45. Horne, D.W., R.S. Holloway, and H.M. Said, Uptake of 5-formyltetrahydrofolate in isolated rat liver 801 mitochondria is carrier-mediated. J Nutr, 1992. 122(11): p. 2204-9. 802 46 T j M t l S i th i th i hibiti t ith di t i d l i 803 46. Tajan, M., et al., Serine synthesis pathway inhibition cooperates with dietary serine an limitation for cancer therapy. Nat Commun, 2021. 12(1): p. 366. py ( ) p Hiller, K., et al., MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal Chem, 2009. 81(9): p. 3429-39. 47. Hiller, K., et al., MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted 805 GC/MS based metabolome analysis. Anal Chem, 2009. 81(9): p. 3429-39. 806 48. Mak, T.W., et al., Glutathione Primes T Cell Metabolism for Inflammation. Immunity, 2017. 46(6): p. 807 1089-1090. 808 49. Dranka, B.P., et al., Assessing bioenergetic function in response to oxidative stress by metabolic 809 profiling. Free Radic Biol Med, 2011. 51(9): p. 1621-35. 810 49. Dranka, B.P., et al., Assessing bioenergetic function in response to oxidative stress by metabolic 809 profiling. Free Radic Biol Med, 2011. 51(9): p. 1621-35. 810 p g ( ) p 50. Kocaturk, B. and H.H. Versteeg, Orthotopic injection of breast cancer cells into the mammary fat pad 811 of mice to study tumor growth. J Vis Exp, 2015(96). 812 813 50. Kocaturk, B. and H.H. Versteeg, Orthotopic injection of breast cancer cells into the mammary fat pad 811 of mice to study tumor growth. J Vis Exp, 2015(96). 812 Figure 1: Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells. (A) Schematic depicting synthesis routes of central metabolic building blocks (lipids, nucleotides, and proteins) and pharmacologic intervention points of the selected drug panel. (B) MDA-MB468 cells were treated with 100 nM Sirolimus (SIR), 1 μM Simvastatin (SIM), 50 nM Rotenone (Rot), galactose (Gal) supplementation, 50 nM Methotrexate (MTX), 1 μM Pemetrexed (PEM), 0.5 mM hydroxyurea (HU), and 100 nM Clofarabine (CLO) for 48 h. Viable cell number was determined by trypan blue staining. Each dot represents an independent experiment; mean ± SD; ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Comparisons to control apart from the labeled are all significant with p<0.0001. (C) MDA-MB468 cells were treated for 48 h as in (B). RNA Extraction, cDNA Synthesis, and qPCR Analysis Cell death induction was assessed by flow cytometric analysis of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3 - 8). 2way ANOVA with Dunnett’s multiple comparisons test. (D) Migration of MDA-MB468 cells in response to 50 nM Rot, Gal, 1 μM SIM, and 100 nM SIR and respective area under curve (AUC); mean ± SEM (n = 3 - 9); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (E) Migration of MDA-MB468 cells in response to 50 nM MTX, 1 μM PEM, 0.5 mM HU, and 100 nM CLO and respective AUC; mean ± SEM (n = 3 - 12); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (F) Correlation of cell migration assessed as AUC over 40h and proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468 cells were treated as in (B) and (D). (G) Correlation of cell migration assessed as AUC over 40h and proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468 cells were treated as in (B) and (E). (H) Migration and invasion of MDA-MB468 cells treated for 24 h with 50 nM MTX was assessed using non-coated (migration) or ECM-Collagen-coated (invasion) Boyden chambers. Each dot represents an independent experiment; mean ± SEM; Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (I) Proliferation of MTX-resistant MDA- MB468 cells upon the indicated concentrations of MTX [nM] measured as cell density; mean ± SEM (n = 4). (J) Migration of parental and MTX-resistant MDA-MB468 cells and respective AUC; mean ± SEM (n = 6 - 15); unpaired t-test with Welch’s correction. For comparison, 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 815 Figure 1: Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells. (A) Schematic 816 depicting synthesis routes of central metabolic building blocks (lipids, nucleotides, and proteins) and pharmacologic intervention points of 817 the selected drug panel. (B) MDA-MB468 cells were treated with 100 nM Sirolimus (SIR), 1 μM Simvastatin (SIM), 50 nM Rotenone (Rot), 818 galactose (Gal) supplementation, 50 nM Methotrexate (MTX), 1 μM Pemetrexed (PEM), 0.5 mM hydroxyurea (HU), and 100 nM 819 Clofarabine (CLO) for 48 h. Viable cell number was determined by trypan blue staining. RNA Extraction, cDNA Synthesis, and qPCR Analysis Graph shows mean ± SEM of five independent experiments each measured in triplicate wells. (C) Quantitative proteomics analysis of SILAC-labeled MDA-MB468 cells treated with and without 50 nM MTX for 72h. The right side of the volcano plot shows the upregulated proteins after MTX treatment (red), while downregulated ones on the left side (blue). Differential expressions were defined as absolute log2 Fold change ≥ 1 with adjusted p- value ≤ 0.05. (D) Mitochondrial ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of MitoSOX mean fluorescence intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (E) Intracellular ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of DCF-DA mean fluorescence intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (F) mRNA expression from the indicated target genes in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. No data shown for SLUG expression at 72 h. (G) mRNA expression from the indicated target genes in MTX-resistant MDA-MB468 cells relative to parental MDA-MB468 cells as measured using real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. (H) Expression of vimentin in MDA-MB468 cells after 240 h 50 nM MTX treatment; β-actin serves as loading control. Quantification of vimentin signal intensity relative to total protein stain. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (I) Expression of vimentin in parental d MTX i t t MDA MB468 ll β ti l di t l Q tifi ti f i ti i l i t it l ti t t t l t i 838 Figure 2: MTX treatment triggers a ROS-driven EMT phenotype. (A) Morphological changes of MDA-MB468 cells in response to 48 h 839 50 nM MTX. Bright-field images are representative of independent experiments. Scale bars correspond to 60 and 300 μm. (B) MID of 840 intracellular ATP upon [U-13C]serine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Graph shows mean ± SEM of five 841 independent experiments each measured in triplicate wells. (C) Quantitative proteomics analysis of SILAC-labeled MDA-MB468 cells 842 treated with and without 50 nM MTX for 72h. RNA Extraction, cDNA Synthesis, and qPCR Analysis Each dot represents an independent experiment; 820 mean ± SD; ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Comparisons to control apart from the labeled are all 821 significant with p<0.0001. (C) MDA-MB468 cells were treated for 48 h as in (B). Cell death induction was assessed by flow cytometric 822 analysis of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3 - 8). 2way ANOVA with Dunnett’s multiple comparisons test. (D) Migration 823 of MDA-MB468 cells in response to 50 nM Rot, Gal, 1 μM SIM, and 100 nM SIR and respective area under curve (AUC); mean ± SEM (n 824 = 3 - 9); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (E) Migration of MDA-MB468 cells in 825 response to 50 nM MTX, 1 μM PEM, 0.5 mM HU, and 100 nM CLO and respective AUC; mean ± SEM (n = 3 - 12); Brown-Forsythe and 826 Welch one-way ANOVA with Dunnett’s multiple comparisons test. (F) Correlation of cell migration assessed as AUC over 40h and 827 proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468 cells were treated as in (B) and (D). (G) Correlation 828 of cell migration assessed as AUC over 40h and proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468 829 cells were treated as in (B) and (E). (H) Migration and invasion of MDA-MB468 cells treated for 24 h with 50 nM MTX was assessed using 830 non-coated (migration) or ECM-Collagen-coated (invasion) Boyden chambers. Each dot represents an independent experiment; mean ± 831 SEM; Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (I) Proliferation of MTX-resistant MDA- 832 MB468 cells upon the indicated concentrations of MTX [nM] measured as cell density; mean ± SEM (n = 4). (J) Migration of parental and 833 MTX-resistant MDA-MB468 cells and respective AUC; mean ± SEM (n = 6 - 15); unpaired t-test with Welch’s correction. For comparison, 834 the time point of 50% wound closure is indicated for both cell lines. 835 836 Figure 2: MTX treatment triggers a ROS-driven EMT phenotype. (A) Morphological changes of MDA-MB468 cells in response to 48 h 50 nM MTX. Bright-field images are representative of independent experiments. Scale bars correspond to 60 and 300 μm. (B) MID of intracellular ATP upon [U-13C]serine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. RNA Extraction, cDNA Synthesis, and qPCR Analysis Graph shows mean ± SEM (n = 3); Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple 862 comparisons test. (M) mRNA expression from the indicated target genes in MDA-MB468 cells in response to 10 mM NAC and 50 nM 863 MTX at 72 h as measured using real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. 864 865 866 MMP9 in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time RT-qPCR. Each dot 857 represents an independent experiment; mean ± SEM. (K) Intracellular ROS levels in response to 24 h 50 nM MTX and 10 mM NAC as 858 measured by flow cytometric quantification of DCF-DA mean fluorescence intensity. Each dot represents an independent experiment. 859 Graph shows mean ± SD; Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple comparisons test. (L) Migratory potential 860 of MDA-MB468 cells in response to 10 mM NAC and 50 nM MTX treatment as time-dependent quantification of relative wound density in 861 IncuCyte and respective AUC. Graph shows mean ± SEM (n = 3); Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple 862 comparisons test. (M) mRNA expression from the indicated target genes in MDA-MB468 cells in response to 10 mM NAC and 50 nM 863 MTX at 72 h as measured using real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. 864 865 866 857 858 859 860 861 862 863 864 865 866 Figure 3: MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis. (A) Schematic depicting interdependence of glycolysis and tricarboxylic acid (TCA) cycle as well as metabolic 13C label pattern from [U-13C]glutamine tracer. (B) Absolute consumption and release (CORE) rates of glutamine and glutamate in the culture medium from MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (C) Enrichment of representative isotopomers of glutamine (Gln), glutamate (Glut), α-ketoglutarate (αKG), fumarate (Fum), malate (Mal), aspartate (Asp), and citrate (Cit) upon [U-13C]glutamine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (D) Schematic depicting interdependence of glycolysis and TCA cycle as well as expected metabolic 13C label pattern from [U-13C]glucose tracer. RNA Extraction, cDNA Synthesis, and qPCR Analysis (E) Absolute CORE rates of lactate and glucose in the culture medium of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (F) Enrichment of representative isotopologues of pyruvate (Pyr), Cit, αKG, Fum, Mal, and Asp upon [U- 13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment with triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (G) Basal cellular respiration in response to 24 h 50 nM MTX treatment was determined in MDA-MB468, LN229, and 4T1 cells as quantification of mitochondrial oxygen consumption rate (OCR). Each dot represents an individual experiment composed of six technical replicates; mean ± SEM. (H) Enrichment of M+3 isotopologue of serine upon [U- 13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (I) Ratio of absolute intracellular NAD+ to NADH in MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (J) M+1 isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon 24 h treatment with 50 nM MTX. Each dot indicates an independent experiment measured in triplicate wells; mean ± SEM. 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 Figure 3: MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis. (A) Schematic depicting interdependence of glycolysis and tricarboxylic acid (TCA) cycle as well as metabolic 13C label pattern from [U-13C]glutamine tracer. (B) Absolute consumption and release (CORE) rates of glutamine and glutamate in the culture medium from MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (C) Enrichment of representative isotopomers of glutamine (Gln), glutamate (Glut), α-ketoglutarate (αKG), fumarate (Fum), malate (Mal), aspartate (Asp), and citrate (Cit) upon [U-13C]glutamine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (D) Schematic depicting interdependence of glycolysis and TCA cycle as well as expected metabolic 13C label pattern from [U-13C]glucose tracer. RNA Extraction, cDNA Synthesis, and qPCR Analysis The right side of the volcano plot shows the upregulated proteins after MTX treatment (red), 843 while downregulated ones on the left side (blue). Differential expressions were defined as absolute log2 Fold change ≥ 1 with adjusted p- 844 value ≤ 0.05. (D) Mitochondrial ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of MitoSOX mean 845 fluorescence intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (E) 846 Intracellular ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of DCF-DA mean fluorescence 847 intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (F) mRNA expression 848 from the indicated target genes in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time 849 RT-qPCR. Each dot represents an independent experiment; mean ± SEM. No data shown for SLUG expression at 72 h. (G) mRNA 850 expression from the indicated target genes in MTX-resistant MDA-MB468 cells relative to parental MDA-MB468 cells as measured using 851 real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. (H) Expression of vimentin in MDA-MB468 cells after 852 240 h 50 nM MTX treatment; β-actin serves as loading control. Quantification of vimentin signal intensity relative to total protein stain. 853 Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (I) Expression of vimentin in parental 854 and MTX-resistant MDA-MB468 cells; β-actin serves as loading control. Quantification of vimentin signal intensity relative to total protein 855 stain. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (J) mRNA expression from 856 MMP9 in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time RT-qPCR. Each dot 857 represents an independent experiment; mean ± SEM. (K) Intracellular ROS levels in response to 24 h 50 nM MTX and 10 mM NAC as 858 measured by flow cytometric quantification of DCF-DA mean fluorescence intensity. Each dot represents an independent experiment. 859 Graph shows mean ± SD; Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple comparisons test. (L) Migratory potential 860 of MDA-MB468 cells in response to 10 mM NAC and 50 nM MTX treatment as time-dependent quantification of relative wound density in 861 IncuCyte and respective AUC. RNA Extraction, cDNA Synthesis, and qPCR Analysis (E) Absolute CORE rates of lactate and glucose in the culture medium of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (F) Enrichment of representative isotopologues of pyruvate (Pyr), Cit, αKG, Fum, Mal, and Asp upon [U- 13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment with triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (G) Basal cellular respiration in response to 24 h 50 nM MTX treatment was determined in MDA-MB468, LN229, and 4T1 cells as quantification of mitochondrial oxygen consumption rate (OCR). Each dot represents an individual experiment composed of six technical replicates; mean ± SEM. (H) Enrichment of M+3 isotopologue of serine upon [U- 13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (I) Ratio of absolute intracellular NAD+ to NADH in MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (J) M+1 isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon 24 h treatment with 50 nM MTX. Each dot indicates an independent experiment measured in triplicate wells; mean ± SEM. 88 Figure 4: Mitochondria protect SOC-dependent serine catabolism in the presence of MTX. (A) Schematic of the cytosolic and 89 mitochondrial compartments of SOC and the pharmacologic intervention points of MTX. (B) Absolute CORE rates of serine and glycine 90 of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents the mean of an individual experiment each measured in 91 triplicate wells; mean ± SEM. (C) MID of extracellular formate upon [U-13C]serine tracer in MDA-MB468 cells in response to 24 h 50 nM 92 MTX. Graph shows mean ± SEM of three independent experiments each measured in triplicate wells; unpaired t-test with Welch’s 93 correction for M+1 isotopologue enrichment. (D) MID of intracellular serine upon [U-13C]serine tracer in MDA-MB468 cells in response to 94 24 h 50 nM MTX; mean ± SEM of four independent experiments each measured in triplicate wells. (E) Ratio of absolute intracellular serine 95 and glycine in response to 24 h 50 nM MTX in MDA-MB468 cells. RNA Extraction, cDNA Synthesis, and qPCR Analysis Each dot represents an independent experiment in triplicate wells; mean 96 ± SEM. (F) MID of intracellular serine upon [U-13C]serine tracer in SHMT1, MFT, SHMT2, and FPGS-depleted HAP1 cells; mean ± SEM 97 of independent experiments each measured in triplicate wells (n = 2 – 8). (G) Ratio of absolute intracellular serine and glycine levels in 98 SHMT1, MFT, SHMT2, and FPGS depleted HAP1 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM. 99 00 8 Figure 4: Mitochondria protect SOC-dependent serine catabolism in the presence of MTX. (A) Schematic of the cytosolic and 9 mitochondrial compartments of SOC and the pharmacologic intervention points of MTX. (B) Absolute CORE rates of serine and glycine 0 of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents the mean of an individual experiment each measured in triplicate wells; mean ± SEM. (C) MID of extracellular formate upon [U-13C]serine tracer in MDA-MB468 cells in response to 24 h 50 nM 2 MTX. Graph shows mean ± SEM of three independent experiments each measured in triplicate wells; unpaired t-test with Welch’s 3 correction for M+1 isotopologue enrichment. (D) MID of intracellular serine upon [U-13C]serine tracer in MDA-MB468 cells in response to 4 24 h 50 nM MTX; mean ± SEM of four independent experiments each measured in triplicate wells. (E) Ratio of absolute intracellular serine 5 and glycine in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate wells; mean 6 ± SEM. (F) MID of intracellular serine upon [U-13C]serine tracer in SHMT1, MFT, SHMT2, and FPGS-depleted HAP1 cells; mean ± SEM 7 of independent experiments each measured in triplicate wells (n = 2 – 8). (G) Ratio of absolute intracellular serine and glycine levels in 8 SHMT1, MFT, SHMT2, and FPGS depleted HAP1 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM. 9 0 Figure 5: Targeting de novo serine synthesis decreases MTX-dependent cancer cell migration. (A) MID of intracellular serine upon [U-13C]glucose tracer in MDA-MB468 cells in response to 24 h treatment with 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, 15 μM BI-4916, and 50 nM MTX as indicated; mean ± SEM of independent experiments each measured in triplicate wells (n = 1 – 9). RNA Extraction, cDNA Synthesis, and qPCR Analysis (B) M+1 isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon treatment as in (A). Each dot indicates an independent experiment measured in triplicate wells; mean ± SEM. (C) Basal cellular respiration in response to 24 h 10 μM WQ-2101 10 μM NCT-502, 30 μM CBR-5884, and 15 μM BI-4916 treatment was determined in MDA-MB468 cells as quantification of mitochondria OCR. Graph shows fold OCR compared to untreated control. Each dot represents an individual experiment composed of six technic a replicates; mean ± SEM. (D) M+2 isotopologues of indicated TCA-metabolites in MDA-MB468 cells using [U-13C]glucose tracer upon treatment as in (B). Each dot indicates an independent experiment measured in triplicate wells; mean ± SEM. (E) Migration of MDA- MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective AUC; mean ± SEM (n = 5); Brown-Forsythe and Welch one- way ANOVA with Dunnett’s multiple comparisons test. (F) Migration of MDA-MB468 cells in response to 50 nM MTX, 15 μM BI-4916, and serine- and glycine starvation and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (G) mRNA expression of the indicated target genes in MDA-MB468 cells in response to 72 h 50 nM MTX, 15 μM BI-4916, and serine- and glycine starvation as measured using real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. (H) MDA-MB468 cells were treated for 48 h with 50 nM MTX and 15 μM BI-4916 under serine- and glycine starvation. Cel death induction was assessed by flow cytometry of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3). 2way ANOVA with Dunnett’s multiple comparisons test. (I) Migration of MTX-resistant MDA-MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (J) Schematic depiction of the cytosolic and mitochondrial compartments of SOC and the pharmacologic intervention points of MTX and inhibitors of PHGDH. 901 Figure 5: Targeting de novo serine synthesis decreases MTX-dependent cancer cell migration. (A) MID of intracellular serine upon 902 [U-13C]glucose tracer in MDA-MB468 cells in response to 24 h treatment with 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, 15 903 μM BI-4916, and 50 nM MTX as indicated; mean ± SEM of independent experiments each measured in triplicate wells (n = 1 – 9). RNA Extraction, cDNA Synthesis, and qPCR Analysis (B) 904 M+1 isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon treatment as in (A). Each dot indicates 905 an independent experiment measured in triplicate wells; mean ± SEM. (C) Basal cellular respiration in response to 24 h 10 μM WQ-2101, 906 10 μM NCT-502, 30 μM CBR-5884, and 15 μM BI-4916 treatment was determined in MDA-MB468 cells as quantification of mitochondrial 907 OCR. Graph shows fold OCR compared to untreated control. Each dot represents an individual experiment composed of six technic al 908 replicates; mean ± SEM. (D) M+2 isotopologues of indicated TCA-metabolites in MDA-MB468 cells using [U-13C]glucose tracer upon 909 treatment as in (B). Each dot indicates an independent experiment measured in triplicate wells; mean ± SEM. (E) Migration of MDA- 910 MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective AUC; mean ± SEM (n = 5); Brown-Forsythe and Welch one- 911 way ANOVA with Dunnett’s multiple comparisons test. (F) Migration of MDA-MB468 cells in response to 50 nM MTX, 15 μM BI-4916, and 912 serine- and glycine starvation and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s 913 multiple comparisons test. (G) mRNA expression of the indicated target genes in MDA-MB468 cells in response to 72 h 50 nM MTX, 15 914 μM BI-4916, and serine- and glycine starvation as measured using real-time RT-qPCR. Each dot represents an independent experiment; 915 mean ± SEM. (H) MDA-MB468 cells were treated for 48 h with 50 nM MTX and 15 μM BI-4916 under serine- and glycine starvation. Cell 916 death induction was assessed by flow cytometry of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3). 2way ANOVA with Dunnett’s 917 multiple comparisons test. (I) Migration of MTX-resistant MDA-MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective 918 AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (J) Schematic depiction 919 of the cytosolic and mitochondrial compartments of SOC and the pharmacologic intervention points of MTX and inhibitors of PHGDH. 920 21 Figure 6: Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility. (A) Absolute formate CORE rates of 22 MDA-MB468 cells in response to MTHFD2 knockdown and treatment for 24 h with 50 nM MTX. RNA Extraction, cDNA Synthesis, and qPCR Analysis Each dot indicates an independent 23 experiment measured in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (B) Proliferation of NucLight Rapid Red 24 stained MDA-MB468 cells harboring a MTHFD2 knockdown in response to 50 nM MTX; mean ± SEM (n = 6). (C) Migration of MDA- 25 MB468 cells in response to MTHFD2 knockdown and 50 nM MTX and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch 26 one-way ANOVA with Dunnett’s multiple comparisons test. (D) MDA-MB468 cells carrying a MTHFD2 knockdown were treated for 48 h 27 with 50 nM MTX. Cell death induction was assessed by flow cytometry of AnnexinV-APC/PI-stained cells; mean ± SD (n = 4). 2way 28 ANOVA with Dunnett’s multiple comparisons test. 29 921 922 923 924 925 926 927 928 929 921 Figure 6: Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility. (A) Absolute formate CORE rates of 922 MDA-MB468 cells in response to MTHFD2 knockdown and treatment for 24 h with 50 nM MTX. Each dot indicates an independent 923 experiment measured in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (B) Proliferation of NucLight Rapid Red 924 stained MDA-MB468 cells harboring a MTHFD2 knockdown in response to 50 nM MTX; mean ± SEM (n = 6). (C) Migration of MDA- 925 MB468 cells in response to MTHFD2 knockdown and 50 nM MTX and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch 926 one-way ANOVA with Dunnett’s multiple comparisons test. (D) MDA-MB468 cells carrying a MTHFD2 knockdown were treated for 48 h 927 with 50 nM MTX. Cell death induction was assessed by flow cytometry of AnnexinV-APC/PI-stained cells; mean ± SD (n = 4). 2way 928 ANOVA with Dunnett’s multiple comparisons test. 929 921 Figure 6: Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility. (A) Absolute formate CORE rates of 922 MDA-MB468 cells in response to MTHFD2 knockdown and treatment for 24 h with 50 nM MTX. Each dot indicates an independent 923 experiment measured in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (B) Proliferation of NucLight Rapid Red 924 stained MDA-MB468 cells harboring a MTHFD2 knockdown in response to 50 nM MTX; mean ± SEM (n = 6). RNA Extraction, cDNA Synthesis, and qPCR Analysis (C) Migration of MDA- 925 MB468 cells in response to MTHFD2 knockdown and 50 nM MTX and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch 926 one-way ANOVA with Dunnett’s multiple comparisons test. (D) MDA-MB468 cells carrying a MTHFD2 knockdown were treated for 48 h 927 with 50 nM MTX. Cell death induction was assessed by flow cytometry of AnnexinV-APC/PI-stained cells; mean ± SD (n = 4). 2way 928 ANOVA with Dunnett’s multiple comparisons test. 929 0 Figure 7: Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo. (A) Experimental layout: 4T1 breast cancer 1 cells transfected with non-targeting control (SCR, n=9) and 4T1 cells with knockdown of Mthfd1l (n=9) were injected into the mammary 2 fat pads of immunocompetent female Balb/c mice (1 injection/mouse in the left mammary fat pad). Primary tumor growth was monitored 3 over time and lung metastases formation was evaluated at the end-point. (B) Primary tumor size was measured at the indicated time 4 points and mean tumor volume was calculated. Data are presented as tumor volume ± SEM at each time point following injection with 5 4T1 SCR or Mthfd1l KD cells. (C) Primary tumor weight was measured at the end-point. Each dot indicates an independent animal; mean 6 ± SEM; unpaired t-test with Welch’s correction. (D) Lung tissue was stained with H&E stain. Representative pictures show microscopic 7 lung metastasis in mice after orthotopic injection of 4T1 SCR and Mthfd1l KD cells. Arrows indicate metastatic nodules. Scale bar 8 corresponds to 100 μm. (E) Macroscopic lung metastases were counted and number of metastases per lung is depicted. Each dot 9 indicates an independent animal; mean ± SEM; unpaired t-test with Welch’s correction. 0 1 Figure 7: Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo. (A) Experimental layout: 4T1 breast cancer cells transfected with non-targeting control (SCR, n=9) and 4T1 cells with knockdown of Mthfd1l (n=9) were injected into the mammary fat pads of immunocompetent female Balb/c mice (1 injection/mouse in the left mammary fat pad). Primary tumor growth was monitored over time and lung metastases formation was evaluated at the end-point. (B) Primary tumor size was measured at the indicated time points and mean tumor volume was calculated. Data are presented as tumor volume ± SEM at each time point following injection with 4T1 SCR or Mthfd1l KD cells. (C) Primary tumor weight was measured at the end-point. Each dot indicates an independent animal; mean ± SEM; unpaired t-test with Welch’s correction. (D) Lung tissue was stained with H&E stain. Representative pictures show microscopic lung metastasis in mice after orthotopic injection of 4T1 SCR and Mthfd1l KD cells. Arrows indicate metastatic nodules. Scale bar corresponds to 100 μm. (E) Macroscopic lung metastases were counted and number of metastases per lung is depicted. Each dot indicates an independent animal; mean ± SEM; unpaired t-test with Welch’s correction. Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementalFiguresKiweleretal.pdf
https://openalex.org/W2918520968
https://journal.umy.ac.id/index.php/jiwp/article/download/5832/4062
Indonesian
null
GERAKAN ISLAM INDONESIA: MENGURAI BELENGGU, MEMBANGUN PERADABAN
Journal of Islamic World and Politics
2,018
cc-by-sa
5,811
GERAKAN ISLAM INDONESIA: MENGURAI BELENGGU, MEMBANGUN PERADABAN* Mohammad Syifa A. Widigdo Magister Studi Islam Universitas Muhammadiyah Yogyakarta Email: syifamin@gmail.com Abstrak “Gerakan Islam Indonesia (GII) pada umumnya tidak mempunyai pengaruh, kontribusi, dan peran global yang berarti meskipun kiprah dan kontribusinya di tingkat nasional tidak perlu disangsikan lagi. Kalaupun ada bagian dari GII yang mempunyai jaringan internasional, mereka berada dalam posisi inferior karena hanya memosisikan dirinya sebagai cabang, proksi, atau simpatisan dari organisasi Islam internasional. Akibatnya, GII tidak saja hanya menjadi gerakan lokal atau gerakan proksi organisasi transnasional, tapi juga tidak sanggup menghadapi tantangan global yang semakin kompleks. Dalam artikel ini, saya berusaha memetakan kelemahan dan keterbatasan GII tersebut dan menyajikan beberapa tawaran jalan keluarnya. Salah tawaran solusinya adalah bahwa Gerakan Islam se-Asia Tengara penting untuk berkolaborasi dan bergerak secara aktif sehingga Islam di Asia Tenggara tidak lagi dianggap sebagai pinggiran. Sebab, Gerakan Islam di Asia Tenggara mempunyai cukup potensi dan modal untuk menjadi salah satu pusat peradaban Islam di dunia.” Kata Kunci: Gerakan Islam, Jaringan Internasional, Asia Tenggara, dan Tantangan Global Tantangan Global Abstract Indonesian Islamic Movementsgenerally do not show a global influence and contribution although in a national level its crucial role and contribution are undeniable. Some of these movements may have an international network  Sebagian (diskusi tentang tantangan dan belenggu Gerakan Islam Indonesia) dari artikel ini pernah dipublikasikan di Harian Umum Republika dengan judul “Peran Global Gerakan Islam Indonesia” pada tanggal 10 Februari 2016. Artikel ini merupakan versi yang lengkap dan mendalam, termasuk diskusi tentang solusi untuk menaklukkan tantangan dan keluar dari belenggu, yang penulis olah dari dari hasil penelitian terhadap Gerakan Islam Indonesia. Vol.2. No.2 July-December 2018 ISSN: 2614-0535 E-ISSN: 2655-1330 Mohammad Syifa A. Widigdo 387 y g Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban but their power and authority are limited since their position is merely as a branch, proxy, or sympathizer of a certain transnational Islamic organization. As a result, these local and proxy movements are unable to cope with global challenges, which are increasingly complex. In this article, I attempt to map weaknesses and limitations of Indonesian Islamic Movements and offer some altenative solutions to cope with such weaknesses and limitations. One of the altenative solutions is for Indonesian Islamic Movements to operate and collaborate more actively with other Islamic movements in Southeast Asia. In this way, Islamic movements in Indonesia in particular, and in Southeast Asia in general, are not deemed peripheral. They have sufficient potentials and capabilities to be one of the centers of Islamic civilization. Key words: Islamic Movement, International networking, Southeast Asia, and Global Challenge September 2013; Chaq, Mei 2013). Tidak mengherankan jika kemudian gerakan Islam diidentikkan dengan gerakan politik yang bertujuan merubah tatanan rezim di tingkat nasional maupun global(Lihat Basyir, November 2016, pp. 339- 362) .Sementara kegiatan dan perjuangan organisasi Islam yang menggunakan cara-cara transformatif dan damai seringkali tidak mendapatkan label sebagai “Gerakan Islam.”Istilah yang dipakai untuk menggambarkan gerakan ini, misalnya, adalah “organisasi September 2013; Chaq, Mei 2013). Tidak mengherankan jika kemudian gerakan Islam diidentikkan dengan gerakan politik yang bertujuan merubah tatanan rezim di tingkat nasional maupun global(Lihat Basyir, November 2016, pp. 339- 362) .Sementara kegiatan dan perjuangan organisasi Islam yang menggunakan cara-cara transformatif dan damai seringkali tidak mendapatkan label sebagai “Gerakan Islam.”Istilah yang dipakai untuk menggambarkan gerakan ini, misalnya, adalah “organisasi 3 Untuk mempelajari lebih lanjut perbedaan antara tajdīd, iṣlāḥ, dan iḥyā’, silahkan baca Abū Khalil, As‟ad and Mahmoud Haddad. “Revival and Renewal.” In The Oxford Encyclopedia of the Islamic World. Oxford Islamic Studies Online, http://www. oxfordislamicstudies.com/article/opr/ t236/e0682 (accessed Apr 30, 2013). PENDAHULUAN Istilah “Gerakan Islam” belakangan cenderung mengalami peyorasi karena seringkali diasosiasikan dengan gerakan-gerakan yang berorientasi politik, radikal, at au meng ha l a l kanc ara - c ara kekerasan untuk mencapai tujuan politik.Peyorasi ini tampak dalam beberapa kajian dan penelitian membahas gerakan Islam, biasanya dikaitkan dengan varian radikal dari gerakan tersebut (Lihat tulisan Ahyar, Mei 2015 pp. 1-26; Ar, 8 Islamic World and Politics Vol.2. No.2 July-December 2018 88 Islamic World and Politics Vol.2. No.2 July-December 2018 388 Islam”(Lihat misalnya Collins, Oct., 2004). Padahalistilah “gerakan” dalam konteks sejarah Islam mengacu pada dua konsep, yakni pembaharuan (tajdīd) dan reformasi (iṣlāḥ), yang meniscayakan makna perubahandan transformasi secara damai dan beradab.3Misalnya, gerakan pembaharuan (tajdīd) Muhammad Abduh (w. 1905) di Mesir di bidang pemikiran melalui jurnal “Al-Urwat al- Wuthqā” dan reformasi (iṣlāḥ) di bidang pendidikan dengan memperkenalkan bidang studi sains di kurikulum Universitas al-Azhar(Hourani, 1983, pp. 130-192).Oleh sebab itu, dalam artikel ini, saya menggunakan istilah “Gerakan Islam” dalam pengertian yang luas, tidak hanya untuk gerakan yang berorientasi politik dan radikal, tapi juga (bahkan utamanya) merujuk pada organisasi dan gerakan keagamaan berorientasi sosial, intelektual, atau kultural, seperti NU (Nahdlatul Ulama), Muhammadiyah, Persis (Persatuan Islam), Hidayatullah, HMI (Himpunan Mahasiswa Islam), Islam”(Lihat misalnya Collins, Oct., 2004). Padahalistilah “gerakan” dalam konteks sejarah Islam mengacu pada dua konsep, yakni pembaharuan (tajdīd) dan reformasi (iṣlāḥ), yang meniscayakan makna perubahandan transformasi secara damai dan beradab.3Misalnya, gerakan pembaharuan (tajdīd) Muhammad Abduh (w. 1905) di Mesir di bidang pemikiran melalui jurnal “Al-Urwat al- Wuthqā” dan reformasi (iṣlāḥ) di bidang pendidikan dengan memperkenalkan bidang studi sains di kurikulum Universitas al-Azhar(Hourani, 1983, pp. 130-192).Oleh sebab itu, dalam artikel ini, saya menggunakan istilah “Gerakan Islam” dalam pengertian yang luas, tidak hanya untuk gerakan yang berorientasi politik dan radikal, tapi juga (bahkan utamanya) merujuk pada organisasi dan gerakan keagamaan berorientasi sosial, intelektual, atau kultural, seperti NU (Nahdlatul Ulama), Muhammadiyah, Persis (Persatuan Islam), Hidayatullah, HMI (Himpunan Mahasiswa Islam), dan PII (Pelajar Islam Indonesia), bahkan perguruan tinggi-perguruan tinggi Islam negeri maupun swasta. Saya melihat adanya beberapa kelamahan dalam “Gerakan IslamIndonesia (GII)” dalam konteks pembaharuan (tajdīd) dan reformasi (iṣlāḥ) tersebut. Di satu sisi, GII yang direpresentasikan oleh organisasi-organisasi Islam yang sudah mapan tidak mempunyai kredensial dan jaringan di tingkat internasional yang membuatnya hanya berpengaruh di tingkat nasional saja. Organisasi-organisasi Islam seperti NU, Muhammadiyah, Persis, Hidayatullah, HMI, dan PIImeskipun mempunyai pengaruh kuat di tingkat nasional, tapi pengaruh visi dan gerakan keislamannya tidak merambah ke tingkat yang lebih luas. PENDAHULUAN Di sisi yang lain, GII yang mempunyai jaringan di tingkat internasional kebanyakan hanya merupakan cabang, proksi, atau simpatisan dari gerakan-gerakan Islam transnasional. Daripada menyebarkan visi berislam yang bercorak keindonesiaan ke tingkat internasional, kelompok-kelompok Islam ini lebih bersemangat untuk menerjemahkan doktrin- doktrin induk organisasinya di tanah Indonesia. Hal ini terjadi, misalnya, dalam kasus gerakan salafi, HTI Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo u, Membangun Peradaban 389 bidang merambah ke dunia muslim, kaum muslim mempunyai respon yang beragam. Sebagian dari mereka menyambut modernisasi dengan antusias. Nilai-nilai yang lahir dari rahim modernitas seperti demokrasi, kebebasan individu, kemerdekaan berfikir, persamaan hak, hak asasi manusia, ide kemajuan (the idea of progress), dan sejenisnya diadopsi dan diinjeksikan ke dunia muslim tanpa melalui proses penyaringan yang ketat. Mereka kemudian sering disebut sebagai muslim liberal. (Hizbut Tahrir Indonesia), berbagai kelompok simpatisan Negara Islam Iraq dan Suriah (NIIS). Dua mainstream Gerakan Islam Indonesia sebagaimana disebutkan di atas, selain bermasalah dalam konteks skala pengaruh dan corak pemikiran, juga mempunyai kelemahan dalam cara pandang dunianya. Walhasil, Gerakan Islam Indonesia tidak saja hanya menjadi gerakan lokal atau gerakan proksi organisasi transnasional, tapi juga tidak sanggup menghadapi tantangan global yang semakin kompleks. Melalui refleksi pendek atas fenomena Gerakan Islam Indonesiakontemporer dan literatur yang saya baca, saya berusaha memetakan kelemahan dan keterbatasan GII tersebut dan menyajikan beberapa tawaran jalan keluarnya. Salah satu jalan keluarnya adalah penggalakan internasionalisasi Gerakan Islam Indonesia, minimal dimulai dari lever Asia Tenggara. Sebagian yang lain melihat modernitas dengan nilai-nilai dibawanya sebagai sebuah ancaman dan karenanya harus ditolak dan dilawan. Modernitas diidentikkan dengan nilai, budaya, dan politik negara Barat yang dinilai tidak cocok dengan Islam. Mereka kemudian melihat Islam tidak hanya sebagai agama tapi juga sebagai ideologi politik, ekonomi, sosial, dan budaya. Islam, bagi mereka, adalah alternatif sistem nilai yang orisinal dan solusi bagi segala permasalahan (al-Islām huwa al-ḥall).Beberapa kalangan bahkan berpendapat bahwa tawaran sistem dan peradaban Islam lebih superior dan bisa menggantikan peradaban Barat modern. Kalim Siddiqui(1983) bahkan menganggap bahwa kohesi peradaban Islam yang dianggap superior itu dapat TANTANGAN Dunia modern yang semakin terbuka dan saling terkoneksi memberikan tantangan yang kompleks baik di sektor ekonomi, politik, sosial, budaya, maupun dalam kehidupan keagamaan. Ketika proses modernisasi di hampir segala 390 Islamic World and Politics Vol.2. No.2 July-December 2018 390 direduksi dalam bentuk negara Islam. Dia mengakatan, “The Islamic movement, as demonstrated in earlier discussion, has the network of mosques, political culture, common memory and shared expectations necessary to hold together until a higher state of cohesion is reached. The highest stage of this cohesion is of course the Islamic state”(Siddiqui, 1983). Meskipun mereka juga menikmati dan menggunakan hasil modernitas seperti teknologi dengan berbagai produknya, namun secara umum mereka tetap resisten terhadap modernitas dengan pelbagai turunan nilai dan sistemnya. Untuk mencapai tujuan-tujuan perlawanan tersebut, sebagian memakai cara- cara yang persuasif dan akademik, namun sayangnya sebagian yang lain menghalalkan dan memakai cara-cara kekerasan. Karena klaim keislamannya, mereka sering disebut sebagai kelompok Islamis. direduksi dalam bentuk negara Islam. Dia mengakatan, “The Islamic movement, as demonstrated in earlier discussion, has the network of mosques, political culture, common memory and shared expectations necessary to hold together until a higher state of cohesion is reached. The highest stage of this cohesion is of course the Islamic state”(Siddiqui, 1983). Meskipun mereka juga menikmati dan menggunakan hasil modernitas seperti teknologi dengan berbagai produknya, namun secara umum mereka tetap resisten terhadap modernitas dengan pelbagai turunan nilai dan sistemnya. Untuk mencapai tujuan-tujuan perlawanan tersebut, sebagian memakai cara- cara yang persuasif dan akademik, namun sayangnya sebagian yang lain menghalalkan dan memakai cara-cara kekerasan. Karena klaim keislamannya, mereka sering disebut sebagai kelompok Islamis. dengan nilain-nilai modern tapi juga berakar dalam tradisi khazanah intelektual Islam. Di sisi yang lain, ketika nilai-nilai tersebut dipisahkan dari akar keagamaan, dijadikan jargon ideologis, dan kemudian dipaksakan melalui dominasi politik, represi ekonomi, dan kekuatan militer, mereka me- nolaknya. Penolakan tersebut, misalnya, terefleksi dalam resistensi mereka terhadap sekularisme se- bagai sebagai faham ideologis, ter- hadap struktur kapitalisme atau neo -liberalisme yang eksploitatif, dan terhadap imperialisme politik- militer negara-negara Barat di negeri-negeri muslim. Persepsi Gerakan Islam ter- hadap tantangan global tergantung pada model penyikapan mereka terhadap fenomena modernitas tersebut. Bagi kaum muslim liberal, tantangan utama yang ada dalam benak mereka dalam sikap konservatif, apologetik, dan resisten dari kalangan muslim sendiri. Bagi mereka, sikap seperti itu malah menarik Islam ke dalam kemunduran dan menghambat potensi partisipasi dan kontribusi Islam bagi peradaban modern. TANTANGAN Watt, hubungan antara Barat dan Islam itu tidak melulu konfliktual, tapi juga saling memberi dan menerima,” The history of Islam and Europe is not only a history of mutual conquests (jihad and crusade), but is also a history of cultural encounters in the course of which decisive process of cultural borrowing took place in both sides”(Tibi, 2012, p. 17; Watt, 1991). Gerakan Islam Indonesia yang memiliki masalah berupa belenggu ideologis dan peimikiran adalah kalangan muslimliberal dan Islamis. Kalangan liberal mengalami kesulitan untuk membayangkan masa depan peradaban tanpa menjadikan Barat sebagai referensi utama.Imajinasi mereka terjerat dan terbatas pada apa yang telah dicapai dan dimiliki oleh Barat. Barangkali mereka memang realitis karena kemajuan peradaban Barat TANTANGAN Sementara bagi kalangan Islamis yang resisten terhadap modernitas, tantangan utama yang menghantui pikiran mereka adalah peradaban Namun demikian, sebagian kaum muslimin yang lain meng- hadapi modernitas dengan sikap kritis dan selektif. Di satu sisi, mereka menghargai nilai-nilai modern tersebut karena dianggap paralel dan ekuivalen dengan semangat ajaran Islam. Mereka bahkan mampu merumuskan nilai- nilai yang tidak saja bersesuaian Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 391 Barat yang menginisiasi dan mengendalikan modernisme. Islam sebagai sebuah agama dan peradaban dianggap berlawanan secara diametral dengan peradaban Barat modern tersebut. Sedangkan bagi kalangan muslim kritis, tan- tangan utamanya adalah memilah bagian-bagian yang positif dari peradaban Barat modern dan khazanah Islam untuk kemudian dijembatani dan disinergikan, dan pada yang bersamaan, menanggalkan bagian-bagian yang negatifdari keduanya. Bagi mereka, masa depan dunia bukanlah konflik antar dua peradaban, Barat dan Islam, melainkan dialog dan sinergi antar berbagai tradisi peradaban di dunia.Dalam bahasa Bassam Tibbi dengan mengutip William M. Watt, hubungan antara Barat dan Islam itu tidak melulu konfliktual, tapi juga saling memberi dan menerima,” The history of Islam and Europe is not only a history of mutual conquests (jihad and crusade), but is also a history of cultural encounters in the course of which decisive process of cultural borrowing took place in both sides”(Tibi, 2012, p. 17; Watt, 1991). respon kaum muslimin terhadap tantangan modernitas sebagaimana dijelaskan di atas. Hanya saja, Gerakan Islam Indonesia (GII) mempunyai- beberapa keter- batasan dan kelemahan yang mem- buatnya tidak maksimal dalam berpartisipasi dan berkontribusi bagi konstruksi peradaban dunia, khususnya peradaban Islam. Keter- batasan dan kelemahan tersebut muncul bukan semata dipicu oleh sebab eksternal seperti proses modernisasi(Tibi, 2001),tapi lebih karena sebab-sebab internal yang saya sebut sebagai belenggu-belenggu yang menghambat dan membatasi kemampuan eksplorasi, kreatifitas, dan kebebasan mereka. Belenggu- belenggu tersebut adalah, pertama, belenggu ideologis danepistemologis (pemikiran), dan kedua, belenggu pergerakan. Barat yang menginisiasi dan mengendalikan modernisme. Islam sebagai sebuah agama dan peradaban dianggap berlawanan secara diametral dengan peradaban Barat modern tersebut. Sedangkan bagi kalangan muslim kritis, tan- tangan utamanya adalah memilah bagian-bagian yang positif dari peradaban Barat modern dan khazanah Islam untuk kemudian dijembatani dan disinergikan, dan pada yang bersamaan, menanggalkan bagian-bagian yang negatifdari keduanya. Bagi mereka, masa depan dunia bukanlah konflik antar dua peradaban, Barat dan Islam, melainkan dialog dan sinergi antar berbagai tradisi peradaban di dunia.Dalam bahasa Bassam Tibbi dengan mengutip William M. BELENGGU Tipologi Gerakan Islam di Indonesia tidak jauh dari tiga bentuk 392 Islamic World and Politics Vol.2. No.2 July-December 2018 392 bisa dilihat secara empirik di abad ke-21 ini. Namun demikian, sikap ideologisdan pemikiran yang membebek kepada Barat dan hanya berorientasi untuk menerjemahkan pencapaian Barat dalam konteks Indonesia seperti itu membuat mereka tidak kreatif untuk mencari referensi lain dalam konteks pembangunan peradaban di masa depan. Hal yang serupa terjadi pada kalangan Islamis. Mereka secara ideologis dan epistemologis menjadikan pencapaian masya- rakat Islam di masa lalu sebagai rujukan utama. Bahkan, mereka tidak bisa melepaskan imajinasi dan pemikiranya dari bayangan kejayaan masa lalu untuk dibang- kitkan kembali di masa kini atau masa depan. Idealisasi tentang kekhalifahan, generasi masa awal Islam yang saleh (salaf al-ṣāliḥ), atau para imam yang terjaga dari dosa (maʿṣūm) mendominasi imajinasi dan aspirasi mereka. Sikap seperti ini tidak saja utopis tapi juga membuat mereka tidak kreatif dalam konteks menerjemahkan nilai-nilai luhur yang terpendam dalam khazanah teks dansejarah Islam dalam konteks kekinian. Mereka lebih terobsesi pada bentuk, institusi, dan struktur dari sejarah masa lalu Islam daripada isi dan nila-nilai luhur yang diwariskannya. Kedua sikap dan pemikiran tersebut di atas, baik dari kalangan liberal maupun Islamis, mempunyai dua konsekuensi yang kontraproduktif bagi pembangunan peradaban masa depan: satu, membelenggu kemerdekaan dan kemampuan Gerakan Islam Indonesia untuk secara kreatif dan orisinal merumus- kan sendiri model per-adabannya, dua, membuat Gerakan Islam Indonesia kedap rasa dan empati sehingga tidak bisa mendengar dan menyelami suasana batin masyarakat Indonesia yang bergelut dengan berbagai pemasalahannya. Oleh sebab itu, hemat saya, satu- satunya yang bisa diharapkan untuk mempimpin agenda pembangunan peradaban masa depan adalah Gerakan Islam Indonesia yang kritis dan transformatif. Gerakan ini tidak saja terbuka terhadapkebijaksanaan (wisdom) dan keutamaan (virtue) baik yang datang dari Barat maupun dari khazanah teks dan sejarah masa lalu Islam, tapi juga, mempunyai kemampuan untuk menyelami suasana batin masyarakat Indonesia karena mereka memang bergelut dengan permasalahan masyarakat dalam aktivitas kesehariannya. Hanya saja, Gerakan Islam bisa dilihat secara empirik di abad ke-21 ini. Namun demikian, sikap ideologisdan pemikiran yang membebek kepada Barat dan hanya berorientasi untuk menerjemahkan pencapaian Barat dalam konteks Indonesia seperti itu membuat mereka tidak kreatif untuk mencari referensi lain dalam konteks pembangunan peradaban di masa depan. Hal yang serupa terjadi pada kalangan Islamis. Mereka secara ideologis dan epistemologis menjadikan pencapaian masya- rakat Islam di masa lalu sebagai rujukan utama. BELENGGU Bahkan, mereka tidak bisa melepaskan imajinasi dan pemikiranya dari bayangan kejayaan masa lalu untuk dibang- kitkan kembali di masa kini atau masa depan. Idealisasi tentang kekhalifahan, generasi masa awal Islam yang saleh (salaf al-ṣāliḥ), atau para imam yang terjaga dari dosa (maʿṣūm) mendominasi imajinasi dan aspirasi mereka. Sikap seperti ini tidak saja utopis tapi juga membuat mereka tidak kreatif dalam konteks menerjemahkan nilai-nilai luhur yang terpendam dalam khazanah teks dansejarah Islam dalam konteks kekinian. Mereka lebih terobsesi pada bentuk, institusi, dan struktur dari sejarah masa lalu Islam daripada isi dan nila-nilai luhur yang diwariskannya. Hanya saja, Gerakan Islam Indonesia yang kritis tersebut mempunyai belenggunya tersendiri. Tidak seperti kalangan liberal dan Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo u Membangun Peradaban 393 Timur Tengah, khususnya Jazirah Arab bagi kalangan Sunnī dan Iran bagi kalangan Syi‟ah, sebagai episentrum gerakan. Kiprah mereka di Indonesia tidak lebih dari sekedar menjadi simpatisan, cabang, atau proksi bagi pusat pergerakan mereka di Timur Tengah. Dalam istilah Bassam Tibi, mereka adalah tumpahan (spill-over) dari pro- blematika dan pergerakan di Timur Tengah yang dianggap sebagai pusat (the core)(Tibi, 2012, p. 22).Oleh sebab itu, tidak mengherankan jika pergerakan mereka umumnya mengandung orientasi politik dan hanya ditujukan untuk men- dapatkan legitimasi, afirmasi, dan pengaminan dari kelompok- kelompok induk mereka di Timur Tengah. Islamis yang mempunyai jaringan internasional kuat karena afinitas ideologis dan epistemologisnya, GII yang kritis dan transformatifbelum ditunjang oleh kredensial, integritas, dan jaringan untuk dapat berpartisipasi dan berkontribusi secara global. Itulah yang menjadi belenggu pergerakannya. Berbeda dengan jenis beleng- gu GII yang bersifat kritis-trans- formatif, GII yang berkarakter liberal dan Islamis memiliki belenggu pergerakannya tersendiri. Mereka tidak terlalu memiliki masalah dengan jaringan. Masalah mereka terletak pada integritas dan kredensial sebagai Gerakan Islam yang mempunyai jiwa keislaman dan keindonesiaan. Gerakan kalangan liberal menempatkan Barat sebagai pusat orientasi per- gerakan dan Indonesia sebagai cabangnya. Pergerakan mereka umumnya berada di level pemikiran dan ditujukan hanya untuk mendapatkan legitimasi, afirmasi, atau anggukan dari Barat bahwa mereka telah menjadi pegerakan Islam yang modern dan liberal di tanah air. Orientasi keislaman dan keindonesiaan seringkali absen dari agenda-agenda pemikiran dalam pergerakannya. Gerakan kaum Islamis memiliki belenggu yang serupa. Mereka menjadikan GII yang berkarakter kritis- transformatif tidak memiliki problem atau belenggu sebagaimana yang dimiliki oleh gerakan liberal dan Islamis dalam konteks kehausan untuk mendapat legitimasi, afirmasi, dan persetujuan dari pihak luar. BELENGGU Mereka telah memiliki legitimasi, afirmasi, dan dukungan sosialmaupun kultural dari masya- rakat Indonesia. Mereka juga telah bergelut dengan teks, sejarah, dan menjadi bagian tak terpisahkan dari kehidupan masyarakat Islam di dunia. Hanya saja, sebagaimana 394 Islamic World and Politics Vol.2. No.2 July-December 2018 394 yang tadi dikemukakan, masalah utama bagi mereka adalah minim-nya tingkat partisipasi dan kontribusi mereka di tingkat global. Masalah ini diperparah dengan belenggu-belenggu yang membatasi pergerakan dan kiprah mereka, seperti krisis kredensial, integritas, dan jaringandi dalam pergerakan mereka. Namun, dalam hal krisis kredensial dan integritas, mereka berbeda dengan kalangan liberal dan Islamis. Jika kalangan liberal mengalamai krisis kredensial dan integritas dalam konteks komitmen keislaman dan kalangan Islamis dalam konteks komitmen keindonesiaan,maka krisis kredensial dan integritas yang dimiliki oleh gerakan Islam kritis berada dalam konteks pergaulan global. Mereka masih dianggap sebagai pemain figuran. Keislaman mereka masih dianggap Islam yang pinggiran dan inferior(Tibi, 2012, p. 22).Bagaimana cara mengatasi belenggu-belenggu gerakan Islam Indonesia yang seperti ini? di Madinah. Kota -kota lain diang- gap sebagai kota pinggiran yang tidak mempunyai otoritas ke- agamaan maupun kekuasaan. Setelah berhasil merebut kekuasaan dari tangan Alī b. Abī Tālib pada tahun 661, Mu‟āwīyah mendirikan Daulah Umayyah yang berpusat di Damaskus, Syria, hingga tahun 750. Pusat kekuasaan dan keagamaan pun berpindah ke sana. Kota-kota lain dianggap sebagai pinggiran dan tidak punya signifikansi kekuasaan dan keagamaan yang setara dengan pusat. Termasuk Mekah dan Madinah, Kufah, dan Basrah. Pesaing-pesaing utama politik yang tinggal di kota-kota pinggiran tersebut dihabisi, seperti Husayn b. „Alī di Kufah pada tahun 680 dan „Abdullāh b. Zubayr di Mekah pada tahun 692. Namun demikian pusat- pusat kajian kagamaan di luar Damaskus mulai merebak. Hasan al- Basri (642-728) mempunyai pusat kajian yang ramai dan berpengaruh di Basrah. „Abdullāh b. „Abbās (w. 687), „Abdullāh b. „Umar (w. 693), dan Mālik b. Anas (711-795) mengembangkan tradisi kesarjanaan tersendiri di Mekkah dan Madinah. Abū Hanīfah (699- 767) merumuskan dan menularkan t d l i h k t di i di p. 22).Bagaimana cara mengatasi belenggu-belenggu gerakan Islam Indonesia yang seperti ini? p. 22).Bagaimana cara mengatasi belenggu-belenggu gerakan Islam Indonesia yang seperti ini? PINGGIRAN MEMBANGUN KREDENSIAL, INTEGRITAS, DAN JARINGAN 767) merumuskan dan menularkan metodologi hukum tersendiri di Kufah. Tradisi keilmuan yang berkembang baik di kota-kota Pada awal Islam, dari zaman Nabi hingga al-Khulafā‟ al- Rāshidūn (622-661), pusat keagamaan dan kekuasaan berada Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 395 perdagangan, kota-kota tersebut juga menjadi pusat-pusat keilmuan yang menjadi tiang penyangga zaman keemasan Islam. „Ulamā‟ di bidang ilmu al-Qur‟an, Hadīth, fiqih, kalām, ta‟awwuf, filsafat, dan kesusastraan kebanyakan berasal dari kota-kota pinggiran tersebut. Demikian pula dengan para ilmuwan di bidang matematika, optik, kimia, astronomi, kedokteran dan ilmu-ilmu rasional lainnya. Mereka banyak yang berasal dan berkarya di wilayah luar pusat kekuasaanitu. Dinamika kesarjana- an dan perdagangan semacam inilah yang membuat kota- kota tersebut tetap memiliki kredensial di bidang kesarjanaan Islam, integritas di bidang ekonomi, dan jaringan yang kuat baik di bidang keilmuan maupun ekonomi. Dengan demikian, daerah-daerah pinggiran tersebut mempunyai daya tawar, tidak dipandang sebelah mata, dan mampu berkontribusi secara signifikan dalam konteks kejayaan peradaban Islam di abad pertengahan(Hodgson, 1974a, 1974b) pinggiran kekuasaan tersebut membuatnya tidak kehilangan signifikansi keagamaan. Kota-kota tersebut tetap mempunyai legitimasi keislaman yang setara, bahkan beberapa lebih kuat, dibandingkan dengan Damaskus. Kecenderungan seperti ini terus terjadi ketika pusat kekuasaan beralih ke Daulah Abbasiyah di tahun 750 hingga 1258. Pusat ke- kuasaan dunia Islam boleh saja berada di Baghdad dan sebagian dibagi dengan Dinasti Fatimiyyah yang berpusat di Kairo (dengan kekuasaan meliputi Afrika Utara dan Mesir) dan Dinasti Umayyah di Andalusia yang berpusat di Kordoba. Namun demikian, kota- kota baru di luar lingkaran pusat kekuasaan juga berkembang. Di wilayah Khurāsān, terdapat Nīshāpūr, Herat, Marw, dan Balkh. Di Asia Tengah, terdapat kota-kota seperti Samarqand, Bukhara, dan Khawarizm. Di Andalusia, selain Cordoba juga ada Granada. Kota- kota di pinggir kekuasaan tersebut tidaklah inferior terhadap pusat- pusat kekuasaan. Sebagian menjadi kota perdagangan yang strategis dan ramai, seperti Samarqand dan Nishapur, salah satunya karena memang berada di jalur perdagangan Silk Road. Namun demikian, selain menjadi kota Narasi sekilas tentang kotadan wilayah pinggiran dala sejarah Islam tersebut memberi gambaran tentang peran dan sumbangan kota-kota itu terhadap pembangunan peradaban. Kota-kota tersebut 396 Islamic World and Politics Vol.2. No.2 July-December 2018 396 tidak hanya menjadi salah satu tiang kuat penyangga peradaban. Beberapa kotabahkan menjadi pusat kesarjanaan, ekonomi, dan politik yang baru, termasuk Baghdad, Kairo, Cordoba, dan Samarqand. PINGGIRAN MEMBANGUN KREDENSIAL, INTEGRITAS, DAN JARINGAN Catatan sejarah ini dapat dijadikan inspirasi dan pijakan bagi negeri- negeri di luar pusat dan sumber diskursus keislaman di masa kini, termasuk Indonesia. Gerakan Islam di Indonesia tidak perlu meminta dan mencari legitimasi keberagamaan dan kesarjanaan Islam dari negeri -negeri di Timur Tengah. Mereka bisa mengakses sendiri sumber-sumber keislaman dari yang primer seperti al-Qur‟an dan Sunnah hingga yang sifatnya literatur kesarjanaan seperti yang ditulis oleh para sarjana dan „ulamā‟. Oleh sebab itu, salah satu hal yang penting dilakukan adalah pembangunan kredensial kesarjanaan Islam di Indonesia, terutama melalui tradisi riset dan publikasi yang kuat. Hal ini penting dilakukan agar para sarjana Islam di Indonesia dapat berdiksusi secara setara dengan koleganya di negara- negara Timur Tengah maupun Barat dan memberikan kontribusi yang penting bagi kesarjanaan Islam di tingkat dunia. Hanya saja, tradisi kesarjanaan Islam saja tidak c k p Pem bangunan kredensial dan integritas keislaman di Indonesia juga perlu ditopang oleh kredensial dan integritas di bidang-bidang yang lain, terutama di bidang ekonomi, ilmu pengetahuan, sosial, serta politik. Negara-negara Islam di kawasan Timur Tengah cenderung menganggap negeri-negeri Islam di luar kawasan mereka sebagai kurang Islam (less Islamic) dan oleh karenanya mereka perlu diislamkan lagi. Anggapan semacam ini akan luntur dengan sendirinya apabila negeri-negeri Muslim di luar Timur Tengah itu mempunyai kredensial dan integritas di bidang ekonomi, ilmu pengetahuan, sosial, serta politik. Pengalaman hubungan antara negara-negara Timur Tengah dengan negara-negara Barat meng- ajarkan tentang pola hubungan yang penuh penghormatan itu. Timur Tengah lebih respek (untuk tidak mengatakan, inferior) terhadap negara-negara Barat daripada negara-negara Muslim di luar kawasan Timur Tengah bukan karena bertambahnya jumlah populasi Muslim atau majunya- kesarjanaan Islam di Barat, melainkan, karena sumber daya ekonomi, keunggulan ilmu pengetahuan, tatanan sosialnya yang Hanya saja, tradisi kesarjanaan Islam saja tidak cukup. Pem- Mohammad Syifa A. Widigdo 39 Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 397 kekuatan politiknya.4Oleh sebab itu, agar peradaban Islam di Indonesia mempunyai daya tawar, posisi sejajar, dan tidak lagi dianggap remeh, figuran, inferior, atau lemah oleh koleganya di Timur Tengah dan di belahan dunia yang lain, maka Gerakan Islam Indonesia dituntutuntuk menjadi peolopor bagi pembangunan bukan saja iklim dan tradisi kesarjanaan yang kuat, tapi juga kekuatan ekonomi, keunggulan institusi pendidikan, keteraturan sosial, dan stabilitas politik. PINGGIRAN MEMBANGUN KREDENSIAL, INTEGRITAS, DAN JARINGAN Dalam konteks Indonesia kini, stabilitas politik Indonesia dan negara-negara yang berpenduduk Muslim di kawasan Asia Tenggara jauh lebih baik daripada kondisi politik di negara-negara Timur Tengah. Gerakan Islam Indonesia tidak perlu untuk terobsesi di sektor politik, apalagi dengan hanya glorifikasi struktur politik masa lalu (baca: khilāfah atau imāmah). Pada dasarnya,yang diperlukan di sektor inihanyalah terusmerawat demokrasi sehingga proses pergantian politik dapat berlangsung secara damai dan problem-problem kerakyatan dapat diselesaikan. Pekerjaan rumahGII kemudian tinggal mengerucutpada tigabidang, yaitu membangun kekuatan ekonomi, mewujudkan tatanan sosial yang adil dan damai, dan keunggulan ilmu pengetahuan. 4 Hal ini tercermin dalam kenyataan bahwa negera-negara Timur Tengah mengirimkan pelajar-pelajar terbaiknya untuk belajar di Barat. Para sarjana dan professional terbaik mereka juga banyak yang berkarir di negera-negara Barat. Bahkan, para pembesar politik maupun tokoh intelektual mereka pun kebanyakan pernah menempuh pendidikan di Barat. Ini menunjukkan betapa mereka mempunyai respek yang besar terhadap kemajuan peradaban dan perkembangan ilmu pengetahuan di Barat. Bukan itu saja, beberapa negara di Timur Tengah, seperti Qatar, UEA, dan Arab Saudi bahkan membuka cabang- cabang perguruan tinggi Barat di negara mereka atau merekrut secara besar- besaran para pengajar lulusan Barat untuk mengajar di perguruan tinggi mereka. Hal yang sama terjadi dalam konteks politik dan ekonomi. Ketergantungan mereka terhadap Barat, terutama Amerika dan sekutunya, sangat besar baik dalam konteks menyelesaikan konflik di wilayahnya, mengkonsumsi minyak yang diproduksinya, atau untuk membangun gedung, hotel, dan toko- toko di kawasannya. Gerakan Islam di Indonesia sebenarnya mempunyai kapasitas yang cukup untuk dapat menger- jakan dan menyelesaikan pekerjaaan rumah tersebut. Untuk hal yang pertama, yakni membangun ke- kuatan ekonomi, gerakan Islam di Indonesia mempunyai lembaga perbankan syariah, keuangan mikro, pengelola zakat atau wakaf, 398 Islamic World and Politics Vol.2. No.2 July-December 2018 398 dan bahkan asosiasi pengusaha yang dapat menjadi aktor utama dalampemberdayaan ekonomi umat dan bangsa. dalam konteks mengembangkan pemahaman keislaman yang progresif dan transormatif di kalangan generasi muda Islam Indonesia. Sementara organisasi non-pemerintah (ornop) atau yayasan, seperti The Wahid Institute, Maarif Institute, Fahmina Institute, dan LSAF (Lembaga Agama dan Filsafat) berperan penting dalam konteks mengadvokasi kelompok- kelompok yang terpinggirkan, hak- hak perempuan, dan pluralisme kepercayaan berdasarkan pema- haman- Islam yang progresif dan transformatif. Sementara untuk hal yang kedua, yaitu mewujudkantatanan sosial yang adil dan damai, gerakan Islam di Indonesia juga memiliki organisasi- organisasi Islam yang mapan untuk dijadikan sebagaiujuk tombak dalam mewujudkan- cita- cita sosial tersebut. 5 Berikut kepanjangan dari beberapa singkatan yang disebut di atas: UIN (Universitas Islam Negeri), IAIN (Institut Agama Islam Negeri), STAIN (Sekolah Tinggi Agama Islam), UII (Universitas Islam Indonesia) di Yogyakarta, Unisma (Universitas Islam Malang), UMI (Universitas Muslim Indonesia) di Makassar, PPIM (Pusat Pengkajian Islam dan Masyarakat). PINGGIRAN MEMBANGUN KREDENSIAL, INTEGRITAS, DAN JARINGAN Dengan demikian, diharapkan, kapasitas dan kompetensi mereka dapat meningkat dan karya-karya mereka dapat memberi sumbangan yang berarti bagi kesarjanaan dan kemanusiaan dalam skala global. pembangunan peradaban. Tidak perlu di tingkat dunia tampaknya masih terlalu muluk dan utopis. Partisipasi dan kontribusi Gerakan Islam di Indonesia dapat dimulai dengan membangut jaringan yang kuat di lingkup negara-negara Asia Tenggara. Mungkin selama ini asosiasi jaringan gerakan Islam di Asia Tenggara dilekatkan dengan kelompok-kelompok radikal yang menghalalkan kekerasan. Jaringan kelompok-kelompok radikal (Islamis) tersebut akan kalah dan terhenti dengan sendirinya jika gerakan Islam yang kritis dan transformatif di Asia Tenggara dapat berkolaborasi dan bersinergi secara struktural maupun kultural di sektor ekonomi, sosial, serta ilmu pengetahuan.Jaringan formal secara ekonomi telah terbuka melalui pemberlakuan Masyarakat Ekonomi Asean (MEA) pada akhir tahun 2015. Masyarakat Asia Tenggara ke depan akan semakin terkoneksi dan terintegrasi antara yang satu dengan yang lain. Kenyataan ini memberikan kesempatan bagi gerakan Islam di Indonesia. Mereka dapat mempelopori kolaborasi dan sinergi elemen-elemen masyarakat dan perguruan tinggi untuk meningkatkan kompetensi diri, menciptakan karya unggul di bidang ilmu pengetahuan dan teknologi, PINGGIRAN MEMBANGUN KREDENSIAL, INTEGRITAS, DAN JARINGAN Organisasi masyarakat (ormas) keagamaan seperti NU, Muhammadiyah, Persis, dan Hidayatullah menjadi aktor penting tidak saja dalam menyebarkan pemahaman- keagamaan yang moderat dan damai, tapi juga menjadi pemain utama dalam membangun hubungan yang saling menghargai dan menghormati antar agama serta membela hak-hak kelompok yang dianggap sesat dan minoritas. Organisasi pelajar dan mahasiswa seperti HMI, PMII (Pelajar Mahasiswa Islam Indonesia), dan IMM (Ikatan Mahasiswa Muhammadiyah)telah menjadi organisasi perkaderan dan perjuangan yang teruji. Mereka tidak saja telah berhasil mendidik generasi muda Indonesia untuk menjadi pemimpin di berbagai sektor ke- hidupan. Lebih dari itu, mereka juga merupakan pemain utama Untuk hal yang ketiga, yakni membangun keunggulan ilmu pengetahuan,- gerakan Islam di Indonesia juga mempunyai banyak institusi pendidikan dan lembaga penilitian yang dapat didorong untuk menjadi pusat-pusat keunggulan ilmu pengetahuan. Selain pesantren, madrasah, dan sekolah Islam, Indonesia juga mempunyai perguruan tinggi Islam yang negeri (i.e. UIN, IAIN, STAIN) maupun swasta (e.g. Perguruan Tinggi Muhammadiyah, UII, Unisma, UMI) yang tersebar di seluruh pelosok negeri. Lembaga- lembaga penelitian yang terkoneksi dengan perguruan tinggi (e.g. PPIM UIN Syarif Hidayatullah) maupun di luar perguruan tinggi (e.g. The Habibie Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 399 pembangunan peradaban. Tidak perlu di tingkat dunia tampaknya masih terlalu muluk dan utopis. Partisipasi dan kontribusi Gerakan Islam di Indonesia dapat dimulai dengan membangut jaringan yang kuat di lingkup negara-negara Asia Tenggara. Mungkin selama ini asosiasi jaringan gerakan Islam di Asia Tenggara dilekatkan dengan kelompok-kelompok radikal yang menghalalkan kekerasan. Jaringan kelompok-kelompok radikal (Islamis) tersebut akan kalah dan terhenti dengan sendirinya jika gerakan Islam yang kritis dan transformatif di Asia Tenggara dapat berkolaborasi dan bersinergi secara struktural maupun kultural di sektor ekonomi, sosial, serta ilmu pengetahuan.Jaringan formal secara ekonomi telah terbuka melalui pemberlakuan Masyarakat Ekonomi Asean (MEA) pada akhir tahun 2015. Masyarakat Asia Tenggara ke depan akan semakin terkoneksi dan terintegrasi antara yang satu dengan yang lain. Kenyataan ini memberikan kesempatan bagi gerakan Islam di Indonesia. Mereka dapat mempelopori kolaborasi dan sinergi elemen-elemen masyarakat dan perguruan tinggi untuk meningkatkan kompetensi diri, menciptakan karya unggul di bidang ilmu pengetahuan dan teknologi, Center) juga ada.5 Yang diperlukan selanjutnya adalah pembangunan kapasitas dan kompetensi mereka agar dapatmendidik, meneliti, dan berkarya di level yang setara, atau bahkan, mengungguli, pusat-pusat keunggulan dunia. Di samping perlu keberpihakan dan keterlibatan pemerintah, mereka juga perlu untuk membuka jaringan dan berkolaborasi dengan pusat-pusat keunggulan dunia. 6 Saya memilih untuk memakai istilah “dereligiusisasi” daripada “sekularisasi” dengan pemikiran bahwa yang paling dikhawatirkan dari proses sekularisasi adalah tersingkirnya aspek-aspek religius dari nafas kehidupan masya- rakat. Penghargaan terhadap etika dan norma agama dihilangkan dan digantikan dengan etika dan norma yang bersifat positivis dan materialistik. SINERGI UNTUK PERADABAN Chicago: The University of Chicago Press. Hodgson, M. G. S. (1974b). The Venture of Islam 2. Chicago: The University of Chicago Press. and History in a World of Civilization (Vol. 1: The Classical Age of Islam). Chicago: The University of Chicago Press. Hodgson, M. G. S. (1974b). The Venture of Islam 2. Chicago: The University of Chicago Press. Hourani, A. (1983). Arabic Thought in the Liberal Age, 1798-1939. Cambridge: Cambridge University Press. SINERGI UNTUK PERADABAN Dengan sumber daya gerakan Islam di Indonesia seperti yang dikemukakan di atas, seharusnya umat Islam Indonesia melalui gerakan -gerakan tersebut di atas mampu mengambil peran aktif dan strategis dalam konteks 400 Islamic World and Politics Vol.2. No.2 July-December 2018 400 dan sekaligus memberi sumbangan penting bagi peradaban dunia. (wasa’iyyah ) dalam beragama, pluralisme keagamaan dan kepercayaan di Asia Tenggara, hak- hak kaum minoritas, pemberdayaan dan pembelaan perempuan, serta ekonomi kerakyatan dan pemberdayaan, maka mereka akan mendapatkan dua manfaat sekaligus. Pertama, mereka akan dapat mencegah dan mengobati penyakit radikalisme dalam gerakan Islam melalui pemahaman keagamaan yang moderat dan, kedua, menangkal penyingkiran nilai, etika, dan norma keagamaan dari masyarakat melalui penyajian Islam di Asia Tenggara yang dapat berdialog dengan modernitas dan realita yang terus berkembang. Di samping itu, kolaborasi dan sinergi pusat-pusat keunggulan yang dipelopori oleh gerakan Islam di Asia Tenggara juga diharapkan dapat mengatasi beberapa tan- tangan yang dihadapi oleh umat Islam selama ini. Tantangan internal masyarakat Asia Tenggara, seperti kemiskinan, menjadi masalah yang diharapkan dapat diatasi dengan pembukaan MEA. Namun tantangan-tantangan lain, terutama ideologi kekerasan yang dibawa oleh sebagian gerakan Islamis dari Timur Tengah dandereligiusasi6pemikiran dan budaya yang diimpor kalangan liberal dari dunia Barat, bukan menjadi domain dan konsentrasi MEA untuk menyelesaikannnya. Dalam hal ini, gerakan Islam di Asia juga dapat mengambil peran penting. Jika organisasi Islam dan pusat-pusat keunggulan Asia Tenggara berkomitmen danterus mengeksplorasi hubungan antara Islam denganpemahaman moderat Pada akhirnya, diharapkan, masyarakat Islam di Asia Tenggara mampu menampilkan peradaban tersendiri. Yaitu, peradaban yang menunjukkan bahwa Islam di Asia Tenggara dapat berjalan beriringan dengan demokrasi politik dan ekonomi, mendorong keunggulan ilmu pengetahuan dan teknologi, menghargai keragaman agama dan kepercayaan, melindungi kaum minoritas, membela hak-hak kaum perempuan, serta dapat hidup harmonis dalam perbedaan sekte, etnis, dan agama. Dengan demikian, jika peradaban Islam di belahan dunia lain, misalnya Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 401 Timur Tengah, ingin belajar tentang hal-hal tersebut di atas, mereka akan pergi belajar ke pengalaman paradaban Islam di Asia Tenggara. Saat itulah, peradaban Islam yang awalnya dianggap pinggiran, akan sejajar atau bahkan mengungguli peradaban yang menganggap dirinya sebagai pusat Islam. and History in a World of Civilization (Vol. 1: The Classical Age of Islam). Daftar Pustaka Ahyar, M. (Mei 2015 ). Membaca Gerakan Islam radikal dan Deradikalisasi Gerakan Islam. Walisongo, 23(1), 1-26. Siddiqui, K. (1983). Struggle for the supremacy of Islam— some critical dimensions. In K. Siddiqui (Ed.), Issues in the Islamic Movement 1981-82. London: The Open Press Limited. Ar, E. H. (September 2013). Pola Gerakan Islam Garis Keras di Indonesia. Jurnal Khatulistiwa – Journal Of Islamic Studies, 3(2). Tibi, B. (2001). Islam Between Culture and Politics. New York: Palgrave. Basyir, K. (November 2016). Ideologi Gerakan Politik Islam di Indonesia. Al-Tahrir, 16(2), 339 - 362. Tibi, B. (2012). Islam in Global Politics: Conflict and cross- civilizational bridging. New York: Routledge. Chaq, M. D. u. (Mei 2013). Pemikiran Hukum Gerakan Islam Radikal. Tafaqquh, 1(1), 16-42. Watt, W. M. (1991). Muslim- Christian Encounters. New York: Routledge. Collins, E. F. (Oct., 2004). Islam and the Habits of Democracy: Islamic Organizations in Post- New Order South Sumatra. Indonesia(78), 93-120. Hodgson, M. G. S. (1974a). The Venture of Islam 1: Conscience
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Cognitive sub-Nyquist hardware prototype of a collocated MIMO radar
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Cognitive Sub-Nyquist Hardware Prototype of a Collocated MIMO Radar Kumar Vijay Mishra, Eli Shoshan, Moshe Namer, Maxim Meltsin, David Cohen, Ron Madmoni, Shahar Dror, Robert Ifraimov, and Yonina C. Eldar the Xampling framework where Fourier coefficients of the received signal are acquired from their low-rate samples (or Xamples) [8, p. 387-388] [10]. Application of Xampling in space and time enables sub-Nyquist sampling without loss of any of the aforementioned radar resolutions. The Xamples are expressed as a matrix of unknown target parameters and the reconstruction algorithm is derived by extending the orthogonal matching pursuit (OMP) [8] to simultaneously solve a system of CS matrix equations. In sub-Nyquist MIMO, the radar antenna elements are randomly placed within the aperture (see [11] for introduction and [3] for recent updates on random arrays), and signal orthogonality is achieved by frequency division multiplexing (FDM). In a conventional MIMO radar, the use of non-overlapping FDM waveforms results in a strong range-azimuth coupling [12–14] in the receiver processing, and therefore, it is common to use orthogonal code signals (i.e. code division multiplexing or CDM). The coupling due to FDM can be reduced by randomizing the carrier frequencies across transmitters [13]. The FDM-based sub-Nyquist MIMO mitigates the range-azimuth coupling by randomizing the element locations in the aperture. the Xampling framework where Fourier coefficients of the received signal are acquired from their low-rate samples (or Xamples) [8, p. 387-388] [10]. Application of Xampling in space and time enables sub-Nyquist sampling without loss of any of the aforementioned radar resolutions. The Xamples are expressed as a matrix of unknown target parameters and the reconstruction algorithm is derived by extending the orthogonal matching pursuit (OMP) [8] to simultaneously solve a system of CS matrix equations. In sub-Nyquist MIMO, the radar antenna elements are randomly placed within the aperture (see [11] for introduction and [3] for recent updates on random arrays), and signal orthogonality is achieved by frequency division multiplexing (FDM). In a conventional MIMO radar, the use of non-overlapping FDM waveforms results in a strong range-azimuth coupling [12–14] in the receiver processing, and therefore, it is common to use orthogonal code signals (i.e. code division multiplexing or CDM). The coupling due to FDM can be reduced by randomizing the carrier frequencies across transmitters [13]. The FDM-based sub-Nyquist MIMO mitigates the range-azimuth coupling by randomizing the element locations in the aperture. I. Introduction Multiple input multiple output (MIMO) radar is a novel radar paradigm [1] that uses an array of several transmit and receive antenna elements, with each transmitter radiating a different waveform. In a collocated MIMO radar [2], the antenna elements are placed close to each other so that the radar cross-section of a target appears identical to all the elements. The waveform diversity in a collocated MIMO is based on the mutual orthogonality - usually in time, frequency or code - of different transmitted signals. The receiver separates and coherently processes the target echoes corresponding to each transmitter. The angular resolution of MIMO is same as a virtual phased array with the same antenna aperture but many more antenna elements than MIMO. In this work, we present the first hardware prototype of a sub-Nyquist MIMO that can demonstrate reduction in both spatial and spectral sampling using real-time analog signals. This implementation follows the recommendations of [9] for signal orthogonality, array structure and reconstruction algorithms. The prototype can be configured either as a filled or thinned array, thereby allowing comparison of Nyquist and sub-Nyquist spatial sampling using the same hardware. Our previous work in [15] presented the hardware realization of spectral sub-Nyquist sampling in radar. In [15], a few randomly chosen, narrow subbands of the received signal spectrum are pre-filtered before being sampled by low-rate analog-to-digital converters (ADCs). Since this implementation uses a bandpass filter and an ADC for each subband, a similar implementation of spectral sub-Nyquist sampling in each channel of a MIMO receiver would require enormous hardware resources. To circumvent such a simplistic and excessively large design, we instead transmit only in those subbands that are sampled by the receiver. This eliminates all pre-filtering band-pass stages and each receiver channel requires only one low-rate ADC to sample all subbands. While a radar achieves high angular resolution by using a large virtual aperture, its range-time resolution can be improved by transmitting signals with large bandwidth. In other words, the conventional processing resolution is limited by the number of elements and the receiver sampling rate. Several methods have been proposed to address the problem of preserving the MIMO radar resolution when either the number of antennas [3] or the number of received samples [4–6] is reduced. Most exploit the fact that the target scene is sparse facilitating the use of compressed sensing (CS) methods [7, 8]. 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Cognitive Sub-Nyquist Hardware Prototype of a Collocated MIMO Radar Abstract—We present the design and hardware implementation of a radar prototype that demonstrates the principle of a sub-Nyquist collocated multiple-input multiple-output (MIMO) radar. The setup allows sampling in both spatial and spectral domains at rates much lower than dictated by the Nyquist sampling theorem. Our prototype realizes an X-band MIMO radar that can be configured to have a maximum of 8 transmit and 10 receive antenna elements. We use frequency division multiplexing (FDM) to achieve the orthogonality of MIMO waveforms and apply the Xampling framework for signal recovery. The prototype also implements a cognitive transmission scheme where each transmit waveform is restricted to those pre-determined subbands of the full signal bandwidth that the receiver samples and processes. Real-time experiments show reasonable recovery performance while operating as a 4×5 thinned random array wherein the combined spatial and spectral sampling factor reduction is 87.5% of that of a filled 8 × 10 array. Keywords—MIMO radar, sub-Nyquist, compressed sensing, collocated, cognitive radar The authors are with the Andrew and Erna Viterbi Faculty of Electrical and Computer Engineering, Technion - Israel Institute of Technology, Haifa, Israel, e-mail: {mishra@ee, elis@ee, namer@ee, maxim.meltsin@ee, davidco@tx, mron@t2, shahard@t2, rifraimov@ef, yonina@ee}.technion.ac.il This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 646804-ERC-COG-BNYQ. K.V.M. acknowledges partial support via Andrew and Erna Finci Viterbi Fellowship. The authors are with the Andrew and Erna Viterbi Faculty of Electrical and Computer Engineering, Technion - Israel Institute of Technology, Haifa, Israel, e-mail: {mishra@ee, elis@ee, namer@ee, maxim.meltsin@ee, davidco@tx, mron@t2, shahard@t2, rifraimov@ef, yonina@ee}.technion.ac.il This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 646804-ERC-COG-BNYQ. K.V.M. acknowledges partial support via Andrew and Erna Finci Viterbi Fellowship. C. Cognitive Transmission Let Bh be the set of all frequencies in the transmit signal spectrum of effective bandwidth Bh. In the cognitive radar transmission, the spectrum ˜Hm(ω) of each of the transmitted waveforms ˜hm(t) is limited to a total of Nb non-overlapping frequency bands Bi, 1 ≤i ≤Nb (Figure 1): xq (t) = M−1 X m=0 L X l=1 αlhm (t −τl) e j2πβmqθl, (3) (3) where αl denotes the complex-valued reflectivity of the lth target and βmq is governed by the array structure. Our goal is to estimate the range-time τl and azimuths θl of each target. ˜Hm(ω) =  γ(ω)Hm(ω), ω ∈ Nb [ i=1 Bi ⊂Bh 0, otherwise (7) A. MIMO Radar Model Let the operating wavelength of the radar be λ and the total number of transmit and receive elements be T and R respectively. The classic approach to collocated MIMO adopts a virtual uniform linear array (ULA) structure [17], where the receive antennas spaced by λ 2 and transmit antennas spaced by R λ 2 form two ULAs (or vice versa, see e.g. [18]). Here, the coherent processing of a total of TR channels in the receiver creates a virtual equivalent of a phased array with TR λ 2-spaced receivers and normalized aperture Z = TR 2 . per channel. Xampling obtains a set κ of arbitrarily chosen Fourier coefficients from low rate samples of the received channel signal such that |κ| = K < N. Let us now consider the mth transmission. Suppose Y m is the K × Q matrix with qth column given by ym,q[k], k ∈κ. The matrix Y m can be expressed as Let us now consider a collocated MIMO radar system that has M < T transmit and Q < R receive antennas. The locations of these antennas are chosen uniformly at random within the aperture of the virtual array mentioned above. The mth transmitting antenna sends a unique pulse sm (t) given by Y m = AmX (Bm)T , (5) (5) where X is a sparse matrix in which the location and values of the non-zero elements correspond to the locations and reflectivity of the targets respectively. The matrices Am and Bm are known functions of radar parameters (T, R, fm, κ, τ, and transmit bandwidth), and each of their columns correspond to a range and an azimuth cell, respectively. The sparse matrix X can be recovered from the set of equations (5) for all 0 ≤m ≤M −1, by solving the optimization problem sm (t) = hm (t)e j2π fct, 0 ≤t ≤τ, (1) (1) where τ denotes the pulse repetition interval (PRI), fc is the common carrier frequency at the RF (radio-frequency) stage, and {hm (t)}M−1 m=0 is a set of narrowband, orthogonal FDM pulses each with the continuous-time Fourier transform (CTFT) minimize X ||X||0 subject to Y m = AmX (Bm)T , 0 ≤m ≤M −1. (6) Hm(ω) = ∞ Z −∞ hm(t)e−jωtdt. (2) (2) (6) An approximate solution to this problem can be obtained through an extension of the matrix OMP method [20]. II. Sub-Nyquist Collocated MIMO Radar With the exception of cognitive transmission, the array and signal models of sub-Nyquist MIMO realized by our prototype closely follow that detailed by [9] and, hence, we only summarize them here. Fig. 1. A conventional radar with signal bandwidth Bh transmits in the band Bh. A cognitive radar transmits only in subbands {Bi}Nb i=1, but with more in-band power than the conventional radar. I. Introduction Limiting the signal transmission to selective subbands allows for more in-band power resulting in an increase in signal-to-noise ratio (SNR). This approach has recently been proposed in the context of sub-Nyquist cognitive radars [16]. Our prototype, therefore, additionally demonstrates application of cognitive transmission in sub-Nyquist MIMO radar. Additionally, a radar that transmits in several disjoint narrow bands has advantages of disguising the transmit frequencies as an effective electronic counter measure (ECM), and also escape radio-frequency (RF) interference from other licensed radiators in the vacant non-transmit subbands. Recently, [9] proposed a sub-Nyquist collocated MIMO radar (sub-Nyquist MIMO hereafter) that can recover the target range and azimuth by simultaneously thinning an antenna array and sampling received signals at sub-Nyquist rates. The recovery algorithm uses In the following section, we briefly review the signal and system model of sub-Nyquist MIMO. We then describe the design philosophy 56 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Fig. 1. A conventional radar with signal bandwidth Bh transmits in the band Bh. A cognitive radar transmits only in subbands {Bi}Nb i=1, but with more in-band power than the conventional radar. of our prototype and its major sub-modules in Section III. Finally, we present results obtained by the prototype in real-time experiments. A. MIMO Radar Model We refer the reader to [9] for full details of this recovery algorithm. Suppose the target scene consists of L non-fluctuating point targets (Swerling-0 model) [19] whose location is given by their range-time τl and direction-of-arrival (DoA) θl, 1 ≤l ≤L. The pulses transmitted by the radar are reflected back by the targets and collected at the receive antennas. When the received waveform is downconverted from RF to baseband, we obtain the following signal at the qth antenna, Suppose the target scene consists of L non-fluctuating point targets (Swerling-0 model) [19] whose location is given by their range-time τl and direction-of-arrival (DoA) θl, 1 ≤l ≤L. The pulses transmitted by the radar are reflected back by the targets and collected at the receive antennas. When the received waveform is downconverted from RF to baseband, we obtain the following signal at the qth antenna, B. Sub-Nyquist Range-Azimuth Recovery (7) The application of Xampling in both space and time enables recovery of range and direction at sub-Nyquist rates. The performance guarantees of this procedure are provided in [9]. The received signal xq(t) is separated into M channels, aligned and normalized. The Fourier coefficients of the received signal corresponding to the channel that processes the mth transmitter echo are given by where γ(ω) = Bh/|Bi| for ω ∈Bi. The total transmit power Pt remains the same such that the power relation between the conventional and cognitive waveforms is ) Z Bh/2 −Bh/2 |Hm(ω)|2 dω = Nb X i=1 Z Bi | ˜Hm(ω)|2 dω = Pt (8) (8) ym,q[k] = L X l=1 αle j2πβmqθle−j 2π τ kτle−j2πfmτl, (4) (4) In a cognitive radar, the sub-Nyquist receiver obtains the set κ of the Fourier coefficients only from the subbands Bi. where −N 2 ≤k ≤−N 2 −1, fm is the (baseband) carrier frequency of the mth transmitter and N is the number of Fourier coefficients 57 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) TABLE I. Technical characteristics of the prototype Parameters Mode 1 Mode 2 Mode 3 Mode 4 #Tx, #Rx 8,10 8,10 4,5 8,10 Element placement Uniform Random Random Random Equivalent aperture 8x10 8x10 8x10 20x20 Angular resolution (sine of DoA) 0.025 0.025 0.025 0.005 Range resolution 1.25 m Signal bandwidth per Tx 12 MHz (15 MHz including guard-bands) Pulse width 4.2 µs Carrier frequency 10 GHz Unambiguous range 15 km Unambiguous DoA 180◦(from -90◦to 90◦) TABLE I. Technical characteristics of the prototype Parameters Mode 1 Mode 2 Mode 3 Mode 4 #Tx, #Rx 8,10 8,10 4,5 8,10 Element placement Uniform Random Random Random Equivalent aperture 8x10 8x10 8x10 20x20 Angular resolution (sine of DoA) 0.025 0.025 0.025 0.005 Range resolution 1.25 m Signal bandwidth per Tx 12 MHz (15 MHz including guard-bands) Pulse width 4.2 µs Carrier frequency 10 GHz Unambiguous range 15 km Unambiguous DoA 180◦(from -90◦to 90◦) Fig. 2. Tx and Rx element locations for the hardware prototype modes over a 6 m antenna aperture. Mode 4’s virtual array equivalent is the 20 × 20 ULA. chosen Fourier coefficients. Therefore, sub-Nyquist radar prototype in [15] opted for sampling random disjoint subsets of κ, with each subset containing consecutive Fourier coefficients. B. System Description Figure 3 shows the sub-Nyquist MIMO prototype, user interface and radar display. The inset graph depicts the signal flow through a simplified block diagram. The user selects the prototype mode from the control interface and passes the control triggers to the transmit waveform generator card, where an FPGA device reads out the pre-stored received waveform from an on-board memory. Two separate digital-to-analog converters (DACs) - one each for I and Q samples - convert the resulting signal to baseband analog domain. The transmit waveform generator is off-the-shelf Xilinx VC707 evaluation board that is custom fit with a 4DSP FMC204 16-bit DAC card. Each of the I and Q analog signals are then passed on to their respective analog pre-processor cards. B. Sub-Nyquist Range-Azimuth Recovery The prototype in [15] used four random Fourier coefficient groups, pre-filtered the baseband signal to corresponding four subbands (or Xampling slices), and sampled each subband via separate low-rate ADC. If we use the same pre-filtering approach as in [15] for each of the eight channels of our sub-Nyquist MIMO prototype, then the hardware design would need a total of 4 × 8 = 32 bandpass filters (BPFs) and ADCs excluding the analog filters to separate transmit channels. We sidestep this requirement by adopting cognitive transmission wherein the analog signal of each channel lives only in certain pre-determined subbands and consequently, a BPF stage is not required. More importantly, for each channel, a single low-rate ADC can subsample this narrow-band signal as long as the subbands are coset bands so that they don’t aliase after sampling [21]. This implementation needs only eight low-rate ADCs, one per channel. Another advantage of this approach is flexibility of the prototype in selecting the Xampling slices. Unlike [15], the number and spectral locations of slices are not permanently fixed, and they can be changed (within the constraints of aliasing due to subsampling). Fig. 2. Tx and Rx element locations for the hardware prototype modes over a 6 m antenna aperture. Mode 4’s virtual array equivalent is the 20 × 20 ULA. The prototype can be configured to operate in various array configurations or modes. When operating at its maximum strength of 8 Tx and 10 Rx elements, it can be programmed as either a ULA (Mode 1) or a random array (Mode 2), each with the equivalent aperture of an 8 × 10 virtual array, i.e., 1.2 m. For the same aperture, the system can be operated as a thinned 4 × 5 array (Mode 3). In this configuration fewer receivers are serialized and the channels corresponding to the removed transmit elements are not processed by the digital receiver. Mode 3, hence, demonstrates the spatial sub-Nyquist sampling. Finally, the prototype can also function as a 8x10 thinned array (Mode 4) which can be viewed as a spatial sub-Nyquist version of a 20x20 virtual array with aperture of 6 m. III. Hardware Design We consider the implementation of a MIMO radar architecture that has a maximum of 8 transmit (Tx) and 10 receive (Rx) antenna elements. As explained in subsequent sections, the spatial and spectral sampling aspects of sub-Nyquist MIMO that we intend to demonstrate manifest only in the receiver processing. Therefore, our radar prototype does not physically radiate the transmit waveforms from an antenna and receive data from actual targets in real-time. Instead, multiple transmit waveforms are pre-computed externally at baseband, their echoes from simulated targets are recorded and the complex samples (in-phase I and quadrature-phase Q pairs) are stored in an on-board memory of a custom-designed waveform generator board. The prototype then processes these pre-recorded signals in real-time. Similarly, we omit the implementation of the up-conversion to RF carrier frequency in the transmitter and the corresponding down-conversion in the receiver from this prototype. We would assume that the physical array aperture and simulated target response correspond to an X-band ( fc = 10 GHz) radar. The choice of radar frequency band also affects the clutter response that we intend to consider in a future extension of this prototype. Figure 2 shows exact details of element locations for all four modes. Table I summarizes the technical parameters of the prototype for all four array configurations. As mentioned before, this system employs FDM-based signal orthogonality with each transmit signal hm(t) chosen to be approximately flat in spectrum, over the extent of 12 MHz (one-sided band). Each of the transmit waveforms is separated from its nearest neighbor by a 3 MHz guard-band. A. Design Philosophy A conventional 8x10 MIMO radar receiver would require simultaneous hardware processing of 80 (or 160 I/Q) data streams. Since a separate sub-Nyquist receiver for each of these 80 channels is expensive, we implement the eight channel analog processing chain for only one receive antenna element and serialize the received signals of all 10 elements through this chain. This approach allows the prototype to implement a number of receivers greater than 10 as the eight-channel hardware only limits the number of transmitters. Given a particular receive element, we intend to extract Fourier coefficient set κ for each of its transmit channels using low-rate ADCs. It has been shown [7, pp. 210-268] that high recovery performance is promised when these coefficients are drawn uniformly at random. An ADC can not, however, individually acquire each of the randomly A custom-built analog pre-processor (APP) splits the 120 MHz baseband analog signal from the waveform generator in 8 channels. The 9 dB attenuation due to 8-channel splitter is compensated with the use of 10 dB amplifier for each channel. The signal corresponding 58 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Fig. 3. Sub-Nyquist MIMO prototype and user interface. The analog pre-processor module consists of two APP cards mounted on opposite sides of a common chassis. The inset shows the simplified block diagram of the system. The subscript r represents received signal samples for rth receiver. Wherever applicable, the second subscript corresponds to a particular transmitter. The square brackets (parentheses) are used for digital (analog) signals. Fig. 3. Sub-Nyquist MIMO prototype and user interface. The analog pre-processor module consists of two APP cards mounted on opposite sides of a common chassis. The inset shows the simplified block diagram of the system. The subscript r represents received signal samples for rth receiver. Wherever applicable, the second subscript corresponds to a particular transmitter. The square brackets (parentheses) are used for digital (analog) signals. Fig. 3. Sub-Nyquist MIMO prototype and user interface. The analog pre-processor module consists of two APP cards m chassis. The inset shows the simplified block diagram of the system. The subscript r represents received signal samples the second subscript corresponds to a particular transmitter. The square brackets (parentheses) are used for digital (analo Fig. 5. Plan Position Indicator (PPI) display of results for Mode 1 and 3. The origin is the location of the radar. A. Design Philosophy Only the first transmitter channel uses a low-pass filter as it is difficult to practically realize a bandpass filter with a passband close to zero. The first five channels use Chebyshev filter design and the rest are elliptic filters, all with a passband ripple of 0.1 dB. Since subsampling raises the out-of-band noise, all of these front-end filters are designed to provide approximately 30 dB stopband attenuation. The imbalance in gain and spectral distortion are corrected by placing tunable equalizers at the end of APP chain. The channelized I/Q analog signals are then digitized using low-rate 16-bit ADCs in a digital receiver card. A digital receiver is realized using a single Xilinx VC707 evaluation board with two eight-channel 4DSP FMC168 digitizer daughter cards, one each for I and Q signals. The digital receiver output is transferred over LAN to a radar display. As shown in Figure 4a, the cognitive radar signal occupies only certain subbands in a 15 MHz band. Here, the sliced transmit signal has eight subbands each of width 375 kHz with the frequency range of 1.63-2, 2.16-2.53, 3.05-3.42, 3.88-4.25, 5.66-6.03, 6.51-6.88, 8.64-9.01 and 12.32-12.69 MHz before subsampling. The resulting coherence [22] Fig. 5. Plan Position Indicator (PPI) display of results for Mode 1 and 3. The origin is the location of the radar. The red dot indicates the north direction relative to the radar. Positive (negative) distances along the horizontal axis correspond to the east (west) of the radar. Similarly, positive (negative) distances along the vertical axis correspond to the north (south) of the radar. The estimated targets are plotted over the ground truth. for this selection of Fourier coefficients is 0.42. The total signal bandwidth is 0.375 × 8 = 3 MHz. This signal is subsampled at 7.5 MHz and, as shown in Figure 4b, there is no aliasing between different subbands. A non-cognitive signal would have occupied entire 15 MHz spectrum requiring a Nyquist sampling rate of 30 MHz. Therefore, use of cognitive transmission enables spectral sampling reduction by a factor of 4 (= 30 MHz/7.5 MHz) for each channel. Depending on whether the guard-bands of non-cognitive transmission are included in the computation or not, the effective signal bandwidth is reduced by a factor of 5 (= 15 MHz/3 MHz) or 4 (= 12 MHz/3 MHz) respectively for each channel. Mode 3 has 50% spatial sampling reduction when compared with Mode 1 or 2. A. Design Philosophy The red dot indicates the north direction relative to the radar. Positive (negative) distances along the horizontal axis correspond to the east (west) of the radar. Similarly, positive (negative) distances along the vertical axis correspond to the north (south) of the radar. The estimated targets are plotted over the ground truth. (a) Before subsampling (b) After subsampling Fig. 4. The normalized one-sided spectrum of one channel of a given receiver (a) before and (b) after subsampling with a 7.5 MHz ADC. Each of the subbands span 375 kHz and is marked with a numeric label. In a non-cognitive processing, the signal occupies the entire 15 MHz spectrum before sampling. (a) Before subsampling (b) After subsampling Fig. 4. The normalized one-sided spectrum of one channel of a given receiver (a) before and (b) after subsampling with a 7.5 MHz ADC. Each of the subbands span 375 kHz and is marked with a numeric label. In a non-cognitive processing, the signal occupies the entire 15 MHz spectrum before sampling. to each transmitter is then filtered using BPFs with 12 MHz passband. Only the first transmitter channel uses a low-pass filter as it is difficult to practically realize a bandpass filter with a passband close to zero. The first five channels use Chebyshev filter design and the rest are elliptic filters, all with a passband ripple of 0.1 dB. Since subsampling raises the out-of-band noise, all of these front-end filters are designed to provide approximately 30 dB stopband attenuation. The imbalance in gain and spectral distortion are corrected by placing tunable equalizers at the end of APP chain. The channelized I/Q analog signals are then digitized using low-rate 16-bit ADCs in a digital receiver card. A digital receiver is realized using a single Xilinx VC707 evaluation board with two eight-channel 4DSP FMC168 digitizer daughter cards, one each for I and Q signals. The digital receiver output is transferred over LAN to a radar display. As shown in Figure 4a, the cognitive radar signal occupies only certain subbands in a 15 MHz band. Here, the sliced transmit signal has eight subbands each of width 375 kHz with the frequency range of 1.63-2, 2.16-2.53, 3.05-3.42, 3.88-4.25, 5.66-6.03, 6.51-6.88, 8.64-9.01 and 12.32-12.69 MHz before subsampling. The resulting coherence [22] to each transmitter is then filtered using BPFs with 12 MHz passband. C. Experimental Results [8] Y. C. Eldar, Sampling Theory: Beyond Bandlimited Systems. Cambridge University Press, 2015. We evaluated the performance of all the modes through hardware simulations. Only one pulse was transmitted in all experiments and all modes were evaluated against the identical target scenarios. We injected the received signal corresponding to the echoes from 10 targets, placed at arbitrary range and azimuths, in the transmit waveform generator. In the first experiment, when the minimum angular spacing (in terms of the sine of DoA) between any two targets was approximately 0.025, the recovery performance of the thinned 4×5 array in Mode 3 was not worse than Modes 1 and 2 until the noise was dramatically increased. Figure 5 shows the detection performance of all the modes for this experiment when the signal SNR = −5 dB; the injected noise is complex white Gaussian. Here, a successful detection (green circle) occurs when the estimated target is within two range cells and one DoA bin of the ground truth (blue circle); otherwise, the estimated target is labeled as a false alarm (magenta circle). In case of high SNR or absence of noise, our criterion for successful detection is sensu stricto, i.e. the estimated target must lie at the exact location of the ground truth for a successful detection. [9] D. Cohen, D. Cohen, Y. C. Eldar, and A. M. Haimovich, “Sub-Nyquist collocated MIMO radar in time and space,” in IEEE Radar Conf., May 2016. [10] O. Bar-Ilan and Y. C. Eldar, “Sub-Nyquist radar via Doppler focusing,” IEEE Trans. on Signal Proc., vol. 62, no. 7, pp. 1796–1811, 2014. [11] Y. Lo, “A mathematical theory of antenna arrays with randomly spaced elements,” IEEE Trans. Antennas Propag, vol. 12, no. 3, pp. 257–268, 1964. [12] O. Rabaste, L. Savy, M. Cattenoz, and J.-P. Guyvarch, “Signal waveforms and range/angle coupling in coherent colocated MIMO radar,” in Int. Conf. on Radar, 2013, pp. 157–162. [13] H. Sun, F. Brigui, and M. Lesturgie, “Analysis and comparison of mimo radar waveforms,” in IEEE Radar Conf., 2014, pp. 1–6. [14] J. Xu, G. Liao, S. Zhu, L. Huang, and H. C. So, “Joint range and angle estimation using MIMO radar with frequency diverse array,” IEEE Trans. Signal Process., vol. 63, no. 13, pp. 3396–3410, 2015. [15] E. Baransky, G. Itzhak, I. Shmuel, N. Wagner, E. Shoshan, and Y. C. Eldar, “A sub-Nyquist radar prototype: Hardware and applications,” IEEE Trans. Aerosp. Electron. References [1] E. Fishler, A. Haimovich, R. Blum, D. Chizhik, L. Cimini, and R. Valenzuela, “MIMO radar: An idea whose time has come,” in IEEE Radar Conf., 2004, pp. 71–78. [2] J. Li and P. Stoica, “MIMO radar with colocated antennas,” IEEE Signal Process. Mag., vol. 24, no. 5, pp. 106–114, 2007. g , , , pp , [3] M. Rossi, A. M. Haimovich, and Y. C. Eldar, “Spatial compressive i f MIMO d ” Si l l 62 2 g , , , pp , [3] M. Rossi, A. M. Haimovich, and Y. C. Eldar, “Spatial compressive sensing for MIMO radar,” IEEE Trans. Signal Process., vol. 62, no. 2, pp. 419–430, 2014. Fig. 6. As in Fig. 5, but for a closely-spaced target scenario. The inset plots show the selected region in each PPI display at a magnified scale. sensing for MIMO radar,” IEEE Trans. Signal Process., vol. 62, no. 2, pp. 419–430, 2014. pp [4] Y. Yu, A. P. Petropulu, and H. V. Poor, “MIMO radar using compressive sampling,” IEEE J. Sel. Topics in Signal Process., vol. 4, no. 1, pp. 146–163, 2010. spatial and spectral sampling reduction in Mode 3, then we use a total one-eighth of the Nyquist sampling rate and one-tenth of the Nyquist signal bandwidth (guard-bands included). The sampling rate reduction is, therefore, seven-eighth or 87.5% in Mode 3. The receiver processes 80 and 20 channels in 8x10 and 4x5 arrays, respectively. So, the hardware resources are reduced by 75% in Mode 3. [5] D. S. Kalogerias and A. P. Petropulu, “Matrix completion in colocated MIMO radar: Recoverability, bounds & theoretical guarantees,” IEEE Trans. Signal Process., vol. 62, no. 2, pp. 309–321, 2014. [6] S. Sun, W. U. Bajwa, and A. P. Petropulu, “MIMO-MC radar: A MIMO radar approach based on matrix completion,” IEEE Trans. Aerosp. Electron. Syst., vol. 51, no. 3, pp. 1839–1852, 2015. [7] Y. C. Eldar and G. Kutyniok, Compressed Sensing: Theory and Applications. Cambridge University Press, 2012. IV. Summary We presented the first hardware prototype of sub-Nyquist MIMO that demonstrates real-time operation of both spatial and spectral reduction in sampling. The thinned 4x5 array achieves the detection performance of its filled array counterparts even though the combined reduction of spatial and spectral sampling is 87.5%. While we did not analyze the performance for Doppler recovery and clutter contamination, the prototype design does not restrict such an evaluation. Future theoretical insights on the selection of best subbands and improved recovery algorithms can further enhance the performance. A. Design Philosophy If we account for both 59 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Fig. 6. As in Fig. 5, but for a closely-spaced target scenario. The inset plots show the selected region in each PPI display at a magnified scale. C. Experimental Results Syst., vol. 50, no. 2, pp. 809–822, 2014. [16] D. Cohen, A. Dikopoltsev, R. Ifraimov, and Y. C. Eldar, “Towards sub-Nyquist cognitive radar,” in IEEE Radar Conf., May 2016. In the second experiment, the minimum angular spacing between the two targets was reduced to 0.02, and the SNR of the injected signal remained at −5 dB. Since the angular resolution of Mode 4 is better than the other three modes (see Table I), all the targets are detected successfully in Mode 4. Mode 1 and 3 showed a false alarm as seen in the inset plots of Figure 6. The Mode 2 also shows successful recovery in the broad sense of our detection criterion. The strict sense location error in Mode 2 is clearly larger than that in Mode 4. However, relatively better performance of Mode 2 over Modes 1 and 3 is not entirely fortuitous here. Figure 2 shows that both Tx and Rx array elements in Mode 2 are distributed such that its virtual array is wider than Modes 1 and 3. Thus, the effective angular resolution for Mode 2 could be better than 1 and 3, but still worse than 4. yq g y [17] C.-Y. Chen, “Signal processing algorithms for MIMO radar,” Ph.D. dissertation, California Institute of Technology, 2009. [18] E. Brookner, “MIMO radars and their conventional equivalents An update,” in IET Int. Radar Conf. IET, 2015, pp. 1–8. [19] M. Skolnik, Radar handbook. McGraw-Hill, 1970. [20] T. Wimalajeewa, Y. C. Eldar, and P. K. Varshney, “Recovery of sparse matrices via matrix sketching,” arXiv preprint arXiv:1311.2448, 2013. [21] K. M. Cohen, C. Attias, B. Farbman, I. Tselniker, and Y. C. Eldar, “Channel estimation in UWB channels using compressed sensing,” in IEEE Int. Conf. on Acoustics, Speech and Signal Processing, 2014, pp. 1966–1970. [22] P. Xia, S. Zhou, and G. B. Giannakis, “Achieving the Welch bound with difference sets,” IEEE Trans. Inf. Theory, vol. 51, no. 5, pp. 1900–1907, 2005. 60
https://openalex.org/W2181412895
https://etj.uotechnology.edu.iq/article_38169_6c0fb55dc2c4f16290019a37514f204e.pdf
English
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A Simplified Method For Estimating The Peak Load In The Large Cold Stores
Maǧallaẗ al-handasaẗ wa-al-tiknūlūǧiyā
2,009
cc-by
5,505
Nomenclature Nomenclature A : surface area that heats transfer through it (m 2 ) if ,fo : Heat transfer coefficient for the external and internal air layers (W/ 2 m C o Nomenclature A : surface area that heats transfer through it (m 2 ) if ,fo : Heat transfer coefficient for the external and internal air layers (W/ 2 m C o Cp : Specific heat of the product (J/kg. C o ) * Mechanical Engineering Department, University of Technology/Baghdad * Mechanical Engineering Department, University of Technology/Baghdad ** College of Engineering, University of Anbar / Anbar *** Mechanical Engineering Department, Technology Institute /Baghdad ** College of Engineering, University of Anbar / Anbar *** Mechanical Engineering Department, Technology Institute /Baghdad A Simplified Method For Estimating The Peak Load In The Large Cold Stores Dr. Ahmed A. M. Saleh* , Dr. Obaid T. Fadhil** & Aziz. J. Aziz*** Received on:28/9/2008 Accepted on:2/4/2009 2009 , 10 . No , 27 .l No , Journal . Tech & . Eng 2009 , 10 . No , 27 .l No , Journal . Tech & . Eng Abstract In this paper a simplified method for estimating the peak load in the large cold stores is found and its occurrence day by utilizing two new variables A & B. The first variable represents the number of days that is necessary for cooling the product (storage) up to the needed temperature according to the daily temperature differences only. The second variable represents the number of days required for cooling the total product (storage) depending upon the daily storage mass only without returning back to the details of distribution of the daily thermal load that is followed in calculating thermal load for cold stores. g By the demonstrative and analysis of the thermal load in cold stores with detailing study for each effective factors in estimating the thermal load characteristic is observed that the factors daily temperature differences (∆T) and storage mass (m) per day affect significantly in estimating the peak load values and day of occurrence. Keywords: Cold store, Peak Load Calculation, Daily Temperature Difference, Daily Storage Mass Daily Storage Mass ﻁﺭﻴﻘﺔ ﻤﺒﺴﻁﺔ ﻟﺘﺨﻤﻴﻥ ﺤﻤل ﺍﻟﺫﺭﻭﺓ ﻓﻲ ﺍﻟﻤﺨﺎﺯﻥ ﺍﻟﻤﺒﺭﺩﺓ ﺍﻟﻜﺒﻴﺭﺓ ﺍﻟﺨﻼﺼﺔ ﺘﻡ ﻓﻲ ﻫﺫﺍ ﺍﻟﺒﺤﺙ ﺇﻴﺠﺎﺩ ﻤﻌﺎﺩﻟﺔ ﻴﻤﻜﻨﻨﺎ ﺒﻭﺍﺴﻁﺘﻬﺎ ﺘﺨﻤﻴﻥ ﺤﻤل ﺍﻟﺫﺭﻭﺓ ﻭ ﻴﻭﻡ ﺤﺩﻭﺜـﻪ ﻓـﻲ ﻤﺨﺎﺯﻥ ﺍﻟﺘﺒﺭﻴﺩ ﺍﻟﻜﺒﻴﺭﺓ ﻭﺫﻟﻙ ﺒﺎﻻﺴﺘﻔﺎﺩﺓ ﻤﻥ ﺍﻟﻤﺘﻐﻴﺭﺍﺕ ﺩﻭﻥ ﺍﻟﺭﺠﻭﻉ ﺍﻟﻰ ﺘﻔﺎﺼﻴل ﺘﻭﺯﻴـﻊ ﺍﻟﺤﻤـل ﺍﻟﺤﺭﺍﺭﻱ . ﻴﻤﺜل ﺍﻟﻤﺘﻐﻴﺭ ﺍﻻﻭل ﻋﺩﺩﺍﻷﻴﺎﻡ ﺍﻟﻤﻁﻠﻭﺒﺔ ﻟﺘﺒﺭﻴﺩﺍﻟﻤﻨﺘﺞ ) ﺍﻟﺨﺯﻴﻥ ( ﺍﻟـﻰ ﺩﺭﺠـﺔ ﺍﻟﺤـﺭﺍﺭﺓ ﺍﻟﻤﻁﻠﻭﺒﺔ ﻁﺒﻘﺎﹰ ﻟﻔﺭﻕ ﺩﺭﺠﺎﺕ ﺍﻟﺤﺭﺍﺭﺓ ﺍﻟﻴﻭﻤﻴﺔ ﻓﻘﻁ، ﺃﻤﺎ ﺍﻟﻤﺘﻐﻴﺭ ﺍﻟﺜﺎﻨﻲ ﻓﻴﻤﺜل ﻋﺩﺩ ﺍﻷﻴﺎﻡ ﺍﻟﻤﻁﻠﻭﺒـﺔ ﺇﻋﺘﻤﺎﺩﺍﻋﻠﻰ ﻤﻘﺩﺍﺭﺍﻟﺨﺯﻥ ﺍﻟﻴﻭﻤﻲ . ﺘﺒﻴﻥ ﻤﻥ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻋﻼﻩ ﺒﺄﻥ ﺍﻟﻌﺎﻤﻠﻴﻥ ﻓﺭﻕ ﺩﺭﺠﺎﺕ ﺍﻟﺤﺭﺍﺭﺓ ﺍﻟﻴﻭﻤﻲ ﻭﻜﻤﻴﺔ ﺍﻟﻜﺘ ﻠﺔ ﺍﻟﻤﺨﺯﻭﻨـﺔ ﻟﻜـل ﻴﻭﻡ )∆ T ، m (؛ ﻟﻬﻤﺎ ﺘﺄﺜﻴﺭ ﻤﻠﻤﻭﺱ ﻓﻲ ﺘﺤﺩﻴﺩ ﻤﻘﺩﺍﺭ ﺤﻤل ﺍﻟﺫﺭﻭﺓ ﻭﺯﻤﻥ ﺤﺩﻭﺜﻪ ﻭﺫﻟـﻙ ﺒﻌﺩ ﺘﺤﻠﻴل ﻭﺩﺭﺍﺴﺔ ﻜل ﺍﻟﻌﻭﺍﻤل ﺍﻟﻤﺅﺜﺭﺓ ﺍﻟﺘﻲ ﺘﺤﺩﺩ ﻤﻭﺍﺼﻔﺎﺕ ﺍﻟﺤﻤل ﺍﻟﺤـﺭﺍﺭﻱ ﻓـﻲ ﺍﻟﻤﺨـﺎﺯﻥ ﺍﻟﻤﺒﺭﺩﺓ ﺍﻟﻜﺒﻴﺭﺓ . air ρ : Air density (kg/ 3 m ) 1. Introduction air ρ : Air density (kg/ 3 m ) 1. Introduction K : Thermal conduction coefficient for materials forming the wall (W/m. C o ) Cold stores are vessels or rooms properly thermal insulation supplied by electrical and mechanical machines that decrease the temperature of the space and its contents to a proper temperature thus; this lowered proper temperature reduces or prevents the factors which help to corrupt the storage products. The suitable temperature for storing depends on two main factors; type of the storage and the duration of store [1]. m : Daily storing mass in aday (kg) mTot: Total capacity mass storage( kg) L N : Number of lighting L N : Number of lighting P N : Number of persons P N : Number of persons m N : Number of equipment m N : Number of equipment NOC: Numbers of the air change per day m OH : operating of hours of the m OH : operating of hours of the equipment per a day These stores are frequently supplied with cooling equipment, which its capacity reaches thousands of tons of cooling, and other completion equipment that support in works of separate, transfer, pouch and so on. Also the initial cost of the cold stores is very expensive. The cooling system forms a very important part from the total cost, thus the equipment selection is very important and it is a real effective factor upon the total coast of the project. L OH : Operating hours of lighting per day (hr/day) P OH : Hours of work in the day m P : Input Power of equipment (W) LP : Power of lighting (W) 1 Q : Heat transfer through wall, roof, and ground (W) 2 Q : The amount of heat transfer due to the air change (W) 3 Q : Thermal load from lighting (W) Jing Xie et al. [2] they used CFD technique to study a two-dimensional mathematical model for the inside a mini type construction cold store (4.5 m × 3.3 m × 2.5 m). The simulation results reflected the characteristics of airflow and temperature distribution, and a several design parameters (corner baffle, the stack mode of food stuffs, etc.) were analyzed. The results of calculation indicated that all these designed parameters influenced the flow and temperature fields. ∆T : Daily temperature reduction C o ∆TTot : Total temperature differences required ( C o ) ∆T : Daily temperature reduction C o ∆TTot : Total temperature differences required ( C o ) y p ∆TTot : Total temperature differences required ( C o ) in h : Enthalpy of the internal air (inside cold store condition) (J/kg) air ρ : Air density (kg/ 3 m ) 1. Introduction 4 Q : Thermal load from equipment (W) 5 Q : Amount of heat from persons (W) ( ) Q6 : Sensible heat (W) Q6 : Sensible heat (W) Q7 : Amount of heat results from respiration (W) P Q : Amount of heat generates from each person (W/person) qres : Heat respiration (J/kg) qres : Heat respiration (J/kg) V : daily air change (m3/day) s V : Volume of cold store ( 3 m ) S. Akdemir [3] studies the cooling load and choosing of cooling system elements for designing of the cold store. He calculated transmissions, infiltration, production, and other source of heat. In U : over all heat transfer coefficients for wall, roofs and ground (W/m2. C o ) g,r, w T ∆ : Temperature difference between two sides of wall, roof and ground ( C o ) https://doi.org/10.30684/etj.27.10.17 2412-0758/University of Technology-Iraq, Baghdad, Iraq This is an open access article under the CC BY 4.0 license http://creativecommons.org/licenses/by/4.0 A Simplified Method For Estimating The Peak Load In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 ∆T : Daily temperature reduction C o ∆TTot : Total temperature differences required ( C o ) air ρ : Air density (kg/ 3 m ) 1. Introduction out h : Enthalpy of the external air (outside condition) (J/kg) in h : Enthalpy of the internal air (inside cold store condition) (J/kg) K : Thermal conduction coefficient for materials forming the wall (W/m. C o ) m : Daily storing mass in aday (kg) mTot: Total capacity mass storage( kg) L N : Number of lighting P N : Number of persons m N : Number of equipment NOC: Numbers of the air change per day m OH : operating of hours of the equipment per a day L OH : Operating hours of lighting per day (hr/day) P OH : Hours of work in the day m P : Input Power of equipment (W) LP : Power of lighting (W) 1 Q : Heat transfer through wall, roof, and ground (W) 2 Q : The amount of heat transfer due to the air change (W) 3 Q : Thermal load from lighting (W) 4 Q : Thermal load from equipment (W) 5 Q : Amount of heat from persons (W) Q6 : Sensible heat (W) Q7 : Amount of heat results from respiration (W) P Q : Amount of heat generates from each person (W/person) qres : Heat respiration (J/kg) V : daily air change (m3/day) s V : Volume of cold store ( 3 m ) U : over all heat transfer coefficients for wall, roofs and ground (W/m2. C o ) g,r, w T ∆ : Temperature difference between ∆T : Daily temperature reduction C o out h : Enthalpy of the external air (outside condition) (J/kg) 2.2.2 Equipment Load The equipment load can be calculated using the following equation: m m m 4 OH . P. N Q = /24 ……. (5) m m m 4 OH . P. N Q = /24 ……. (5) 2.2.5 Respiration load It is the amount of heat generates from product due to the vitality chemical reactions in any plan and it can calculate using the following equation [5]: g,r, w 1 T A U Q ∆ ⋅ ⋅ = ……(1-a) o n n 2 2 1 1 i f/1 K / X ...... K / X K / X f/1 U /1 + + + + = ……(1-b) 3600 24 q. m Q res 7 × = ……….. (8) (8) 2.1.1 Heat transfer through wall, roof, and ground 3600 24 T . C . m Q p 6 × ∆ = Can be calculated using the following equation (Searle engineers hand book 1988) [5]: PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 addition the amount of the cooling fluid was also calculated. addition the amount of the cooling fluid was also calculated. 2.2.3 Persons (workers) The thermal load per day in the cold stores can be divided into two main parts: it is calculated using the following equation: 24 / OH . Q . N Q P p P 5 = ……..(6) 24 / OH . Q . N Q P p P 5 = ……..(6) 2.2 Internal thermal load This thermal load generated inside the cold store and it contains the following: Erol, N. [4] stated that the determination of total input cooling load would not be possible, for this reason there may be some deviation in the cooling load and the focus point must be minimizing the deviation. He explained that the ambient temperature of cold store, the situation of store product before entering the cold store, the daily working hours and the determination of which product will be stored are important for calculation of the cooling load. 2.2.1 Lighting load It is the thermal energy generated from lighting used inside a store. It can be calculated using the following equation [5]: 24 / OH . P. N Q L L L 3 = …… (4) 2. Elements of thermal load in the cold stores 2.2.3 Persons thermal load (workers) 2.2.3 Persons thermal load (workers) load it is calculated using the following equation: The external thermal load is due to transfer of the heat from external to internal space of the store. It consists of: 2.1.1 Heat transfer through wall, roof, and ground The external thermal load is due to transfer of the heat from external to internal space of the store. It consists of: 2.1.1 Heat transfer through wall, roof, and ground 2.1.2 Air change heat gain 3. Factor affects thermal load 5. Case Study-Sample of Calculation Input data 5. Case Study-Sample of Calculation Input data p - Dimension: (25 × 15 × 5) m - Store Room Temperature: 4 C o - Store Room Temperature: 4 C o - Product : Potato - Capacity : 550 ton - Product Initial Temperature : 35 C o - Outside Temperature : 50 C o & RH= 20% 2 - Wall U-value : 0.34 W/m2. C o - Wall U-value : 0.34 W/m2. C o (for 10 mm Polystyrene board) (for 10 mm Polystyrene board) Peak load calculation (traditional (for 10 mm Polystyrene board) Peak load calculation (traditional method) Due to the probability of miss-match of all components loads in one day so it can not be able to find the peak load directly although the correct choice of the daily storing mass and the daily temperature difference was used. Due to the probability of miss-match of all components loads in one day so it can not be able to find the peak load directly although the correct choice of the daily storing mass and the daily temperature difference was used. ) - Q1 : Wall & Roof , use equation -1 ) - Q1 : Wall & Roof , use equation -1 q Q1)wall = 0.34 × 400 × (50 – 5) = 6256 W/day Q1)Roof = 0.34 × 375 × (50 – 5) = 5865 W/day Q1 = 6256 + 5865 = 12121 W/day q Q1)wall = 0.34 × 400 × (50 – 5) = 6256 W/day Q1)Roof = 0.34 × 375 × (50 – 5) = 5865 W/day Q1 = 6256 + 5865 = 12121 W/day 3. Factor affects thermal load It can be calculated using the following equation [5]: In the revision of the previous thermal loads which are used for design any cold store; it can be noticed that: ….. (2) ( ) 24 3600 h h . V . Q in out air 2 × − ρ = • T he internal thermal load forms the larger part of the overall load and the heat from product has a larger part of the internal load (specific heat and respiration heat of potato are 3.56 kJ/kg. C o , 270 kJ/kg and for (V) Is calculated from the knowledge of store's volume and the number of air change in a day as shown below (Air- Conditioning & Refrigeration Institute 1987) [6]. NOC . V V s = …….(3) NOC . V V s = NOC . V V s = PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 meat 3.2 kJ/kg. C o , 231 kJ/kg ) returning to ASHRAE Handbook 1998 [7]. The other loads take different ratio from overall load. meat 3.2 kJ/kg. C o , 231 kJ/kg ) returning to ASHRAE Handbook 1998 [7]. The other loads take different ratio from overall load. T T A Tot ∆ ∆ = (day) …….(9) (day) * Factor -B: It represents number of days required to fill the store by products depending on the daily storing mass (m) only, i.e. : It is clear that there are two important factors in calculation of the product load, the daily storing mass (m) and the daily temperature difference ∆T which affect directly in the value of the load, so the wrong selection of those two factors may give a very far result from the actual and then choose incompatible equipment to the real load (expensive unnecessarily cost). The time is taken by the product to reach the optimum storage temperature (sometimes called the pull- down time) will be limited by the overall refrigeration capacity of the equipment (M.Eltawil, D.Samuel and O.Singhal 2006 [8]. m m B Tot = (day) …..(10) m m B Tot = (day) …..(10) 5. Case Study-Sample of Calculation 4. Determination of the peak load and starting day To simplifying the analysis of the calculation, it’s important to redefine some variables: * External load = Q1 + Q2 (W) * Base load=Q3+Q4+Q5 (W) * Internal load = Q6+ Q7 (W) And two new factors: * External load = Q1 + Q2 (W) * Base load=Q3+Q4+Q5 (W) * Internal load = Q6+ Q7 (W) And two new factors: = 3287.7 W/day - Q3 : Lighting, use equation - 4 ( operating hour = 10) And two new factors: * Factor -A : It represents number of days necessary for cooling the product (storage) up to the required temperature depending upon the daily temperature differences (∆T) only i.e. : y - Q4 : Equipment, use equation- 5 ( fan power 4×3 kW & heater of 8 kW) Q4 = 20 × 1000 × 18 / 24 = 1500 W/day PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com 6. Daily load distribution Curves A collection of different cases of storing were taken depending upon storing masses and the daily temperature difference which covered most of the expected states in the loads of cold stores. Table -2 shows those chosen cases and the two factors A and B. y - Q6 : Product(sensible), use equation - 7 ( Cp of product= 3.56 kJ/kg. C o ) 3600 24 ) 4 35 ( 10 56 .3 10 550 Q 3 3 6 × − × × × × = Figures (1) to (5) represent the distribution of the daily load during the first period of storing for cases in the Table-2. = 702523 W/day - Q7 : Respiration(latent), use equation - 8 3600 24 10 270 10 550 Q 3 3 7 × × × × = = 1718750 W/day = 702523 W/day - Q7 : Respiration(latent), use equation - 8 10 270 10 550 3 3 × × × 7. Results and Discussion The graphical analysis and study of the load component distribution of previous figures could build Table-3 which represents a summary of the obtained results and offers simple equations to calculate peak load for the most cases without returning back to the details of loads distribution or graphical analysis or backup figures that is required to calculate throughout the first days of storing reaching to last days with algebraic sum to determine the peak load and day of it. y Table -1 shown the summary of calculation y Table -1 shown the summary of calculation y Table -1 shown the summary of calculation PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 - Q5 : Person, use equation - 6 ( operating hour = 10 ) Q3 = 10 × 240 × 8 / 24 = 800 W/day - Q6 : Product(sensible), use equation - 7 ( Cp of product= 3.56 kJ/kg. C o ) 3600 24 ) 4 35 ( 10 56 .3 10 550 Q 3 3 6 × − × × × × = = 343750 W 6. Daily load distribution Curves Load calculation with ∆T and ∆m ( new method) Load calculation with ∆T and ∆m ( new method) - Let ∆T per day = 2 C o Therefore number of days required (Factor- A) = (35-4)/2 = 15.5 day y - Mass charging per day (m) = 110 ton Therefore number of days required (Factor- B) = 550/110 = 5 day In general, these are the obtained simplified equations Peak load = Base Load + External oad y - Load calculation Q1=0.34×2× (400+375) (where ∆T=2 ○C ) = 527 W 24 3600 1000 87 .4 5. 2962 27 .1 Q2 × × × × = - Load calculation Q1=0.34×2× (400+375) (where ∆T=2 ○C ) = 527 W + B×Q7 + minimum of (A or B) ×Q6 + B Q7 + minimum of (A or B) Q6 Peak load Day = maximum of (A or B) In order to verify the validity of the above equation, it can be applied on the case study example 24 3600 1000 87 .4 5. 2962 27 .1 Q2 × × × × = • Base load= Q3 + Q4 + Q5 = 17.36 kW/day • Base load= Q3 + Q4 + Q5 = 17.36 kW/day = 212 W Q3, Q4, Q5 same as before Q6 using equation-7 and equation-8, with ∆T=2 ○C and m=110 ton • External load = Q1 + Q2 = 15.41 kW/day • External load = Q1 + Q2 = 15.41 kW/day • B × Q7 = 5 × 343.7 = 1718.5 kW/day • B × Q7 = 5 × 343.7 = 1718.5 kW/day y • Min(A, B) × Q6 = 5 × 9.05 = 45.3 kW/day • Min(A, B) × Q6 = 5 × 9.05 = 45.3 kW/day 3600 24 2 10 56 .3 10 110 Q 3 3 6 × × × × × = = 9064.8 W 3600 24 10 270 10 110 Q 3 3 7 × × × × = 3600 24 2 10 56 .3 10 110 Q 3 3 6 × × × × × = = 9064.8 W 3600 24 10 270 10 110 Q 3 3 7 × × × × = y Total load = 17.36 + 15.41 + 1718.5 Total load = 17.36 + 15.41 + 1718.5 +45.3 =1796.57( kW/day) and the peak load day is 20. +45.3 =1796.57( kW/day) and the peak load day is 20. +45.3 =1796.57( kW/day) and the peak load day is 20. PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load In The Large Cold Stores A Simplified Method For Estimating The Peak Load In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 Eng. & Tech. Journal ,Nol.27, No. 10, 2009 The result of the total peak load above is about 1796 kW/day, whenever, the peak load calculated with traditional method listed in table-1 is 2454 kW/day. i.e. the actual peak load (suggested) is about 26% less than the other (traditional). calculation and the probability of wrong estimation of the load in large cold stores which causes a loss of money by choosing over size machine to overcome the load due to the overestimate of the cooling load. References MarAjans, Istanbul, Turkey, 1993 MarAjans, Istanbul, Turkey, 1993 [5] Searle engineers hand book, cold room calculation, A division of Hall – Thermo tank product, London office,1988 [6]Air-Conditioning & Refrirration Institute, Refrigeration and Air- conditioning, Second Edition, Prentice- Hall, Inc., Englewood Cliffs, 1987 [7] ASHRAE Handbook, Refrigeration, American Society of Heating, Refrigeration and Air-Conditioning Engineers, Inc. SI Edition. 1998 [8] M.Eltawil, D.Samuel and O.Singhal, Potato storage Technology and store Design Aspect, Agricultural Engineering International, the CIGRE journal Invited Overview No. 11, Vol. VIII, April, 2006. [7] ASHRAE Handbook, Refrigeration, American Society of Heating, Refrigeration and Air-Conditioning Engineers, Inc. SI Edition. 1998 In general, the peak load will decrease (practically is preferable) when the value of A and B is large; if there are no technical limitations. References [1] Design of Room cooling Facilities structural and energy Requirements, North Carolina State University, Department of Biological and Agricultural Engineering, Post harvest commodity series, A6-414-2,1988 [1] Design of Room cooling Facilities structural and energy Requirements, North Carolina State University, Department of Biological and Agricultural Engineering, Post harvest commodity series, A6-414-2,1988 [2] Jing Xie, Xiao-Hua Qu, Jun-yesh and Da-wen sun, Effect Of Design Parameter on Flow and Temperature Field of a Cold store by CFD simulation, Journal of Food Engineering, Volume 77, Issue 2, November 2005 [3] S. Akdemir, Designing of Cold Store and Choosing of Cooling system Elements, Journal of Applied Sciences 8(5):788-794, 2008 [1] Design of Room cooling Facilities structural and energy Requirements, North Carolina State University, Department of Biological and Agricultural Engineering, Post harvest commodity series, A6-414-2,1988 [2] Jing Xie, Xiao-Hua Qu, Jun-yesh and Da-wen sun, Effect Of Design Parameter on Flow and Temperature Field of a Cold store by CFD simulation, Journal of Food Engineering, Volume 77, Issue 2, November 2005 [3] S. Akdemir, Designing of Cold Store and Choosing of Cooling system Elements Journal of Applied Sciences [2] Jing Xie, Xiao-Hua Qu, Jun-yesh and Da-wen sun, Effect Of Design Parameter on Flow and Temperature Field of a Cold store by CFD simulation, Journal of Food Engineering, Volume 77, Issue 2, November 2005 The final peak load equation shows that the last term is the major factor affected on the result of the peak load calculation therefore the relation between 1/A or ∆T/ ∆T Tot and 1/B or m/m Tot can be plotted against the increasing in the load as a percentage of product load (Q6), as shown in figure (6). [3] S. Akdemir, Designing of Cold Store and Choosing of Cooling system Elements, Journal of Applied Sciences 8(5):788-794, 2008 [4] Erol N., Fundamental method for refrigeration and air-conditioning, Istanbul Technical university, Alummi Association of vocational school of Maritime, [4] Erol N., Fundamental method for refrigeration and air-conditioning, Istanbul Technical university, Alummi Association of vocational school of Maritime, Figure (6) shows the increasing of 1/A (increases the ratio of daily temperature difference to the total of temperature difference(∆T/∆T Tot )causes increasing the peak load while the value of 1/B is constant until it reaches its maximum value when A=B. As mentioned above the load ratio increases when 1/B is significantly increased with constant value of 1/A. 24 3600 1000 87 .4 5. 2962 27 .1 Q2 × × × × = From Table-3 and figures (1) to (5), in some cases it appears that peak load duration might not be exceeded more than one day and then reduced gradually, that means the possibility to pass (avoid) this period of highly load in this day by changing the storing method. Table-3 enables the store manager or staff to determine previously the value and time of peak load to take the necessary technical arrangements. 7. Conclusions The result of applying the present prediction method of peak load estimation reveals the significant difference between the two method of PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 Table (1) Summary of load calculation Q Load type kW/day 1 2 External Load 12.121 3.29 3 4 5 Base Load 1.56 15.0 0.8 6 7 Internal (Product) Load 702.5 1718.7 TOTAL 2454 Table (1) Summary of load calculation Table (2) The chosen cases for percentage of the daily storing mass and the temperature difference Table (2) The chosen cases for percentage of the daily storing mass and the temperature difference Case ∆T/∆T Tot % Factor-A (day) m/m Tot % Factor-B (day) 1 25 4 10 10 2 10 10 25 4 3 50 2 50 2 4 33 3 10 10 5 20 5 50 2 Table (3) A summary of the result case Peak load day Nominal load starting day Peak load (W) Nominal Load (W) A=B A or B A+B Base load + External Load + B*Q7+A*Q6 Base load + External Load + B*Q7 A>B A A+B Base load + External Load + B*Q7+B*Q6 Base load + External Load + B*Q7 B>A B A+B Base load + External Load + B*Q7+A*Q6 Base load + External Load + B*Q7 Table (3) A summary of the result PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Loa In The Large Cold Stores 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Respiration Load External Load Base Load Figure (1) Load distribution case No. 1 (A= 4 , B= 10) 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Respiration Load External Load Base Load Figure (2) Load distribution case No .2 (A=10, B=4) Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Load In The Large Cold Stores 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Respiration Load External Load Base Load Figure (1) Load distribution case No. 1 (A= 4 , B= 10) Figure (1) Load distribution case No. 1 (A= 4 , B= 10) 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Respiration Load External Load Base Load Figure (2) Load distribution case No .2 (A=10, B=4) 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Respiration Load External Load Base Load 0 20 40 60 80 100 120 140 Load ( not to scale ) Figure (2) Load distribution case No .2 (A=10, B=4) PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Load In The Large Cold Stores 0 10 20 30 40 50 60 70 80 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale) Product Load Resipration Load External Load Base Load Figure (3) Load distribution case No. 3 (A=2, B=2) Figure (4) Load distribution case No. 4 (A= 3 , B=10) 0 20 40 60 80 100 120 140 160 180 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Respiration Load External Load Base Load Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Load In The Large Cold Stores 0 10 20 30 40 50 60 70 80 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale) Product Load Resipration Load External Load Base Load Figure (3) Load distribution case No. 3 (A=2, B=2) Figure (3) Load distribution case No. 3 (A=2, B=2) Figure (4) Load distribution case No. 4 (A= 3 , B=10) 0 20 40 60 80 100 120 140 160 180 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Respiration Load External Load Base Load Figure (4) Load distribution case No. 4 (A= 3 , B=10) PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Loa In The Large Cold Stores 0.0 20.0 40.0 60.0 80.0 100.0 1/A % Product Load Increse % 1/B=100 % 1/B=75 % 1/B=50 % 1/B=25 % 1/B=16.5 % 1/B=12.5 % 1/B=10 % 100 75 50 25 10 12 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Resipration Load External Load Base Load Figure (5) Load distribution case No. 5 (A= 5 , B=2) Figure (6) Percentage of product load (Q6) increasing with respect to 1/A & 1/B Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Load In The Large Cold Stores 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Day Load ( not to scale ) Product Load Resipration Load External Load Base Load Figure (5) Load distribution case No. 5 (A= 5 , B=2) 0.0 20.0 40.0 60.0 80.0 100.0 1/A % Product Load Increse % 1/B=100 % 1/B=75 % 1/B=50 % 1/B=25 % 1/B=16.5 % 1/B=12.5 % 1/B=10 % 100 75 50 25 10 12 Figure (5) Load distribution case No. 5 (A= 5 , B=2) Figure (6) Percentage of product load (Q6) increasing with respect to 1/A & 1/B 0.0 20.0 40.0 60.0 80.0 100.0 1/A % Product Load Increse % 1/B=100 % 1/B=75 % 1/B=50 % 1/B=25 % 1/B=16.5 % 1/B=12.5 % 1/B=10 % 100 75 50 25 10 12 Figure (5) Load distribution case No. 5 (A= 5 , B=2) Figure (6) Percentage of product load (Q6) increasing with respect to 1/A & 1/B PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com
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Rendering near-field speckle statistics in scattering media
ACM transactions on graphics
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Rendering Near-Field Speckle Statistics in Scattering Media Rendering Near-Field Speckle Statistics in Scattering Media CHEN BAR, Department of Electrical Engineering, Technion, Israel IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA ANAT LEVIN, Department of Electrical Engineering, Technion, Israel OD = 1 OD = 5 OD = 10 Tissue Sensor Image of illum i1 Image of illum i2 Fig. 1. Rendering near-field speckle patterns. We propose an efficient and physically-accurate algorithm that can simulate speckle patterns produced by coherent illumination sources located or focused very close to the material. One of the important properties of speckle is the memory effect: small translations of the illuminator produce shifted, highly-correlated speckle patterns. In the figure, we used our algorithm to simulate light scattering in a medium with realistic tissue parameters, at thicknesses of 50 µm, 250 µm, 500 µm, and a mean free path of 50 µm, equivalent to optical depths (OD) of 1, 5 and 10, respectively. In each case the figure presents two speckle images obtained with a small shift of the input illumination, leading to correlated shifted speckle patterns (e.g., the shift is visible in the insets of the first two columns). As expected, when the thickness increases, more scattering is present, and thus the memory-effect correlation becomes weaker (the correlation is less visible in the inset of the third column). CHEN BAR, Department of Electrical Engineering, Technion, Israel IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA ANAT LEVIN, Department of Electrical Engineering, Technion, Israel OD = 1 CHEN BAR, Department of Electrical Engineering, Technion, Israel IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA ANAT LEVIN, Department of Electrical Engineering, Technion, Israel OD 1 CHEN BAR, Department of Electrical Engineering, Technion, Israel IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA ANAT LEVIN, Department of Electrical Engineering, Technion, Israel OD = 1 Image of illum i1 Image of illum i2 Fig. 1. Rendering near-field speckle patterns. We propose an efficient and physically-accurate algorithm that can simulate speckle patterns produced by coherent illumination sources located or focused very close to the material. One of the important properties of speckle is the memory effect: small translations of the illuminator produce shifted, highly-correlated speckle patterns. In the figure, we used our algorithm to simulate light scattering in a medium with realistic tissue parameters, at thicknesses of 50 µm, 250 µm, 500 µm, and a mean free path of 50 µm, equivalent to optical depths (OD) of 1, 5 and 10, respectively. Rendering Near-Field Speckle Statistics in Scattering Media In each case the figure presents two speckle images obtained with a small shift of the input illumination, leading to correlated shifted speckle patterns (e.g., the shift is visible in the insets of the first two columns). As expected, when the thickness increases, more scattering is present, and thus the memory-effect correlation becomes weaker (the correlation is less visible in the inset of the third column). We introduce rendering algorithms for the simulation of speckle statistics observed in scattering media under coherent near-field imaging conditions. Our work is motivated by the recent proliferation of techniques that use speckle correlations for tissue imaging applications: The ability to simulate the image measurements used by these speckle imaging techniques in a physically-accurate and computationally-efficient way can facilitate the widespread adoption and improvement of these techniques. To this end, we draw inspiration from recently-introduced Monte Carlo algorithms for rendering speckle statistics under far-field conditions (collimated sensor and illumination). We derive variants of these algorithms that are better suited to the near-field conditions (focused sensor and illumination) required by tissue imaging applications. Our approach is based on using Gaussian apodization to approximate the sensor and illumination aperture, as well as von Mises-Fisher functions to approximate the phase function of the scattering material. We show that these approximations allow us to derive closed-form expressions for the focusing operations involved in simulating near-field speckle patterns. As we demonstrate in our experiments, these approximations accelerate speckle rendering simulations by a few orders of magnitude compared to previous techniques, at the cost of negligible bias. We validate the accuracy of our algorithms by reproducing ground truth speckle statistics simulated using wave-optics solvers, and real-material measurements available in the literature. Finally, we use our algorithms to simulate biomedical imaging techniques for focusing through tissue. Authors’ addresses: Chen Bar, Department of Electrical Engineering, Technion, Haifa, Israel; Ioannis Gkioulekas, Robotics Institute, Carnegie Mellon University, Pittsburgh, PA, USA; Anat Levin, Department of Electrical Engineering, Technion, Haifa, Israel. CCS Concepts: • Computing methodologies →Computational photography; Rendering. Additional Key Words and Phrases: Monte Carlo rendering, speckle, memory effect ACM Reference Format: Chen Bar, Ioannis Gkioulekas, and Anat Levin. 2020. Rendering Near-Field Speckle Statistics in Scattering Media. ACM Trans. Graph. 39, 6, Article 187 (December 2020), 26 pages. https://doi.org/10.1145/3414685.3417813 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 ACM 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. © 2020 Association for Computing Machinery. 0730-0301/2020/12-ART187 $15.00 https://doi.org/10.1145/3414685.3417813 ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 1 INTRODUCTION A core challenge in medicine is the development of technologies for imaging deep inside biological tissues at high spatial resolutions. What makes this type of imaging possible is the fact that, when a light source illuminates tissue, a significant amount of light enters, travels inside, and re-emerges out of the tissue. What makes this type of imaging difficult is the fact that, when inside the tissue, light scatters multiple times. Thus, the fundamental challenge that needs to be solved to enable imaging inside tissue is inverting the multiple scattering process, in order to extract the information that light carries about the tissue it interacted with. 187:2 • Chen Bar, Ioannis Gkioulekas, and Anat Levin In recent years, several imaging techniques have emerged that address this challenge by taking advantage of the fact that images of tissue under coherent (e.g., laser) illumination contain significant speckles: These are pseudo-random, high-frequency spatial varia- tions in the intensity of the captured images. The statistical proper- ties of these speckle patterns is a classical research area within optics, having been the subject of several textbooks [Erf 1978; Goodman 2007; Jacquot and Fournier 2000; Kaufmann 2011]. This research has revealed that, despite its random appearance, a speckle has strong statistical properties that provide rich information about the underlying scattering material (e.g., tissue). Perhaps best known among them is the memory effect (ME) property, illustrated in Fig. 1, which describes how speckle fields remain correlated under small changes in imaging conditions. The memory effect is at the core of speckle-based techniques for tissue imaging applications such as fluorescence imaging and adaptive optics focusing inside tissue. importance sampling scheme for starting the volume path trac- ing process that takes into account the focused beams inside the medium. In our experiments, we validate the physical accuracy of our algorithms by showing that they can reproduce speckle corre- lation statistics simulated using wave-equation solvers, as well as speckle correlation measurements of real materials that are publicly available in the literature [Osnabrugge et al. 2017]. Our algorithms match this synthetic and measured groundtruth more accurately than existing simulation techniques (e.g., multi-slice layer based algorithms [Schott et al. 2015]) and analytical models (e.g., tilt-shift memory effect [Osnabrugge et al. 2017]) from optics. Additionally, we show that our algorithms allow us to simulate focusing-inside- tissue applications, which require near-field conditions. 1.1 Why render near-field speckle statistics? Speckle statistics have strong potential for applications in the con- text of tissue imaging, where scattering by cells and other variations of the local index of refraction in the tissue drastically degrade image contrast. For example, several papers have suggested using speckle correlations to detect incoherent fluorescence sources inside the tissue. As Katz et al. [2014] have observed, due to the memory effect, the auto-correlation of random speckle images, together with a phase retrieval algorithm, can help remove the effect of scattering and reveal the location of the sources under the skin. Unfortunately, this idea has been successfully demonstrated mostly in the far- field setting, with the sources located at a large distance outside the scattering medium, rather than inside it. The only successful demonstration of this idea in the near-field we are aware of is by Chang et al. [2018], whose experiments were able to recover fluo- resent particles spanning a small spatial range of 10 µm. We argue that this state of affairs is due to the limited exploration of near-field speckle statistics, an issue we hope our paper will help address. For this, we draw inspiration from Bar et al. [2019], who intro- duced a Monte Carlo framework for simulating speckle correlations in a way that combines physical accuracy and computational ef- ficiency. Unfortunately, despite offering orders-of-magnitude ac- celeration compared to previous physically-accurate simulation techniques (e.g., wave equation solvers), the algorithms of Bar et al. [2019] are primarily intended for simulating far-field speckle sta- tistics, and remain impractical for the near-field case. For example, as we show in our experiments, using their framework to simulate current techniques for focusing inside scattering [Judkewitz et al. 2014] can take several days on a large cluster. These performance characteristics significantly constrain the scope of investigations that can be performed using these algorithms (e.g., evaluation of effectiveness of existing techniques under different imaging param- eters or for tissue samples of different optical parameters). Another important application of speckle techniques in tissue imaging is the use of adaptive optics [Mertz et al. 2015] to focus light at points deep inside tissue. Achieving this type of focusing requires using a coherent wavefront of a shape specific to the tissue sample being imaged. Determining the exact wavefront is challeng- ing, and typically involves using external information or a guiding star [Horstmeyer et al. 2015]. 1 INTRODUCTION For the small volumes we simulate, our algorithm is already 3000× faster than an adaptation of the far-field algorithm [Bar et al. 2019]; and this performance difference will only increase for real-sized volumes. We believe that the ability to simulate near-field speckle statistics outside the lab will accelerate ongoing research on speckle-based techniques for tissue imaging applications. To fortify this effort, we have made our implementation publicly available [Bar et al. 2020]. l g g p p g Unfortunately, most previous studies of speckle statistical proper- ties and of the memory effect, have been performed under imaging conditions that are not suitable for tissue imaging. Typically, most studies assume that both the light sources and the sensors are out- side and at a large distance from the scattering volume, a set of conditions referred to as far-field imaging. By contrast, tissue imag- ing applications require both sources and sensors to be focused very close to the tissue (e.g., confocal microscopy), or even located inside it (e.g., fluorescent particles), a set of conditions known as near-field imaging. Far-field imaging simplifies analysis, simulation, and experiments relating to speckles. However, inferences drawn for the far-field case do not necessarily generalize to the near-field one. Our goal is to develop physically-accurate and efficient render- ing algorithms that can help improve our understanding of speckle statistical properties under near-field conditions. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 1.1 Why render near-field speckle statistics? Once this wavefront is found, the memory effect can be used to scan an area inside tissue, e.g., by shifting and tilting the wavefront to focus at neighboring points [Os- nabrugge et al. 2017]. In our experiments, we simulate this approach, and show preliminary investigations on the effectiveness of using the tilt-shift memory effect. By enabling researchers to perform such investigations in simulation, without the need for lab experiments, we hope that our paper can help expand the scanning range and operational capabilities of techniques for focusing inside tissue.fi With these considerations in mind, we develop a computationally- efficient algorithm for simulating near-field (focused) speckle sta- tistics inside scattering media. To this end, we extend the Monte Carlo rendering algorithm of Bar et al. [2019] in several ways that make it better-suited for near-field simulations. Our innovations are three-fold: First, we derive a path-integral expression for near- field speckle statistics. Second, we approximate optical apertures and material phase functions using von Mises-Fisher functions, to obtain analytical expressions for connecting paths traced inside a volume to near-field sources and sensors. Third, we develop an p p q g Furthermore, the ability to efficiently render speckle patterns can facilitate the widespread adoption of data-driven approaches in Rendering Near-Field Speckle Statistics in Scattering Media • 187:3 limitation is due to the fact that our algorithms use volume path trac- ing to sample light paths, and is thus shared with standard Monte Carlo volume rendering algorithms for simulating incoherent inten- sity [Novak et al. 2018]. As in the intensity case, techniques based on the diffusion approximation [Jensen et al. 2001] would be better- suited for simulating speckle statistics at volumes of larger optical depths. However, we note that near-field speckle correlations decay as a function of optical depth much faster than intensity does; and are essentially non-existent for volumes of optical depths signif- icantly larger than what we simulate. Consequently, biomedical imaging techniques based on speckle correlations are typically only applicable for optical depths below the diffusive regime, coincid- ing with the range of optical depths we emphasize in our experi- ments. Considering that neither single-scattering [Narasimhan et al. 2006] nor diffusion approximations [Feng et al. 1988] are effective in this range, our algorithms provide a suitable simulation tool for researchers investigating these biomedical imaging techniques. tissue imaging. 1.2 Limitations Our algorithms are subject to a few limitations that suggest im- portant directions for future research. First, they assume that the simulated scattering medium is homogeneous, meaning that its optical scattering parameters are the same at all spatial locations inside the volume. Our algorithms additionally assume that the medium is exponential, meaning that it comprises uncorrelated scat- terers [Bitterli et al. 2018; d’Eon 2018; Jarabo et al. 2018]; and scatters light isotropically, meaning that its phase function is rotationally- invariant [Jakob et al. 2010]. These assumptions are commonly used in biomedical imaging research and applications to approximate how biological tissues scatter light at optical and infrared wave- lengths. Therefore, our algorithms can be used to simulate imaging applications involving such materials. Second, our algorithms are primarily geared towards transmission mode imaging configurations, where illumination and sensing hap- pen at opposite sides of a scattering volume. This is a consequence of our use of von Mises-Fisher functions to approximate the imaging aperture and material phase function. When using these approxi- mations to simulate reflection mode imaging configurations, where illumination and sensing are on the same side of a scattering volume, accuracy will depend on the exact material parameters. In particu- lar, when simulating materials with phase functions that have very little back-scattering, accuracy suffers because of numerical issues. We provide a detailed analysis in App. A.4. In practical terms, this means that our algorithms can be used to simulate tissue imaging applications such as fluorescence imaging and focusing through tissue, both of which correspond to transmissive configurations; but should be used with caution for applications such as confocal microscopy, which corresponds to a reflective configuration.fi Analytical models for speckle statistics. Quantifying differences in speckle characteristics between the near-field and far-field cases can be done using analytical tools that approximate speckle statistics with closed-form mathematical expressions. Most available such tools are for the far-field case [Akkermans and Montambaux 2007; Baydoun et al. 2016; Berkovits and Feng 1994; Dougherty et al. 1994; Feng et al. 1988; Freund and Eliyahu 1992; Fried 1982], though re- cently tools have been introduced for the near-field case [Judkewitz et al. 2014; Osnabrugge et al. 2017]. For example, Osnabrugge et al. [2017] derived a tilt-shift model for the memory effect, assuming that the underlying scattering material is optically thin and very forward-scattering. 2 RELATED WORK Speckle in computational imaging. Speckle statistics have found wide applicability in computational imaging. Example applications include motion tracking [Jacquot and Rastogi 1979; Jakobsen et al. 2012; Smith et al. 2017], looking around the corner [Batarseh et al. 2018; Freund 1990; Katz et al. 2012], and seeing through [Abooka- sis and Rosen 2004; Bertolotti et al. 2012; Katz et al. 2014; Rosen and Abookasis 2003; Takasaki and Fleischer 2014] or focusing through [Choi et al. 2011; Edrei and Scarcelli 2016; Katz et al. 2010, 2012; Lai et al. 2015; Mosk et al. 2012; Nixon et al. 2013; Rueckel et al. 2006; van Putten et al. 2011; Vellekoop and Aegerter 2010; Vellekoop et al. 2012, 2010; Vellekoop and Mosk 2007; Yaqoob et al. 2008] tissue and other scattering layers. Most of these imaging techniques rely on the memory effect of speckles, and therefore are based on spatial correlations between speckle images. Alternatively, imaging tech- niques such as diffusing wave spectroscopy [Pine et al. 1988], laser speckle contrast imaging [Boas and Yodh 1997], and dynamic light scattering [Goldburg 1999] use temporal speckle correlations [Berne and Pecora 2000; Dougherty et al. 1994] to estimate flow (e.g., blood flow [Durduran et al. 2010]) and liquid composition parameters. 1.1 Why render near-field speckle statistics? The use of such approaches is in part motivated by analogous successes in the far-field case, where it has been demonstrated that machine learning algorithms can improve the performance of memory-effect-based imaging around the corner and through scattering [Li et al. 2018; Metzler et al. 2020, 2018]. The successful deployment of machine learning algorithms requires large, physically-accurate datasets. Previously, the lack of physically- accurate simulation tools meant that datasets had to be collected using lab measurements, an approach that is not scalable. We hope that our rendering tools can help reduce the data collection over- head, making machine learning approaches tractable. Last but not least, accurate speckle rendering algorithms can be useful for inverse rendering problems involving speckle mea- surements. In particular, prior work has shown that differentiable rendering techniques can be used to recover accurate scattering pa- rameters of real-world materials from incoherent intensity measure- ments [Gkioulekas et al. 2013]. We expect that our algorithms can be combined with modern differentiable rendering techniques [Nimier- David et al. 2020; Zhang et al. 2020, 2019]. In turn, this has the potential to enable recovering accurate high-resolution models of important material classes, such as biological tissue, from measure- ments of speckle fields and speckle correlations. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 1.2 Limitations Deriving closed-form expressions requires re- strictive assumptions (single scattering, diffusion, or Fokker-Planck limits), which typically do not apply to the predominantly-turbid Third, our algorithms become inefficient for very optically-deep (e.g., more than 10 times the mean free path) volumes, where light is expected to perform a large number of scattering events. This 187:4 • Chen Bar, Ioannis Gkioulekas, and Anat Levin 𝐨𝐨 𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒 ෡𝒊𝒊𝟏𝟏 ෡𝒊𝒊𝟐𝟐 ෞ 𝒗𝒗𝟏𝟏 ෞ 𝒗𝒗𝟐𝟐 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺 𝒊𝒊𝟏𝟏 𝒊𝒊𝟐𝟐 𝒗𝒗𝟐𝟐 𝒗𝒗𝟏𝟏 (a) Directional (b) Focused ෡𝒊𝒊𝟏𝟏 ෡𝒊𝒊𝟐𝟐 ෞ 𝒗𝒗𝟏𝟏 ෞ 𝒗𝒗𝟐𝟐 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺 𝒊𝒊𝟏𝟏 𝒊𝒊𝟐𝟐 𝒗𝒗𝟐𝟐 𝒗𝒗𝟏𝟏 (c) Directional paths (d) Focused paths Fig. 2. Transmissive imaging configurations. Previous work [Bar et al. 2019] simulated speckle statistics by directional sources and sensors, as in (a). In contrast this work considers speckles by focused camera and sources, as in (b). (c-d) demonstrate what is implied when evaluating such speckle correlations using a Monte Carlo algorithm. In the directional formulation, one draws 4 directional connections from each path toward the illumination and viewing directions. In contrast, to simulate speckles through a focused lens, one needs to trace all paths via the aperture. 𝐨𝐨 𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒 ෡𝒊𝒊𝟏𝟏 ෡𝒊𝒊𝟐𝟐 ෞ 𝒗𝒗𝟏𝟏 ෞ 𝒗𝒗𝟐𝟐 (a) Directional ෡𝒊𝒊𝟏𝟏 ෡𝒊𝒊𝟐𝟐 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺 𝒊𝒊𝟏𝟏 𝒊𝒊𝟐𝟐 𝒗𝒗𝟐𝟐 𝒗𝒗𝟏𝟏 (b) Focused 𝒊𝒊𝟏𝟏 𝒊𝒊𝟐𝟐 tissue scattering. Developing efficient and physically-accurate ren- dering tools for near-field speckle statistics can help assess the accu- racy of these approximations, and facilitate their broader adoption in application regimes where they are accurate. Rendering wave-optics effects. Monte Carlo rendering techniques within computer graphics have typically focused on simulating inco- herent light transport. More recently, there have been a few works on simulating wave optics effects, such as diffraction and speckle due to rough surface geometry [Bergmann et al. 2016; Cuypers et al. 2012; Sadeghi et al. 2012; Stam 1999; Sur et al. 2018; Werner et al. 2017; Yan et al. 2018; Yeh et al. 2013]. Variants of volumetric path tracing for simulating speckle in scattering have appeared in op- tics [Lu et al. 2004; Mout et al. 2016; Pan et al. 1995; Sawicki et al. 2008; Schmitt and Knüttel 1997; Xu 2004], though these typically can- not estimate second-order statistics (e.g., the memory effect). Bar et al. [2019] addressed this shortcoming by introducing a Monte Carlo algorithm that uses simultaneous path connections to multiple light sources and sensors, to correctly model speckle correlations between them. 3 BACKGROUND ON SPECKLE STATISTICS We begin by providing background on speckle statistics and on their Monte Carlo modeling for the far-field case. We use this background in Sec. 4 to contrast the far-field and near-field cases, which helps highlight the challenges involved in rendering the latter case, and positions our work relative to the prior art of Bar et al. [2019]. relative to MFP. For example, a volume has optical depth OD = 2 if its thickness is equal to 2 · MFP, meaning that light travelling through the volume undergoes on average two scattering events.i Notation and setting. We use bold letters for vectors (e.g., points o, i, v), with a circumflex for unit vectors (e.g., directions ˆω,ˆi, ˆv). We consider scattering volumes V ∈R3 that satisfy the assump- tions underlying classical radiative transfer for isotropic [Jakob et al. 2010] exponential media [Bitterli et al. 2018; d’Eon 2018; Jarabo et al. 2018]: Each volume comprises a set of scatterers, whose locations in the volume are statistically independent. These scatterers are assumed to be small enough relative to the wavelength of light to be considered infinitesimal points. They are also assumed to be spherically symmetric, and thus scatter incident light waves in a rotationally-invariant way. We model speckle fields arising in such volumes due to incident illumination that we assume to be monochromatic, fully-coherent and unpolarized. These fields are a function of the volumes’ bulk properties, which we describe next. The scattering amplitude function s(cosθ) describes how a field interacts with a scatterer: if a scatterer is illuminated from direction ˆi, the complex scattered field u at direction ˆv is uˆi ˆv = s(ˆi · ˆv). The phase function is defined as ρ(cosθ) ≡|s(cosθ)|2. It is commonly characterized by an anisotropy parameter −1 ≤д ≤1, equal to the average cosθ: д = 0 corresponds to scattering equally in all direc- tions, and д = 1 to fully forward scattering. Tissue is characterized by very forward scattering (д > 0.9) [Cheong et al. 1990]. These parameters are a function of wavelength, and the scatterers’ shape, size, and refractive index. For spherical scatterers, they can be computed using Mie theory [Bohren and Huffman 1983; Frisvad et al. 2007]. The three coefficients also depend on the density ς, equal to the expected number of scatterers in a unit volume. 1.2 Limitations Unfortunately, their algorithm is primarily tailored to far-field imaging and becomes inefficient for the near-field case, as discussed in Sec. 3. The difference between the two cases is reminiscent of the challenges in rendering depth-of-field effects in incoherent light transport, where special rendering algorithms are required due to the need to sample many rays on the aperture plane [Barsky and Kosloff 2008; Kolb et al. 1995; Soler et al. 2009]. (a) Directional𝒊𝟏𝒊𝟐 ෞ 𝒗𝒗𝟏𝟏 ෞ 𝒗𝒗𝟐𝟐 (c) Directional paths 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺 𝒗𝒗𝟐𝟐 𝒗𝒗𝟏𝟏 (d) Focused paths 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝒗𝟏 (c) Directional paths (d) Focused paths Fig. 2. Transmissive imaging configurations. Previous work [Bar et al. 2019] simulated speckle statistics by directional sources and sensors, as in (a). In contrast this work considers speckles by focused camera and sources, as in (b). (c-d) demonstrate what is implied when evaluating such speckle correlations using a Monte Carlo algorithm. In the directional formulation, one draws 4 directional connections from each path toward the illumination and viewing directions. In contrast, to simulate speckles through a focused lens, one needs to trace all paths via the aperture. Fig. 2. Transmissive imaging configurations. Previous work [Bar et al. 2019] simulated speckle statistics by directional sources and sensors, as in (a). In contrast this work considers speckles by focused camera and sources, as in (b). (c-d) demonstrate what is implied when evaluating such speckle correlations using a Monte Carlo algorithm. In the directional formulation, one draws 4 directional connections from each path toward the illumination and viewing directions. In contrast, to simulate speckles through a focused lens, one needs to trace all paths via the aperture. 3 BACKGROUND ON SPECKLE STATISTICS We assume that scattering volumes are spatially homogeneous, meaning that scatterers are uniformly distributed, or equivalently, that the bulk parameters are the same everywhere inside a volume. Bulk material properties. We use a statistical description of the optical properties of scattering volumes. In particular, the scattering and absorption coefficients σs and σa model, respectively, the portion of energy that is scattered and absorbed upon interaction with a scatterer. Their sum is the extinction coefficient σt ≡σa + σs, and its inverse is the mean free path, MFP = 1/σt , which is the average distance in the volume light travels between two scattering events. Given a volume V, it is common to express its geometric dimensions ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 3.1 Modeling and rendering far-field speckle statistics Transmissive far-field imaging. We focus on the geometry illus- trated in Fig. 2(a): Scatterers are placed at a configuration of locations Rendering Near-Field Speckle Statistics in Scattering Media • 187:5 than wave-equation solvers, and scalable to much larger volumes. We proceed to review this Monte Carlo rendering approach. O = {o1, o2, . . .} inside the volume V, each sampled independently from the others, using the volume density ς. This configuration is imaged using light sources and sensors that are on opposite sides of the volume, a setting we refer to as transmission mode imaging. Additionally, in this background section, we assume that the volume is illuminated by a directional plane wave ˆi, and imaged with a direc- tional sensor ˆv. We refer to these conditions together as the far-field imaging conditions. If we know the exact scatterer locations, and incoming and outgoing directions, we can solve the wave equation to obtain the complex-valued scattered field uˆi,O ˆv , arising from the interaction of the incident illumination with the scattering volume. Covariance rendering. Bar et al. [2019] derive their algorithm from a path integral expression for speckle covariance, obtained by considering the correlation of fields that travel along all possible pairs of paths from ˆi1 to ˆv1, and from ˆi2 to ˆv2. They showed that this expression can be simplified to use only pairs of paths that coincide everywhere, except for their connections to ˆi1, ˆv1,ˆi2, ˆv2. We review this simplified formulation. Consider the space P of sub- paths ®xs = o1 →· · · →oB, B ≥1, where each vertex ob ∈V; we denote by ˆωb ≡œ ob, ob+1 the direction of the b-th edge of the sub-path. These vertices correspond to the shared part of two full paths ®x1 = ˆi1→o1→. . .→oB →ˆv1, ®x2 = ˆi2→o1→. . .→oB →ˆv2, formed by connecting the sub-path to ˆi1, ˆv1 and ˆi2, ˆv2. Then, the speckle covariance of Eq. (2) can be expressed as: Defining speckle statistics. For any volume with a given scatterer configuration O, the scattered field typically contains large fluctu- ations with a semi-random noise structure known as speckle (see, e.g., Fig. 1). We can characterize speckle using the first and second- order statistics of fields due to different volumes with the same bulk material properties. 3.1 Modeling and rendering far-field speckle statistics In particular, we can define the speckle mean, Cˆi1,ˆi2 ˆv1,ˆv2 = ∫ P cˆi1,ˆi2 ˆv1,ˆv2(®xs) d®xs, (3) (3) mˆi ˆv ≡EO h uˆi,O ˆv i , (1) (1) where the far-field path contribution function cˆi1,ˆi2 ˆv1,ˆv2 equals the cor- relation of the fields that travel along ®x1, ®x2. For B ≥2, this equals: where the far-field path contribution function cˆi1,ˆi2 ˆv1,ˆv2 equals the cor- relation of the fields that travel along ®x1, ®x2. For B ≥2, this equals: and the speckle covariance, Cˆi1,ˆi2 ˆv1,ˆv2 ≡EO h uˆi1,O ˆv1 · uˆi2,O ˆv2 ∗i −mˆi1 ˆv1 · mˆi2 ˆv2 ∗ , (2) cˆi1,ˆi2 ˆv1,ˆv2(®xs) = f (®xs) · υ(ˆi1→o1)s(ˆi1 · ˆω1) · υ(oB→ˆv1)s( ˆωB−1 · ˆv1) · υ(ˆi2→o1)∗s(ˆi2 · ˆω1)∗ · υ(oB→ˆv2)∗s( ˆωB−1 · ˆv2)∗, (4) cˆi1,ˆi2 ˆv1,ˆv2(®xs) = f (®xs) · υ(ˆi1→o1)s(ˆi1 · ˆω1) (2) · υ(oB→ˆv1)s( ˆωB−1 · ˆv1) · υ(ˆi2→o1)∗s(ˆi2 · ˆω1)∗ · υ(oB→ˆv1)s( ˆωB−1 · ˆv1) where (·)∗is complex conjugation.uˆi1,O ˆv1 ,uˆi2,O ˆv2 are two speckle fields generated by the same scatterer configuration O, when illuminated by two monochromatic, mutually-coherent incident waves from ˆi1,ˆi2, and measured at two sensors ˆv1, ˆv2. The expectation EO is taken with respect to all scatterer configurations O sampled from the same density ς. As we detail in App. A.1, the speckle mean mˆi ˆv can be computed using a closed-form expression and is typically zero. Th f f d li th kl i Cˆi1 ˆi2 · υ(oB→ˆv2)∗s( ˆωB−1 · ˆv2)∗, (4) and for B = 1: and for B = 1: cˆi1,ˆi2 ˆv1,ˆv2(®xs) = σs · υ(ˆi1→o1)υ(o1→ˆv1)s(ˆi1 · ˆv1) · υ(ˆi2→o1)∗υ(o1→ˆv2)∗s(ˆi2 · ˆv2)∗. (5) · υ(ˆi2→o1)∗υ(o1→ˆv2)∗s(ˆi2 · ˆv2)∗. (5) (5) Therefore, we focus on modeling the speckle covariance Ci1,i2 ˆv1 ˆv2.i v ,v The definition of Eq. (2) suggests a straightforward approach for computing this covariance: randomly sample many scatterer con- figurations O from the material bulk parameters, solve the wave equation numerically to compute uˆi1,O ˆv1 ,uˆi2,O ˆv2 , and use averaging to approximate the expectation in Eq. (2). Unfortunately, while exact wave-equation solvers exist [Thierry et al. 2015; Treeby and Cox. 2010; Yee 1966], their computational complexity is prohibitive, typi- cally making them intractable for volumes of width larger than a few dozen wavelengths. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 3.1 Modeling and rendering far-field speckle statistics This computational cost is further exacerbated by the need to use the solvers multiple times for averaging.i In the above, f (®xs) is the standard radiometric throughput of ®xs, augmented by scattering coefficients at the first and last vertex, f (®xs) = (σs)B B−1 Ö b=1 e−σt ∥ob+1−ob ∥ ∥ob+1 −ob ∥2 ρ( ˆωb−1 · ˆωb). (6) (6) Finally, υ(·) is the complex volumetric throughput, defined as: υ( ˆω→o) = e−1 2 σtd( ˆω→o)eik( ˆω ·o), (7) υ(o→ˆω) = e−1 2 σtd(o→ˆω)e−ik( ˆω ·o), (8) (7) (7) (8) (8) (8) For an alternative, note first that, when ˆi1 = ˆi2 = ˆi, ˆv1 = ˆv2 = ˆv and mˆi ˆv = 0, Cˆi,ˆi ˆv,ˆv reduces to the intensity Iˆi ˆv. This intensity is typ- ically modeled in computer graphics using the radiative transfer equation, or its integral form, the volume rendering equation. The latter gives rise to Monte Carlo volume rendering algorithms, which compute intensity using as input only the volume’s bulk mate- rial properties [Novak et al. 2018]. Bar et al. [2019] derived analo- gous Monte Carlo volume rendering algorithms for computing the speckle covariance Cˆi1,ˆi2 ˆv1,ˆv2 for any directions ˆi1,ˆi2, ˆv1, ˆv2. Like their intensity counterparts, these algorithms take as input bulk material parameters, and not particle positions. Bar et al. [2019] showed that their approach is physically accurate, orders-of-magnitude faster where k ≡2π/λ is the wavenumber and λ the wavelength of the illumination; and d( ˆω →o),d(o →ˆω) denote the distance a ray entering or leaving, respectively, o at direction ˆω, travels inside the scattering volume V. Fig. 2(c) visualizes these terms. The covariance rendering algorithm of Bar et al. [2019] uses a Monte Carlo path sampling approach to evaluate the speckle covariance integral of Eq. (3). This algorithm takes advantage of the presence of the radiometric throughput term in Eq. (4), and samples sub-paths ®xs using standard volumetric path tracing. Then, for each sampled sub-path, the endpoints o1, oB are connected to the far-field illuminations ˆi1, ˆi2 and sensors ˆv1, ˆv2, to compute the complex volumetric throughput terms in Eqs. (4) and (5). ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 4 NEAR-FIELD SPECKLE STATISTICS As mentioned in the introduction, many important applications require imaging speckle fields using a sensor that is focused at some point, rather than being at infinity; and using illumination that is better modeled as a point than as a directional source. Both of these points can be near or even inside the scattering volume. To distin- guish them from the far-field case of the previous section, we refer to these conditions together as the near-field imaging conditions. Scattered fields formed under far-field and near-field conditions often have very different characteristics, as we discuss in the rest of the section. As in Sec. 3.1, we focus on transmission mode imag- ing configurations, where illumination and sensing are on opposite sides of the volume. The imaging configuration is shown in Fig. 2(b). N = 2W sin(Θmax) λ . (12) (12) For some representative numbers, we consider simulating a rel- atively thin volume of thickness 400 µm. We assume that we im- age a spatial area of size 100 µm × 100 µm using a numerical aper- ture sin(Θmax) = 0.5 and illumination wavelength λ = 0.5 µm. We need to simulate a volume that is at least as wide as the de- focused beam, as light contributing to the scattered field can be due to scattering anywhere inside the beam (see Fig. 3). For the dimensions mentioned, the defocus blur has size 400 µm at the far edge of the target, suggesting that we need to simulate a volume of widthW = 100 µm+400 µm = 500 µm. Then Eq. (12) suggests using N = 1000 samples on each coordinate axis of the four directions we integrate in Eq. (11). Therefore, to simulate the near-field covari- ance, we need (1000)8 samples, which is prohibitively large. We note that real near-field scenes used in, e.g., Osnabrugge et al. [2017] are bigger and would require an even more far-field samples. Modeling near-field speckle statistics. We use the notation uˆi ˆv,uiv to indicate scattered fields due to far-field and near-field imaging conditions, respectively, with the circumflex distinguishing between directional to point sources/sensors. If we know the scattered field uˆi ˆv for all illumination and sensing directions ˆi, ˆv, we can compute the scattered fielduiv by integrating over the sensor and illumination apertures (Fig. 2(d)) [Goodman 1968; Mertz 2019]. 3.1 Modeling and rendering far-field speckle statistics d Anat Levin 187:6 • Chen Bar, Ioannis Gkioulekas, and Anat Levin We note that when ˆi1 = ˆi2 = ˆi, ˆv1 = ˆv2 = ˆv, the above algorithm becomes equivalent to the standard volumetric path tracing algo- rithm used in computer graphics to render intensity Iˆi ˆv [Novak et al. 2018]. A key observation by Bar et al. [2019] is that, in the case of covariance Cˆi1,ˆi2 ˆv1,ˆv2, each sampled sub-path ®xs needs to contribute to two pairs of input-output directions (blue and green connections in Fig. 2(c)). Using the same sub-paths for both (ˆi1, ˆv1), (ˆi2, ˆv2) pairs is necessary to account for the correlation of fields from different illuminator-viewpoint combinations, and this correlation cannot be modeled using paths sampled independently for each pair. transform with respect to the variables ˆi and ˆv, and thus uˆi ˆv is often referred to as the Fourier field [Goodman 1968; Mertz 2019].i 1 2i The near-field speckle covariance Ci1,i2 v1,v2 can be defined analo- gously to the far-field covariance in Eq. (2). Combining this defini- tion with Eq. (9), we can relate near-field and far-field covariances: Ci1,i2 v1,v2 = ∫∫∫∫ av1(ˆv1)ai1(ˆi1)av2(ˆv2)∗ai2(ˆi2)∗Cˆi1,ˆi2 ˆv1,ˆv2dˆi1dˆi2dˆv1dˆv2. (11) (11) Why is rendering near-field covariance difficult? Eq. (11) provides a conceptually simple way to compute near-field covariance statistics: We sample, or discretize, the domains of ˆv1, ˆv2,ˆi2,ˆi1, estimate the corresponding far-field covariance values as in Sec. 3.1, and form their weighted average using the weights in Eq. (11). Before concluding this section, we mention that Bar et al. [2019] present a variant of the above-described algorithm that simulates scattered fields uˆi1 ˆv1, uˆi2 ˆv2 in a way that accurately reproduces their correlations. In the rest of the paper, we show how to adapt both of these rendering algorithms to the near-field case. We present most of our theory in Secs. 4-6 in the context of covariance rendering, then adapt it in Sec. 7 for field rendering. In practice, this approach is computationally impractical, because of the very large number of samples of the far-field covarianceCˆi1,ˆi2 ˆv1,ˆv2 we need to compute. This number is determined by the width W of the volume V and the numerical aperture Θmax, and not by the size of the sensor. As we show in App. A.2, Nyquist sampling rate implies the number of samples per axis of integration should be: ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 4 NEAR-FIELD SPECKLE STATISTICS Concretely: gg qi p Our discussion so far has been about covariance rendering, but similar arguments apply for field rendering, where using Eq. (9) would require N 4 samples, or (1000)4 for the above example. We dis- cuss the field rendering case in detail in Sec. 7, but for demonstration, we compare in Fig. 3 speckle images rendered by our proposed field rendering algorithm and using Eq. (9). Due to memory constraints, the far-field directions were sampled at 10% of the Nyquist sampling rate; this aliased sampling results in clear replica artifacts in the cor- responding images. Even under these aliased sampling conditions, rendering using Eq. (9) was 100× slower than our algorithm, while requiring 30 GB GPU memory. These challenges cannot be allevi- ated by using a large number of samples for single scattering and fewer samples for multiple scattering, as is often done in intensity rendering [Belcour et al. 2014]: even though intensity images from multiple scattering have low spatial frequency, coherent multiple scattering still creates high-frequency speckle patterns. ui v = ∫ ˆi∈S2 ∫ ˆv∈S2 ai(ˆi)av(ˆv)uˆi ˆv dˆv dˆi, (9) (9) where S2 is the unit sphere and, assuming an ideal lens, av(ˆv) ≡m(ˆv)eik(ˆv·v), ai(ˆi) ≡m(ˆi)e−ik ˆi·i . (10) (10) The functions m(ˆi) and m(ˆv) denote illumination and viewing aperture amplitudes. Typically, these are binary functions indicating which directions pass through an aperture of a finite extent. We indicate the width of these masks using the angle Θmax between the optical axis and the propagating direction that most deviates from this axis, and we refer to sin Θmax as the numerical aperture (NA) (Fig. 3(a)). The term exp(ik(ˆv · v)) is the phase accumulated in direction ˆv when focusing at the point v; and analogously for the term exp(−ik(ˆi · i)). Using paraxial optics approximations, it is also possible to express the two integrals of Eq. (9) as a double Fourier ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. Rendering Near-Field Speckle Statistics in Scattering Media • 187:7 F.F. image with i1 F.F. image with i2 N.F. image with i1 N.F. image with i2 𝜽 𝟒𝟎𝟎𝝁𝒎 𝟓𝟎𝟎𝝁𝒎 𝟒𝟎𝟎𝝁𝒎 image area 𝟏𝟎𝟎𝝁𝒎 NA: 𝐬𝐢𝐧𝜽= 𝟎. 𝟓 viewing objective (a) (b) (c) Fig. 3. Near-field challenges. Complex volumetric throuput : Complex volumetric throuput : Aperture function focused at ˆi : Aperture function × throughput : Scattering amplitude function: Convolved aperture function: (throughput up to first scatterer) 𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐨𝐨𝟏𝟏) ∫ i∈S𝐢 (a) ˜ai(ˆi, o1) (b) ϒ( ˆω1, o s ϒ( ˆω|o1, i) = ˜ai( ˆω|o1) ∗s ˜av( ˆω|o1) ˜av( ˆω|o1) · ϒ( ˆω|o1, i) (c) (a) ˜ai(ˆi, o1) (b) ϒ( ˆω1, o1, i) ˜av( ˆω|o1) · ϒ( ˆω|o1, i) ˜av( ˆω|o1) ˜av( ˆω|o1) ˜ai( ˆω|o1) Fig. 4. Notation summary and visualization. (a) The aperture function ˜ai(ˆi, o1) includes an apodization mask m(ˆi), a complex wave focusing at i and a conjugate wave focusing at o1, the first point on the sampled path (attenuation exp(−1/2σtd( ˆω →o)) is not visualized). (b) The aperture function convolved angulary with the scattering amplitude function leads to ϒ( ˆω1, o1, i), the throughput up to the first point and direction. That is, to compute the field propagating from o1 at direction ˆω1 we integrate over all directions ˆi in the aperture. For each direction we consider the value of the aperture function in this direction, times the amount of energy scattered from ˆi to ˆω1. (c) Visualizing the pipeline of the single-scattering covariance in Eq. (18). Illumination aperture ˜ai is convolved with scattering function s to generate ϒ( ˆω, o1, i), which is then multiplied with the viewing aperture ˜av. For visualization the real component of the involved spherical functions is projected onto the 2D ˆωx , ˆωy plane (north hemisphere only). where now the contribution of each sub-path is determined by the near-field path contribution function ci1,i2 v1,v2. For B ≥2, this equals: 4 NEAR-FIELD SPECKLE STATISTICS Note the ME shift demonstrated in the insets.𝒎𝐢 𝒎𝒎( ̂𝐢𝐢) 𝐨𝐨𝟏𝟏 𝐢𝐢 𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐢𝐢) 𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐨𝐨𝟏𝟏) (a) ˜ai(ˆi, o1) Complex volumetric throuput : υ(ˆi→o) = e−1 2 σtd( ˆω→o)eik( ˆω ·o), Aperture function focused at ˆi : ai(ˆi)=m(ˆi)e−ik ˆi·i Aperture function × throughput : ˜ai(ˆi, o1) = ai(ˆi)υ(ˆi→o) Scattering amplitude function: s( ˆω1 · ˆω2) Convolved aperture function: ϒ( ˆω, o1, i) = ∫ ˆi∈S2 ˜ai(ˆi, o1)s(ˆi · ˆω) (throughput up to first scatterer) 𝒎𝒎( ̂𝐢𝐢) 𝐨𝐨𝟏𝟏 𝐢𝐢 𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐢𝐢) 𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐨𝐨𝟏𝟏) 𝐨𝐨𝟏𝟏 ෡𝐢𝐢𝟏𝟏 𝒔𝒔( ̂𝐢𝐢ȉ ෝ𝝎𝝎) ෡𝐢𝐢𝟐𝟐 ෡𝐢𝐢𝟑𝟑 ෞ 𝛚𝛚𝟏𝟏 (a) ˜ai(ˆi, o1) (b) ϒ( ˆω1, o1, i) ˜ai( ˆω|o1) s ϒ( ˆω|o1, i) = ˜ai( ˆω|o1) ∗s ˜av( ˆω|o1) ˜av( ˆω|o1) · ϒ( ˆω|o1, i) (c) Fig. 4. Notation summary and visualization. (a) The aperture function ˜ai(ˆi, o1) includes an apodization mask m(ˆi), a complex wave focusing at i and a conjugate wave focusing at o1, the first point on the sampled path (attenuation exp(−1/2σtd( ˆω →o)) is not visualized). (b) The aperture function convolved angulary with the scattering amplitude function leads to ϒ( ˆω1, o1, i), the throughput up to the first point and direction. That is, to compute the field propagating from o1 at direction ˆω1 we integrate over all directions ˆi in the aperture. For each direction we consider the value of the aperture function in this direction, times the amount of energy scattered from ˆi to ˆω1. (c) Visualizing the pipeline of the single-scattering covariance in Eq. (18). Illumination aperture ˜ai is convolved with scattering function s to generate ϒ( ˆω, o1, i), which is then multiplied with the viewing aperture ˜av. For visualization the real component of the involved spherical functions is projected onto the 2D ˆωx , ˆωy plane (north hemisphere only). 𝐨𝐨𝟏𝟏 ෡𝐢𝐢𝟏𝟏 𝒔𝒔( ̂𝐢𝐢ȉ ෝ𝝎𝝎) ෡𝐢𝐢𝟐𝟐 ෡𝐢𝐢𝟑𝟑 ෞ 𝛚𝛚𝟏𝟏 (b) ϒ( ˆω1, o1, i) 4 NEAR-FIELD SPECKLE STATISTICS Consider an imaging setup as in (a) where one wants to image a 100 µm × 100 µm area through a 400 µm thick tissue with a 0.5N A objective. The resulting imaging cone is 400 µm wide. As scattering can arise from anywhere inside the imaging cone, for realistic simulation the medium should be at least as wide as the defocus cone, requiring us to simulate a slab of wider than 500 µm. Assuming e.g. λ = 0.5 µm, this results in N = 1000 samples in each axis. (b) Two speckle images generated by two nearby illuminators, rendered using far-field covariances, sampled at 10% of the Nyquist limit revealing aliasing. The simulation still runs 100× slower than our near-field approach, and occupies as much as 30 GB GPU global memory. (c) Aliasing-free speckle images by our suggested near field approach. Note the ME shift demonstrated in the insets.𝒎𝐢 N.F. image with i1 N.F. image with i2 (c) N.F. image with i2 c) 𝜽 𝟒𝟎𝟎𝝁𝒎 𝟓𝟎𝟎𝝁𝒎 𝟒𝟎𝟎𝝁𝒎 image area 𝟏𝟎𝟎𝝁𝒎 NA: 𝐬𝐢𝐧𝜽= 𝟎. 𝟓 viewing objective (a)i F.F. image with i1 (b F.F. image with i2 b) N.F. image with i (c (b) (c) (a)i Fig. 3. Near-field challenges. Consider an imaging setup as in (a) where one wants to image a 100 µm × 100 µm area through a 400 µm thick tissue with a 0.5N A objective. The resulting imaging cone is 400 µm wide. As scattering can arise from anywhere inside the imaging cone, for realistic simulation the medium should be at least as wide as the defocus cone, requiring us to simulate a slab of wider than 500 µm. Assuming e.g. λ = 0.5 µm, this results in N = 1000 samples in each axis. (b) Two speckle images generated by two nearby illuminators, rendered using far-field covariances, sampled at 10% of the Nyquist limit revealing aliasing. The simulation still runs 100× slower than our near-field approach, and occupies as much as 30 GB GPU global memory. (c) Aliasing-free speckle images by our suggested near field approach. 4.1 Near-field covariance path integral where now the contribution of each sub-path is determined by the near-field path contribution function ci1,i2 v1,v2. For B ≥2, this equals: To overcome the computational challenges of evaluating near-field speckle covariance, we first derive for it a path-integral expression, which will absorb the directional integrations of Eq. (11) into the path contribution function. Then, in Sec. 5, we introduce an approx- imation that allows us to compute this path contribution function analytically. This completely removes the need for directional inte- gration, drastically reducing computational complexity.i ci1,i2 v1,v2(®xs) = f (®xs) · ϒ( ˆω1, o1, i1)ϒ( ˆωB−1, oB, v1) · ϒ( ˆω1, o1, i2)∗ϒ( ˆωB−1, oB, v2)∗, ( (14) where ϒ denotes integration over the aperture of terms in Eq. (4): where ϒ denotes integration over the aperture of terms in Eq. (4): denotes integration over the aperture of terms in Eq. ( ϒ( ˆω1, o1, i) ≡ ∫ ˆi∈S2 ˜ai(ˆi, o1)s(ˆi · ˆω1) dˆi, (15) ϒ( ˆωB−1, oB, v) ≡ ∫ ˆv∈S2 ˜av(ˆv, oB)s( ˆωB−1 · ˆv) dˆv, (16) (15) By combining Eq. (3) and Eq. (11), we can express the near-field covariance as a path integral on the same space of sub-paths P: and ˜a denotes weighed aperture functions a: and ˜a denotes weighed aperture functions a: Ci1,i2 v1,v2 = ∫ P ci1,i2 v1,v2(®xs) d®xs, (13) (13) ˜ai(ˆi, o1) ≡ai(ˆi)υ(i→o1), ˜av(ˆv, oB) ≡av(ˆv)υ(oB→ˆv). (17) (17) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 187:8 • Chen Bar, Ioannis Gkioulekas, and Anat Levin Similarly, for B = 1, the contribution function equals: in a narrow function that is sharply peaked around µr, whereas γr = 0 results in a uniform function over the sphere. Von Mises- Fisher functions have found use in various applications in computer graphics [Han et al. 2007], though we emphasize that, compared to this prior work, we use complex functions (γi , 0). ci1,i2 v1,v2(®xs) = σs(o1) · ∫ ˆv1∈S2 ˜av1(ˆv1, o1)ϒ(ˆv1, o1, i1) dˆv1 · ∫ ˆv2∈S2 ˜av2(ˆv2, o1)ϒ(ˆv2, o1, i2) dˆv2 ∗ . (18) (18) Von Mises-Fisher functions can be thought of as a generalization of the Gaussian distribution to the unit sphere, with variance σ2 = 1 γ [Mardia and Jupp 2000]. 4.1 Near-field covariance path integral Intuitively, we can see that this is the case by considering that, when we constrain ∥ˆµ∥= ∥ˆω∥= 1, We use ˜ai(ˆi|o1), ϒ( ˆω1|o1, i) to denote versions of these functions with respect to only their first argument, conditioned on fixed val- ues for their other arguments. These are complex functions on the unit sphere S2. In particular, ϒ( ˆω1|o1, i) can be thought of as a convo- lution of the aperture function ˜ai(ˆi|o1) with the scattering amplitude function s(ˆi · ˆω). We summarize and visualize these terms in Fig. 4. e−γ 2 ∥ˆω−ˆµ ∥2 ∝e−γ ( ˆµ· ˆω), (22) e−γ 2 ∥ˆω−ˆµ ∥2 ∝e−γ ( ˆµ· ˆω), (22) where ∝denotes equality up to a multiplicative scale. where ∝denotes equality up to a multiplicative scale. With Eqs. (14) and (18) at hand, conceptually we can compute the near-field covariance Ci1,i2 v1,v2 using a Monte Carlo rendering al- gorithm exactly analogous to the one proposed by Bar et al. [2019] for the far-field case: First, we sample sub-paths ®xs using standard volumetric path tracing. Second, we compute the path contribution function for each sampled path, and accumulate the results. We note however that, unlike the far-field case, in the near-field case the path contribution function cannot be computed analytically, as it requires spherical integration for evaluating ϒ. Theoretically, this could be done using a second-stage Monte Carlo integration procedure, by importance sampling one or more directions in the aperture plane. However, as we show experimentally in Sec. 8, be- cause these integrals have complex integrands with highly-varying phases, Monte Carlo estimates have very high variance. Considering that this second-stage Monte Carlo integration procedure needs to be performed separately for each sampled sub-path ®xs, it quickly results in an overwhelming computational overhead, making this overall rendering procedure intractable. In the next section, we side- step this overhead by deriving closed-form approximations to these integration and convolution operations, which can be computed analytically without the need for Monte Carlo integration. 5.1 Working with von Mises-Fisher functions Fitting with von Mises-Fisher functions. We aim to approximate the spherical functions that appear in the near-field path contribution function ci1,i2 v1,v2(®xs) using von Mises-Fisher functions.ii We first consider the aperture function ˜ai(ˆi), defined in Eqs. (10) and (17). We reproduce the definition here for convenience: ˜ai(ˆi)=m(ˆi)e−ik(ˆi·i)υ(i→o1)=m(ˆi)e−ik(ˆi·i)−1 2 σtd( ˆω→o)+ik( ˆω ·o). (23) (23) We want to express ˜ai(ˆi) as a von Mises-Fisher function. To achieve this, we first choose to approximate the aperture mask m(·) as a real von Mises-Fisher function. Denoting the optical axis of the system by ˆµa (usually this is the z-axis ˆµa = ˆz = [0, 0, 1]), we have, m( ˆω) ≈e−γaeγa( ˆµa · ˆω). (24) (24) This approximation is a form of apodization: a binary aperture, which completely blocks or transmits fields propagating in different directions, is replaced by a non-binary mask, which attenuates the amplitude of transmitted fields by an increasing amount at larger propagation angles. Such non-binary apertures are typical of sys- tems that use short-focal-length lenses with strong aberrations. On the illumination side, these non-binary apertures are also represen- tative of the Gaussian profiles of laser beams. As we show in Sec. 8, even when the underlying aperture is binary, using the apodiza- tion of Eq. (24) produces accurate speckle statistics for transmissive imaging configurations. We note that the mean width of the non- binary aperture equals σa = 1/√γa, and should be set to match the width of the true binary aperture. Additionally, we can use γa = 0 to model isotropic point sources (e.g., fluorescent particles). ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 5 NEAR-FIELD USING VON MISES-FISHER FUNCTIONS g=0.97 Liquid Clay Coffee Whole Milk Full fitting - 0 - 0 - 0 - 0 - 0 - 0 Lobe zoom - /5 0 /5 - /20 0 /20 - /20 0 /20 - -0.8 - /20 0 /20 - /20 0 /20 2 mixtures 3 mixtures 4 mixtures 6 mixtures G.T. Fig. 5. Phase function fitting. Visualizing the von Mises-Fisher mixture fit of some Henyey Greenstein phase functions as well as real world phase functions measured by [Gkioulekas et al. 2013]. A small number of mixture components provides a good fit even when the phase function includes a back lobe (e.g. liquid clay). The top row shows a full [−π , π] range, and the lower row zooms around the central forward/backward lobe of the function for better visualization. H.G. g=0.2 H.G. g=0.9 H.G. g=0.97 Full fitting - 0 - 0 - 0 Lobe zoom - /5 0 /5 - /20 0 /20 - /20 0 /20 2 mixtures 3 mixtures Liquid Clay Coffee Whole Milk - 0 - 0 - 0 - -0.8 - /20 0 /20 - /20 0 /20 4 mixtures 6 mixtures G.T. ome Henyey Greenstein phase functions as well as real world phase functions H.G. g=0.97 4 mixtures 6 mixtures G.T. Fig. 5. Phase function fitting. Visualizing the von Mises-Fisher mixture fit of some Henyey Greenstein phase functions as well as real world phase functions measured by [Gkioulekas et al. 2013]. A small number of mixture components provides a good fit even when the phase function includes a back lobe (e.g. liquid clay). The top row shows a full [−π , π] range, and the lower row zooms around the central forward/backward lobe of the function for better visualization. of Banerjee et al. [2005] to approximate s as a mixture of von Mises- Fisher functions centered at ˆi: of Banerjee et al. [2005] to approximate s as a mixture of von Mises- Fisher functions centered at ˆi: where βo = q µ + γs ˆωo, co = βo −γs βo (µ · ˆωo). (32) (32) s(ˆi, ˆv) ≈ Õ m πmeγs,m(ˆi·ˆv). (28) (28) We discuss the selection of ˆωo in App. A.3, but the simplest strategy is to select it as the direction at the center of the viewing aperture. We compare approximated and exact convolutions in App. A.4. 5 NEAR-FIELD USING VON MISES-FISHER FUNCTIONS In this section, we present two main technical results. First, we show how to use mixtures of von Mises-Fisher functions to approximate the various spherical functions that appear in the near-field path con- tribution function ci1,i2 v1,v2(®xs) of Eqs. (14) and (18). Second, we show how this approximation allows us to derive closed-form expressions for the convolution and other integral terms in ci1,i2 v1,v2(®xs). Overall, this allows us to compute covariance contribution analytically for each sampled sub-path ®xs, avoiding Monte Carlo integration. The phase terms in Eq. (23) already form a complex von Mises- Fisher function. Thus, to complete our treatment of ˜ai(ˆi), we need to add the attenuation term, for which we assume that the attenuation is approximately constant over the aperture: Von Mises-Fisher functions. We begin with background on complex von Mises-Fisher functions, defined as [Mardia and Jupp 2000]: e−0.5σtd( ˆω→o) ≈e−0.5σtd( ˆµa→o). (25) (25) h( ˆω) = η · e(µ· ˆω), (19) (19) Putting things together, we approximate the aperture function as: Putting things together, we approximate the aperture function as: s together, we approximate the aperture function as: ˜ai( ˆω, o) ≈η · eµ· ˆω, (26) ˜ai( ˆω, o) ≈η · eµ· ˆω, (26) (26) where ˆω ∈S2, and µ = µr + iµi is a complex three-dimensional vector parameterizing the von Mises-Fisher function. We define: where ˆω ∈S2, and µ = µr + iµi is a complex three-dimensional vector parameterizing the von Mises-Fisher function. We define: with γr = ∥µr∥, γi = ∥µi ∥, γ = ∥µ∥= γr −γi + 2i(µr · µi), (20) ˆµ = µ/γ, ˆµr = µr/γr, ˆµi = µi/γi. (21) η = e−γa−1 2 σtd( ˆµa→o1), µ = γa ˆµa + ik(o1 −i). (27) (27) (21) We approximate the viewing aperture function similarly. We approximate the viewing aperture function similarly. The value |h( ˆω)| is maximized when ˆω = ˆµr. The scaleγr is inversely proportional to the support of the function: Large γr values result The value |h( ˆω)| is maximized when ˆω = ˆµr. The scaleγr is inversely proportional to the support of the function: Large γr values result We now turn our attention to the scattering amplitude function s in Eqs. (14) and (18). We use the expectation-maximization algorithm Rendering Near-Field Speckle Statistics in Scattering Media • 187:9 H.G. g=0.2 H.G. g=0.9 H.G. 5 NEAR-FIELD USING VON MISES-FISHER FUNCTIONS Note that, since the scattering amplitude function is approximated by a mixture of von Mises-Fisher functions, we need to compute the convolution with each mixture element separately. In Fig. 5, we show fits of this kind for Henyey-Greenstein phase func- tions, as well as real-world phase functions measured by Gkioulekas et al. [2013]; in all cases, the phase function is accurately approxi- mated using a small number of mixture components. Integration and convolution of von Mises-Fisher functions. The approximations we derived facilitate computing the covariance in- tegrals Eqs. (14) and (18). In particular, these evaluation can now be done analytically, without the need for Monte Carlo integration, using the properties of von Mises-Fisher functions.i 5.2 Visualizing convolutioni The function ϒ( ˆω, o1, i), defined in Eq. (15) as the convolution of the aperture function with the scattering function, equals the through- put of a path up until the first scattering event. This function encodes the contribution of a path starting at node o1 and emerging from it at direction ˆω. This function is a fundamental building block of the near-field correlation, and as such we study and visualize its struc- ture. In the next section, we use this to devise importance sampling schemes for accelerating our Monte Carlo rendering algorithms. We consider first the spherical integration in Eq. (18). By approx- imating the integrand as a von Mises-Fisher function, as described above, we can compute this integral analytically using: ∫ ˆω ∈S2 ηeµ· ˆω = η · 4π sinh(√µ) √µ = η · 2π e √µ −e−√µ √µ . (29) (29) Spatial structure. The aperture function ˜ai(ˆi; o1) is complex, and its phase depends on the distance between the focus point i and location o1 of the first scatterer in the path (Fig. 4(a)). When the phase variation is rapid, blurring this complex function will reduce the magnitude to zero. We want to understand for which o1 positions the throughput contribution ϒ( ˆω, o1, i) is not zero. This will be valuable for defining an importance sampling strategy that avoids sampling o1 in areas receiving no energy. To this end we define In this equation, √µ equals: √µ ≡ q µ2x + µ2y + µ2z. (30) (30) where each term in the summation involves complex square power rather than squared amplitude, and thus √µ is a complex number. We now consider the spherical convolution in Eq. (14). We use the same approach as for integration, and replace the two functions that are being convolved with their approximation in terms of von Mises-Fisher functions we derived above. The resulting convolution of two von Mises-Fisher functions is also available through a simple analytical form. We note that, even though the exact convolution result is not itself a von Mises-Fisher function, it can be accurately approximated as one, as is necessary to facilitate subsequent com- putation steps in our rendering algorithm. In particular, in App. A.3, we show that the convolution can be approximated as: e(o|i) ≡ ∫ ˆω ∈S2 |ϒ( ˆω, o, i)| . (33) (33) Fig. 6 visualizes the shape of e(o|i) for an x −z volume slice. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 5.2 Visualizing convolutioni This is similar to the optics concept of a Gaussian beam [Yariv 1997], focused at i. Considering that our illumination is a beam focused at i, it is expected that scattering points o1 that are not located in the area of the illumination beam will not receive light. However, while the shape of a focused beam depends only on the aperture width γa, e(o|i) also depends on the width of the scattering function γs. In particular, assuming for ease of notation that the aperture axis is Fig. 6 visualizes the shape of e(o|i) for an x −z volume slice. This is similar to the optics concept of a Gaussian beam [Yariv 1997], focused at i. Considering that our illumination is a beam focused at i, it is expected that scattering points o1 that are not located in the area of the illumination beam will not receive light. However, while the shape of a focused beam depends only on the aperture width γa, e(o|i) also depends on the width of the scattering function γs. In particular, assuming for ease of notation that the aperture axis is ∫ ˆψ ∈S2 e(µ· ˆψ) · eγs( ˆω · ˆψ) ≈2π βo e γs βo (µ· ˆω)+co, (31) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 187:10 • Chen Bar, Ioannis Gkioulekas, and Anat Levin North South North South North South North South North South γa = 16; γs = 2 ˜a -0.5 0 0.5 -0.5 0 0.5 -0.5 0 0.5 -0.5 0 0.5 -0.5 0 0.5 s -0.2 0 0.2 -0.2 0 0.2 -0.2 0 0.2 -0.2 0 0.2 -0.2 0 0.2 ˜a ∗s -0.1 0 0.1 -0.01 0 0.01 -0.005 0 0.005 -0.01 0 0.01 -0.001 0 0.001 γa = 16; γs = 2 ˜a -0.5 0 0.5 -0.5 0 0.5 -0.5 0 0.5 -0.5 0 0.5 -0.5 0 0.5 s -10 0 10 -10 0 10 -10 0 10 -10 0 10 -10 0 10 ˜a ∗s -0.5 0 0.5 -0.5 0 0.5 -0.5 0 0.5 -0.2 0 0.2 -0.1 0 0.1 Fig. 6. Visualizing spatial and angular throughput components. Left: e(o|i), the energy of path starting points, as an x −z slice through the volume. This resembles the shape of a Gaussian beam, which is narrow at the focus plane and wide at out-of-focus depths. 5.2 Visualizing convolutioni Right: the angular part of the throughput, ϒ( ˆω |o, i) = ˜a ∗s as a function of direction ˆω, for a subset of o positions marked with corresponding colors on the Gaussian beams. These are functions on the 3D sphere, and we show the north (forward) and south (backward) hemispheres projected on the ˆωx −ˆωy plane. The directions with high throughput shift for points o at the periphery of the beam (e.g., orange and pink points). We display only the real part of these complex spherical functions aligned with the north pole ˆµa = ˆz, we show in App. A.5 that: aligned with the north pole ˆµa = ˆz, we show in App. A.5 that: Mises-Fisher functions, showing good agreement. The directions with highest power after convolution can shift, and may not be located at the center of the sphere (e.g., the orange and pink points in lower panel). This happens at the periphery of the beam. Below we use the directional density ϒ( ˆω|i, o) for importance sampling. e(o|i) = G(oxy |oz, i) = β(z)e −∥oxy −ix,y ∥2 2w(z)2 , (34) with with w(z;γs,γa) = s γa + γs k2 + z2 γa , (35) β(z) = e−1 2 σtd( ˆz→oz)π k2γ 2a(γs + γa) 2w(z;γs,γa)2 eγa+γs . (36) (35) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 5.2 Visualizing convolutioni The panels visualize two scattering functions defined by γs = 2 (wide) and γs = 100 (narrow), showing that the beam waist is wider for narrow forward-scattering phase functions. Right: the angular part of the throughput, ϒ( ˆω |o, i) = ˜a ∗s as a function of direction ˆω, for a subset of o positions marked with corresponding colors on the Gaussian beams. These are functions on the 3D sphere, and we show the north (forward) and south (backward) hemispheres projected on the ˆωx −ˆωy plane. The directions with high throughput shift for points o at the periphery of the beam (e.g., orange and pink points). We display only the real part of these complex spherical functions. ˜a ∗s γa = 16; γs = 2 ˜a -0.5 0 0.5 s -10 0 10 ˜a ∗s -0.5 0 0.5 -0.5 0 0.5 -10 0 10 -0.1 0 0.1 -0.5 0 0.5 -10 0 10 -0.5 0 0.5 Fig. 6. Visualizing spatial and angular throughput components. Left: e(o|i), the energy of path starting points, as an x −z slice through the volume. This resembles the shape of a Gaussian beam, which is narrow at the focus plane and wide at out-of-focus depths. The panels visualize two scattering functions defined by γs = 2 (wide) and γs = 100 (narrow), showing that the beam waist is wider for narrow forward-scattering phase functions. Right: the angular part of the throughput, ϒ( ˆω |o, i) = ˜a ∗s as a function of direction ˆω, for a subset of o positions marked with corresponding colors on the Gaussian beams. These are functions on the 3D sphere, and we show the north (forward) and south (backward) hemispheres projected on the ˆωx −ˆωy plane. The directions with high throughput shift for points o at the periphery of the beam (e.g., orange and pink points). We display only the real part of these complex spherical functions. Fig. 6. Visualizing spatial and angular throughput components. Left: e(o|i), the energy of path starting points, as an x −z slice through the volume This resembles the shape of a Gaussian beam, which is narrow at the focus plane and wide at out-of-focus depths. The panels visualize two scattering functions defined by γs = 2 (wide) and γs = 100 (narrow), showing that the beam waist is wider for narrow forward-scattering phase functions. 7 FIELD RENDERING So far we focused on evaluating speckle covariance. A complemen- tary question is how to directly render speckle images. Suppose, for example, that we want to generate Ni images from Ni different illu- mination points, where each image includes Nv sensor points. This requires sampling Ni,v = Ni · Nv complex numbers. One approach for this is to evaluate the corresponding covariance matrix of size Ni,v × Ni,v, and then use it to sample values. However, for large Ni,v, the covariance matrix can be impractically large. Instead, we seek an algorithm with complexity O(Ni,v) rather than O(N 2 i,v). g p y , i,v For this, we follow Bar et al. [2019], who note that the covariance matrix as given by Eq. (3), (14) and (18) is essentially an infinite summation of rank-1 matrices, decomposed over the path space Cik ,im vk ,vm = ∫ P f (®xs)b(ik, vk)b(im, vm)∗d®xs, (43) for all (k,m) pairs k,m ∈{1, . . . , Ni,v}, with b(ik, vk) = ( ∫ ˆv∈S2 ˜avk (ˆv, o1)ϒ(ˆv, o1, ik), B = 1, ϒ( ˆω1, o1, ik)ϒ( ˆωB, oB, vk), B ≥2. (44) Cik ,im vk ,vm = ∫ P f (®xs)b(ik, vk)b(im, vm)∗d®xs, (43) Cik ,im vk ,vm = ∫ P f (®xs)b(ik, vk)b(im, vm)∗d®xs, (43) for all (k,m) pairs k,m ∈{1, . . . , Ni,v}, with b(ik, vk) = ( ∫ ˆv∈S2 ˜avk (ˆv, o1)ϒ(ˆv, o1, ik), B = 1, ϒ( ˆω1, o1, ik)ϒ( ˆωB, oB, vk), B ≥2. (44) (43) b(ik, vk) = ( ∫ ˆv∈S2 ˜avk (ˆv, o1)ϒ(ˆv, o1, ik), B = 1, ϒ( ˆω1, o1, ik)ϒ( ˆωB, oB, vk), B ≥2. (44) (44) Despite the fact that this rank-one decomposition is over- complete, we can use it to sample from the covariance matrix. We sample sub-paths ®xs ∼p(®xs), using the same importance function as in Sec. 6. The field is updated using b(ik, vk), resulting in Bar et al. [2019] sample sub-paths from a distribution p(®xs) ∝f (®xs), where the first node o1 and direction ˆω1 are sampled uniformly. This uniform sampling strategy can be problematic, because the term c (Eq. (37)) includes the throughput ϒ of the start and end segments. In particular, as discussed in Sec. 5.2, the spatial part of ϒ has a shape similar to a Gaussian beam (Fig. 6). The first scattering point is sampled from We can importance sample the last segment when iluminators are more than camera pixels, or importance sample both the first and last segment in bidirectional algorithms. as in standard volumetric path tracing. The sampling scheme is summarized in Alg. 1, and we provides details in App. A.6. We note that we choose to importance sample the first segment of the path rather than the last one because, for most imaging configurations in this paper, we had a small number of illuminators and a large number of camera pixels. We can importance sample the last segment when iluminators are more than camera pixels, or importance sample both the first and last segment in bidirectional algorithms. The first scattering point is sampled from ALGORITHM 1: Monte Carlo rendering of covariance Ci1,i2 v1,v2. ◃Initialize covariance estimate. Set C = 0. for iteration = 1 : N do ◃Sample a subpath: ◃Sample first vertex from the sum of Gaussian beams. Sample beam index j ∈{1, 2} Sample point o1 ∼e(o1 |ij) . ◃Update covariance with single scattering path. Update C += 1 p(o1) ∫ ˆv1∈S2 ˜av1(ˆv1, o1)ϒ(ˆv1, o1, i1) · ∫ ˆv2∈S2 ˜av2(ˆv2, o1)ϒ(ˆv2, o1, i2) ∗ . ◃Sample first direction from the angular throughput. Sample direction ˆω1 ∼|ϒ( ˆω1 |o1, ij)|2. ◃Continue tracing the subpath: ◃Sample second vertex of subpath. Sample distance d ∼σt e−σt d. Set point o2 = o1 + d · ˆω1. Set b = 2. while ob inside medium do ◃Update covariance with next-event estimation. Update C += 1 p(o1)p( ˆω1|o1)ϒ( ˆω1, o1, i1)ϒ( ˆωb−1, ob, v1) ϒ( ˆω1, o1, i2)∗ϒ( ˆωb−1, ob, v2)∗ ◃Sample next vertex of subpath: ◃Sample direction from phase function. Sample direction ˆωb ∼|s( ˆωb−1 · ˆωb)|2. ◃Sample free path. Sample distance d ∼σt e−σt d. ◃Create next vertex of subpath. Set point ob+1 = ob + d · ˆωb. ◃Account for absorption. Sample scalar a ∼Unif[0, 1]. if a > σs/σt then ◃Terminate subpath at absorption event. break end Set b = b + 1. end end ◃Produce final covariance estimate. Update C = 1 N C. return C. ALGORITHM 1: Monte Carlo rendering of covariance Ci1,i2 v1,v2. p(o1) ∝1 2  |e(o1|i1)|2 + |e(o1|i2)|2 . (40)i (40) We implement this sampling by first uniformly sampling one of he two beams in the summand, and then sampling o1 from the Gaussian beam G(oxy |oz, ij) of Eq. (34), where j ∈1, 2 denotes the index of the sampled beam. The first direction is sampled from p( ˆω1|o1) ∝|ϒ( ˆω1|o1, ij)|2. (41) p( ˆω1|o1) ∝|ϒ( ˆω1|o1, ij)|2. (41) The rest of the path is sampled from (41) The rest of the path is sampled from p(o2 →, . . . , →oB |o1, ˆω1) ∝f (o1 →, . . . , →oB), (42) (42) as in standard volumetric path tracing. The sampling scheme is summarized in Alg. 1, and we provides details in App. A.6. We note that we choose to importance sample the first segment of the path rather than the last one because, for most imaging configurations in this paper, we had a small number of illuminators and a large number of camera pixels. 6 IMPORTANCE SAMPLING In Sec. 4.1 the covariance is expressed as an integral over path space, where each path contributes a termc(®xs). For convenience we repeat here the definition for paths of length B ≥2: (36) and z ≡oz −iz. For every z plane, e(o|i) is a planar Gaussian with standard deviation equal to w(oz −iz). The Gaussian is narrowest when oz −iz = 0, that is, when o1 is at the same depth as i. The beam expands at depths away from the focus depth. c(®xs) = f (®xs) · ϒ( ˆω1, o1, i1)ϒ( ˆωB−1, oB, v1) · ϒ( ˆω1, o1, i2)∗ϒ( ˆωB−1, oB, v2)∗. (37) (37) We use Monte Carlo approximation of this integral, by sampling N sub-paths ®xs,n from a distribution p(®xs,n) and computing We use Monte Carlo approximation of this integral, by sampling N sub-paths ®xs,n from a distribution p(®xs,n) and computing Angular structure. Fig. 6 also provides a visualization of the an- gular part of the throughput, namely the variation of ϒ( ˆω|o, i) as a function of ˆω, in a few positions of the first point o. For that, we display the aperture function ˜ai(ˆi|o) as a spherical function (i.e., a function of direction), before and after convolution with s. We com- pare the exact convolution against the approximation based on von C = 1 N Õ n c(®xs,n) p(®xs,n). (38) (38) The quality of this estimator depends on the sampling distribution p, and estimation variance reduces when p closely approximates c. The quality of this estimator depends on the sampling distribution p, and estimation variance reduces when p closely approximates c. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. Rendering Near-Field Speckle Statistics in Scattering Media • 187:11 The first scattering point is sampled from 7 FIELD RENDERING As the beam assigns zero weight to most points in space, uniform sampling of the starting node o1 will produce many paths with zero contribution. When the phase function is narrow, a similar argument holds for the first direction ˆω1. We address this issue by deriving importance sampling strategies for the start node and direction. u(ik, vk) = 1 √ N Õ n b(ik, vk |®xs,n) s f (®xs,n) p(®xs,n) e2πiζn, (45) (45) where ζn ∈[0, 1] is a random phase ensuring E[b(ik, vk |®xs,n)e2πiζn ] = 0. (46) where ζn ∈[0, 1] is a random phase ensuring ζn [ , ] p g E[b(ik, vk |®xs,n)e2πiζn ] = 0. (46) E[b(ik, vk |®xs,n)e2πiζn ] = 0.f E[b(ik, vk |®xs,n)e2πiζn ] = 0. (46)f (46) As different paths are sampled independently, and given the zero- mean property, contributions from different sub-paths are uncorre- lated. Namely, for n1 , n2: To this end, we sample the path from a distribution E h b(ik, vk |®xs,n1)e2πiζn1 · b(im, vm|®xs,n2)∗e−2πiζn2 i = 0, (47) (47) (39) p(o1 →. . .→oB) = p(o2 →. . .→oB | ˆω1, o1)p( ˆω1|o1)p(o1). (39) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 187:12 • Chen Bar, Ioannis Gkioulekas, and Anat Levin д = 0.9 , σa = 0.6 д = 0.9 , σa = 0.25 д = 0.2 , σa = 0.25 𝒊𝟐 𝒊𝟏 𝒗𝟐 ation objective wing objective  N.F. F.F. Binary N.F. F.F. Binary N.F. F.F. Binary ∆= 0λ ∆= λ ∆= 2λ 0 0.01 0.02 0.03 0.04 0.05 0 1 2 3 10-3 0 0.5 1 10-4 (a) (b) (c) (d)i д = 0.9 , σa = 0.6 д = 0.9 , σa = 0.25 д = 0.2 , σa = 0.25 𝒊𝟐 𝒊𝟏 𝒗𝟏 𝒗𝟐 illumination objective viewing objective   N.F. F.F. Binary N.F. F.F. Binary N.F. F.F. Binary ∆= 0λ ∆= λ ∆= 2λ 0 0.01 0.02 0.03 0.04 0.05 0 1 2 3 10-3 0 0.5 1 10-4 (a) (b) (c) (d) Fig. 7. Comparisons with far field rendering. (a) Imaging setup. We use illumination and viewing pairs defined by ∆= i2x,y −i1x,y = v2x,y −v1x,y and τ = v1x,y −i1x,y = v2x,y −i2x,y. (This setup is also used in Fig. 8-11.) (b-d) We compare covariances rendered using our near-field (N.F.) algorithm against those obtained by rendering far-field (F.F.) correlations and applying focusing. 7 FIELD RENDERING 1 shows speckle images rendered with this algorithm. We note that in Eq. (45), the same set of paths is used to update all illuminators and sensors. As a result, the speckle images due to different illuminators in Fig. 1 are correlated, shifted versions of each other, corresponding to the memory effect property. Rendering each of these images independently, using a standard Monte Carlo volume rendering approach, would fail to reproduce these correlations. We denote by i1x,y, i2x,y, v1x,y, v2x,y the x −y coordinates of the corresponding 3D points on the focus plane. We denote by ∆≡ i2x,y −i1x,y the 2D displacement between the illumination direc- tions, and by τ ≡v1x,y −i1x,y the 2D displacement between the illumination and viewing directions (Fig. 7(a)). Classical memory effect theory [Feng et al. 1988] states that strong correlations ex- ist between fields for illumination and viewing pairs satisfying i2x,y −i1x,y = v2x,y −v1x,y = ∆, for small ∆values. With this in mind, in our experiments, we evaluate and visualize correlations of the form: 7 FIELD RENDERING We also compare with covariances rendered with a binary aperture of equivalent width to our apodized aperture. As binary apertures cannot be well-approximated using von Mises-Fisher functions, we only render them using the far-field algorithm. The volume has size 50λ × 50λ × 20λ. We evaluate two aperture widths in (b,c) and two phase functions in (c,d). Each square visualizes correlation as a function of τ , and different rows correspond to different x-axis illuminator shifts ∆= i2x −i1x . We see close agreement in all cases. h k k i 1 2 д = 0.9 , σa = 0.25 д = 0.2 , σa = 0.25 N.F. F.F. Binary N.F. F.F. Binary 0 1 2 3 10-3 0 0.5 1 10-4 (c) (d) д = 0.9 , σa = 0.6 N.F. F.F. Binary ∆= 0λ ∆= λ ∆= 2λ 0 0.01 0.02 0.03 0.04 0.05 0 (b)i 𝒊𝟐 𝒊𝟏 𝒗𝟏 𝒗𝟐 illumination objective viewing objective   ∆= 0λ ∆= λ ∆= 2λ (a) (a) (b) (c) Fig. 7. Comparisons with far field rendering. (a) Imaging setup. We use illumination and viewing pairs defined by ∆= i2x,y −i1x,y = v2x,y −v1x,y and τ = v1x,y −i1x,y = v2x,y −i2x,y. (This setup is also used in Fig. 8-11.) (b-d) We compare covariances rendered using our near-field (N.F.) algorithm against those obtained by rendering far-field (F.F.) correlations and applying focusing. We also compare with covariances rendered with a binary aperture of equivalent width to our apodized aperture. As binary apertures cannot be well-approximated using von Mises-Fisher functions, we only render them using the far-field algorithm. The volume has size 50λ × 50λ × 20λ. We evaluate two aperture widths in (b,c) and two phase functions in (c,d). Each square visualizes correlation as a function of τ , and different rows correspond to different x-axis illuminator shifts ∆= i2x −i1x . We see close agreement in all cases. sensing focus points v1, v2 are all located at the same depth plane, at the back plane of the volume. This configuration corresponds, for example, to the fluorescent imaging setting, where a fluorescent par- ticle is deep inside a medium such as tissue, and a camera attempts to observe it by focusing through the scattering at the illuminator’s plane. for all (k,m) pairs. Therefore, we see that E h u(ik, vk) · u(im, vm)∗i equals the desired covariance of Eq. (43). Fig. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 8 EVALUATIONfi 7, our near-field approach is 400× faster than the tabulated far-field approach, and 3000× faster than the Monte Carlo approach. The performance advantage will become even larger for volumes with sizes corresponding to realistic lab experiments. All algorithms were implemented and run on a V100 NVIDIA GPU. Table 1. Runtimes of different algorithms for achieving RMSE < 0.01. 10λ × 10λ × 4λ 25λ × 25λ × 10λ 50λ × 50λ × 20λ N.F. 0.46 s 2.12 s 6 s F.F. Tab. 31 s 221 s 2.4 × 103 s F.F. M.C. 199 s 1.47 × 103 s 2.04 × 104 s Table 1. Runtimes of different algorithms for achieving RMSE < 0.01. 10λ × 10λ × 4λ 25λ × 25λ × 10λ 50λ × 50λ × 20λ N.F. 0.46 s 2.12 s 6 s F.F. Tab. 31 s 221 s 2.4 × 103 s F.F. M.C. 199 s 1.47 × 103 s 2.04 × 104 s note that, in Sec. 4, we presented the formal approach for estimat- ing near-field covariance using far-field rendering, based on the far-field covariance Eq. (11) rather than fields. In these experiments, we opt for the field-based approach, because the number of far-field samples it requires scales more favorably with the width W of the simulated volume (W 4 scaling for the field-based approach, W 8 scaling for the covariance-based approach).i Table 2 reports RMSE from equal-time comparisons of the three approaches using volumes of different sizes. Error was measured against a reference rendering produced by running the far-field approach till convergence. For each volume size, we report RMSE for both a small and a long rendering time. The latter allows us to quantify the bias of the near-field approach. While any such bias is too small to be noticeable in the visual comparisons of Fig. 7, the nu- merical difference between the near-field and far-field covariances does not fully converge to zero. We anticipate that most of the bias is due to the assumption that volumetric attenuation is constant for all directions through the aperture (see Eq. (25)). In the second row of Table 2 we compare our near-field approach against a refer- ence evaluated using the far-field approach but with an equivalent constant directional attenuation, showing smaller error. 8 EVALUATIONfi We consider the case where the illumination focus points i1, i2 and Rendering Near-Field Speckle Statistics in Scattering Media • 187:13 Table 2. Equal-time comparisons of different algorithms. 10λ × 10λ × 4λ 25λ × 25λ × 10λ 50λ × 50λ × 20λ 1 s 60 s 5 s 500 s 15 s 6000 s N.F. 0.0089 0.0076 0.0096 0.0093 0.0135 0.0110 N.F. biased ref. 0.0053 0.0021 0.0050 0.0018 0.0080 0.0045 F.F. Tab. 0.0396 0.0078 0.0672 0.0056 0.0915 0.0051 F.F. M.C. 0.1140 0.0198 0.2024 0.0207 0.3317 0.0632 Table 2. Equal-time comparisons of different algorithms. N.F. F.F. Aliasing F.F. M.C. eq. time F.F. M.C. ×10 ∆= 0λ ∆= λ ∆= 2λ (a) (b) (c) (d) (e) Fig. 8. Acceleration of far-field rendering. (a) N.F (1× runtime). (b) F.F. with dense discretization of aperture integral (400× runtime). (c) F.F. with sparser discretization of aperture integral (100× runtime) (d, e) F.F. with Monte Carlo sampling of aperture integral (1× and 10× runtime). N.F. F.F. Aliasing F.F. M.C. eq. time F.F. M.C. ×10 ∆= 0λ ∆= λ ∆= 2λ (a) (b) (c) (d) (e)i covariance is computed when evaluating the integral of Eq. (11). The number of random samples is selected to achieve the lowest possible error for equal runtime. As the integrand is complex, using Monte Carlo approximation results in very high variance. At the cost of increased runtime, Fig. 8(e) demonstrates a better estimate. (a) (e) Fig. 8. Acceleration of far-field rendering. (a) N.F (1× runtime). (b) F.F. with dense discretization of aperture integral (400× runtime). (c) F.F. with sparser discretization of aperture integral (100× runtime) (d, e) F.F. with Monte Carlo sampling of aperture integral (1× and 10× runtime). Runtime and bias evaluation. We compare the runtime of our near-field approach against two versions of the far-field approach: a tabulated version, computing all far field directions before applying near field transformation, as in Fig. 8(b); and a Monte Carlo version, randomly sampling directions, as in Fig. 8(d-e). Table 1 shows the runtime each approach requires to converge to a root-mean-square- error (RMSE) relative to a reference rendering below 0.01. In all cases, our near-field approach has the best performance, and the performance improvement increases as the widthW of the simulated volume increases. For the volume size we use for the results in Fig. 8 EVALUATIONfi We now evaluate the efficiency and accuracy of our proposed ren- dering algorithms. We compare our algorithms against three alterna- tives: First, we compare with an approach that uses the Monte Carlo rendering algorithm of Bar et al. [2019] to produce far-field esti- mates, and then converts them to near-field estimates using Eqs. (2) and (9). We also consider a few variants of this approach that benefit from various acceleration techniques. Second, we compare with groundtruth estimates produced by a wave-equation solver. Third, we compare with estimates produced using a layered propagation approach popular in the optics literature. Additionally, we show experiments evaluating performance improvements due to the im- portance sampling scheme of Sec. 6. In Sec. 9, we show experiments relating to the tilt-shift memory effect [Osnabrugge et al. 2017], in- cluding experiments validating our algorithms against groundtruth measurements of real materials. C(∆,τ) = C i1 x,y,i1 x,y+∆ i1x,y+τ,i1x,y+τ+∆, (48) (48) for different displacements ∆and τ. For sufficiently wide volumes, C(∆,τ) is approximately invariant to i1. Comparison with far-field approach. We compare first with the simulation approach based on the far-field rendering algorithms of Bar et al. [2019]. To reduce the computational burden, we compare with an approach based on a combination of Eqs. (2) and (9), rather than Eq. (11): We first discretize the illumination and viewing aper- tures into a set of directions ˆi and ˆv. For each set of (ˆi, ˆv) values, we use the implementation provided by Bar et al. [2019] to sample far-field scattered waves uˆi ˆv, which we subsequently convert to the near-field scattered waves using Eq. (9). By running the field ren- dering algorithm of Bar et al. [2019] multiple times, we end up with multiple samples of near-field scattered waves, corresponding to different scatterer configurations. Finally, we use these near-field scattered waves to approximate the covariance as in Eq. (2). We Experimental configurations. Our experiments focus on illumina- tion and imaging configurations that are known from literature to produce strongly-correlated fields. We describe these configurations using Fig. 7(a) as reference: As we focus on transmission mode imag- ing, the focused illumination is placed at the back of the sample, and the focused sensor is placed at the front. We refer to App. A.4 for an evaluation of our technique under reflective imaging conditions. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 8 EVALUATIONfi V = 500λ × 500λ × 100λ OD = 4 , д = 0.97 ∆= 0λ ∆= 3λ ∆= 6λ The left part of Fig. 11 shows correlations valuesC(∆,τ) (Eq. (48)), as a function of τ for a few ∆and д values, simulated using µ-diff and our technique. Note that, as these simulations are in 2D, τ is a scalar, and thus C(∆,τ) is a 1D curve. We observe that the plots computed with our technique closely match those produced by the wave-equation solver, demonstrating the accuracy of our technique. At the same time, for this small example our technique is three orders of magnitude faster, and can scale to much larger volumes. Fig. 9. The benefit of importance sampling. The figure compares uni- form sampling vs. importance sampling (I.S.) of the first scatterer on the path o1, vs. importance sampling of both the first scatterer and the first direction o1, ˆω1. Top panel: a small target whose size is only 20λ ×20λ ×20λ. Fig. 9. The benefit of importance sampling. The figure compares uni- form sampling vs. importance sampling (I.S.) of the first scatterer on the path o1, vs. importance sampling of both the first scatterer and the first direction o1, ˆω1. Top panel: a small target whose size is only 20λ ×20λ ×20λ. For such small targets uniform sampling converges as well, although re- quiring a larger number of path samples (compare results with 103 samples to 105 samples), demonstrating that our importance sampling strategy is consistent. Middle panel: When expanding the size of the target volume, convergence of uniform sampling is very slow. As the phase function is rather wide, importance sampling of the first direction does not improve much. Lower panel: If we also use a very forward scattering phase function, convergence is much accelerated by importance sampling the first direction. Comparison with multi-slice layered propagation. We compare additionally with the multi-slice beam propagation method [Schott et al. 2015], which is a popular numerical approach in optics for simulating wave propagation. This approach accounts for multiple- scattering effects by approximating the simulated volume as a se- quence of planar slices orthogonal to the optical axis; layers are modeled as infinitesimally-thin 2D phase masks, separated by free space. The phase mask of each layer is selected to scatter light with an angular spread matching the phase function of the simulated volume. 8 EVALUATIONfi In our experiments, to keep the number of far-field samples man- ageable, we simulate a relatively small volume of size 50λ×50λ×20λ with mean free path MFP = 10λ, leading to an optical depth OD = 2. We consider two choices of aperture width, σa = 0.6 and σa = 0.25, and both wide and narrow forward-scattering Henyey-Greenstein phase functions (low and high д values, respectively). When using the far-field approach, we simulate both von Mises-Fisher-apodized and binary aperture masks m(·) of the same width, to quantify the ef- fect of our apodization approximation on accuracy (binary apertures can only be evaluated at the far-field, at increased computational complexity, as the von Mises-Fisher approximation does not ap- ply). We show the results in Fig. 7. We observe that, in all cases, our near-field rendering algorithm produces very similar results to the far-field approaches. We also note that the far-field approach produces very close results when using apodization and binary masks, indicating that our apodization approximation does not in- troduce significant bias. At the same time, our near-field approach is orders-of-magnitude faster, as we quantify in detail below. The benefit of importance sampling. To evaluate the effect of the importance sampling scheme we introduced in Sec. 6, we compare in Fig. 9 covariance estimates produced by our Monte Carlo al- gorithm, using three different sampling strategies: first, uniform sampling of both the first path vertex and direction; second, impor- tance sampling of the first vertex and uniform sampling of the first direction; and third, importance sampling of both the first vertex and direction. We observe first that, when run till convergence (e.g., in the top panel where the sample is small and the phase function has significant side-scattering), all three combinations converge to the same result. This confirms that importance sampling does not introduce any addiional bias. We additionally observe that the estimate using importance sampling of both the first vertex and In Fig. 8, we additionally compare against two accelerated variants of the far-field approach. We reuse the configuration of Fig. 7(d), this time with a wider τ grid. First, we render far-field covariance only for a (fixed) regular subset of directions. As the number of samples is significantly lower than that required by the Nyquist sampling rate in Eq. (12), aliased replicas appear. 8 EVALUATIONfi Second, we use a Monte Carlo strategy that randomly samples the directions at which far-field 187:14 • Chen Bar, Ioannis Gkioulekas, and Anat Levin V = 20λ × 20λ × 20λ V = 500λ × 500λ × 100λ OD = 2 , д = 0.5 OD = 4 , д = 0.97 log10(RMSE) 103 104 105 106 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 103 104 105 106 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 Sample number Sample number Fig. 10. Convergence plots. log(RMSE) of different sampling strategies as a function of sample number, for the first and third volumes in Fig. 9. 103 Samples 105 Samples Uniform I.S. o1 I.S. o1, ˆω1 Uniform I.S. o1 I.S. o1, ˆω1 V = 20λ × 20λ × 20λ OD = 2 , д = 0.5 ∆= 0λ ∆= 3λ ∆= 6λ V = 500λ × 500λ × 100λ OD = 4 , д = 0.5 ∆= 0λ ∆= 3λ ∆= 6λ V = 500λ × 500λ × 100λ OD = 4 , д = 0.97 ∆= 0λ ∆= 3λ ∆= 6λ Fig. 9. The benefit of importance sampling. The figure compares uni- form sampling vs. importance sampling (I.S.) of the first scatterer on the path o1, vs. importance sampling of both the first scatterer and the first direction o1, ˆω1. Top panel: a small target whose size is only 20λ ×20λ ×20λ. For such small targets uniform sampling converges as well, although re- quiring a larger number of path samples (compare results with 103 samples to 105 samples), demonstrating that our importance sampling strategy is consistent. Middle panel: When expanding the size of the target volume, convergence of uniform sampling is very slow. As the phase function is rather wide, importance sampling of the first direction does not improve much. Lower panel: If we also use a very forward scattering phase function, convergence is much accelerated by importance sampling the first direction. V = 20λ × 20λ × 20λ OD = 2 , д = 0.5 log10(RMSE) 103 104 105 106 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 Sample number V = 500λ × 500λ × 100λ OD = 4 , д = 0.97 103 104 105 106 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 Sample number Sample number Sample number Sample number Sample number p p Fig. 10. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 8 EVALUATIONfi Convergence plots. log(RMSE) of different sampling strategies as a function of sample number, for the first and third volumes in Fig. 9. p p Fig. 10. Convergence plots. log(RMSE) of different sampling strategies as a function of sample number, for the first and third volumes in Fig. 9. Comparison with a wave-equation solver. Bar et al. [2019] demon- strated the accuracy of their far-field covariance rendering algo- rithm by comparing against numerical wave-equation solvers. For additional validation, we also compare directly with the µ-diff solver [Thierry et al. 2015] they use. The solver takes as input a configuration of scatterer locations, and uses numerical techniques to solve the wave equation and output an estimate of the complex scattered field. To compute covariance, we run the solver for multi- ple scatterer instantiations sampled from the same distribution, then use the results to estimate the speckle covariance of Eq. (2). We note that this simulator only works in 2D; thus, for these comparisons only, we restrict our Monte Carlo rendering algorithm to 2D as well. The left part of Fig. 11 shows correlations valuesC(∆,τ) (Eq. (48)), as a function of τ for a few ∆and д values, simulated using µ-diff and our technique. Note that, as these simulations are in 2D, τ is a scalar, and thus C(∆,τ) is a 1D curve. We observe that the plots computed with our technique closely match those produced by the wave-equation solver, demonstrating the accuracy of our technique. At the same time, for this small example our technique is three orders of magnitude faster, and can scale to much larger volumes. Comparison with a wave-equation solver. Bar et al. [2019] demon- strated the accuracy of their far-field covariance rendering algo- rithm by comparing against numerical wave-equation solvers. For additional validation, we also compare directly with the µ-diff solver [Thierry et al. 2015] they use. The solver takes as input a configuration of scatterer locations, and uses numerical techniques to solve the wave equation and output an estimate of the complex scattered field. To compute covariance, we run the solver for multi- ple scatterer instantiations sampled from the same distribution, then use the results to estimate the speckle covariance of Eq. (2). We note that this simulator only works in 2D; thus, for these comparisons only, we restrict our Monte Carlo rendering algorithm to 2D as well. 8 EVALUATIONfi (a) Setup: two beams with spatial displacement ∆a and angular displacement θ enter the volume. We compute the correlation between the speckle patterns measured at the b plane as a function of both tilt and shift, assuming that the input displacement ∆a is selected to maximize Eq. (50). The bottom images visualize the results. (b,c) Analytic and measured correlation, reproduced from Osnabrugge et al. [2017]. (d-e) Correlation computed by our algorithm for two different phase functions with the same average cosine д = 0.98. Fig. 12. The tilt-shift memory effect. (a) Setup: two beams with spatial displacement ∆a and angular displacement θ enter the volume. We compute the correlation between the speckle patterns measured at the b plane as a function of both tilt and shift, assuming that the input displacement ∆a is selected to maximize Eq. (50). The bottom images visualize the results. (b,c) Analytic and measured correlation, reproduced from Osnabrugge et al. [2017]. (d-e) Correlation computed by our algorithm for two different phase functions with the same average cosine д = 0.98. C(∆a, ∆b,θ) ≈δ(∆b −∆a −Lθ)e −L3k2 2ℓtr  θ 2 12 +  θ 2 + ∆b L 2 , (50) (50) where δ(·) is the Dirac delta function, and ℓtr is the transport mean free path ℓtr ≡MFP/(1 −д). Their derivation is based on three simplifying assumptions: it uses a layered representation similar to that of the multi-slice layered propagation technique in Sec. 8; at each layer, it assumes forward-only propagation; and it uses a differential equation to integrate over multiple scattering planes. propagation technique, as the latter requires for each layer a high- resolution discretization of the wave, resulting in large dense arrays that need to be convolved to model propagation between layers. Additionally, Osnabrugge et al. [2017] show measurements of the function C(∆a, ∆b,θ) for a tissue phantom of thickness L = 258 µm, made of silica microspheres immersed in agarose gel. By combining Mie theory [Frisvad et al. 2007] with the dispersion and sizing properties of the materials used for fabrication, the authors estimate for the phantom an anistropy parameterд = 0.98 and mean free path MFP = 296 µm. In Fig. 12(b,c), we replicate from their paper (using data provided by the authors) the measured correlationC(∆a, ∆b,θ), as well as the analytical prediction using the model of Eq. (50). 8 EVALUATIONfi Our simulator is physically accurate, producing results in close agreement with those of the solver, while being orders of magnitude faster.t Our approach Multi-slice simulator д = 0.98 д = 0.3 д = 0.98 д = 0.3 C(∆,τ) -20 0 20 0 0.02 0.04 0.06 0.08 0.1 -20 0 20 0 0.002 0.004 0.006 0.008 0.01 -20 0 20 0 0.02 0.04 0.06 0.08 0.1 -20 0 20 0 0.002 0.004 0.006 0.008 0.01 = 0 = = 2 Wave solver = 0 = = 2 Monte-carlo / Multi-slice τx τx τx τx Fig. 11. Wave optics alternatives. We compare covariances obtained with our approach, against those by a wave-equation solver, and a multi-slice simulator from optics. Our simulator is physically accurate, producing results in close agreement with those of the solver, while being orders of magnitude faster. The multi-slice approach is valid for a very forward-scattering phase function (д = 0.98) and small optical depth. For a wider phase function (д = 0.3), the multi-slice approach produces inaccurate results, due to incorrect modeling of back and side scattering angles. The comparison is performed in 2D due to the limitations of the solver. The simulated volume is 200λ × 70λ wide with OD = 3.i Multi-slice simulator д = 0.98 д = 0.3 20 0 20 -20 0 20 0 0.002 0.004 0.006 0.008 0.01 = 0 = = 2 Wave solver = 0 = = 2 Monte-carlo / Multi-slice τx τx h h b l d l l l 20 20 20 Fig. 11. Wave optics alternatives. We compare covariances obtained with our approach, against those by a wave-equation solver, and a multi-slice simulator from optics. Our simulator is physically accurate, producing results in close agreement with those of the solver, while being orders of magnitude faster. The multi-slice approach is valid for a very forward-scattering phase function (д = 0.98) and small optical depth. For a wider phase function (д = 0.3), the multi-slice approach produces inaccurate results, due to incorrect modeling of back and side scattering angles. The comparison is performed in 2D due to the limitations of the solver. 8 EVALUATIONfi This technique has high computational efficiency compared to exact wave-equation solvers, but cannot model back-scattering, and has worse accuracy for wide scattering angles. direction results in reduced noise in all cases. The improvement becomes more pronounced as the width of the volume increases (second panel in Fig. 9); this is because, as the size of the Gaussian beam relative to the volume decreases, uniform vertex sampling will result in more paths starting in points of the volume that do not receive any light. Finally, the improvement achieved by using importance sampling increases even further as the phase function becomes more forward-scattering (third panel in Fig. 9); in this case, it is necessary to importance sample the first direction as well, oth- erwise the majority of path-starting directions will have near-zero contribution. Fig. 10 additionaly shows convergence plots for the first and third volumes in Fig. 9. The right part of Fig. 11 shows correlation estimates from this technique, using the same experimental settings as in our com- parisons with the wave-equation solver. We observe that, for very forward-scattering phase functions, both our Monte Carlo algo- rithm and the layered propagation technique closely match the groundtruth produced by the solver. However, for phase functions with significant side-scattering, the accuracy of the layered propaga- tion technique is significantly worse than that of ours. Additionally, our Monte Carlo algorithm is significantly faster than the layered Rendering Near-Field Speckle Statistics in Scattering Media • 187:15 Our approach Multi-slice simulator д = 0.98 д = 0.3 д = 0.98 д = 0.3 C(∆,τ) -20 0 20 0 0.02 0.04 0.06 0.08 0.1 -20 0 20 0 0.002 0.004 0.006 0.008 0.01 -20 0 20 0 0.02 0.04 0.06 0.08 0.1 -20 0 20 0 0.002 0.004 0.006 0.008 0.01 = 0 = = 2 Wave solver = 0 = = 2 Monte-carlo / Multi-slice τx τx τx τx Fig. 11. Wave optics alternatives. We compare covariances obtained with our approach, against those by a wave-equation solver, and a multi-slice simulator from optics. 8 EVALUATIONfi The correlation is displayed as a function of (∆b,θ) alone, with ∆a selected according to the Dirac delta relationship in Eq. (50).i 8 EVALUATIONfi The simulated volume is 200λ × 70λ wide with OD = 3.i   2  2 a plane b plane (a) Setup   2  2 a plane b plane (a) Setup θ[rad] -5 0 5 -0.0319 -0.0158 0 0.0158 0.0319 -5 0 5 -0.0319 -0.0160 0 0.0160 0.0319 -5 0 5 -0.0319 -0.0158 0 0.0158 0.0319 -5 0 5 -0.0319 -0.0158 0 0.0158 0.0319 ∆[µm] ∆[µm] ∆[µm] ∆[µm] (b) Analytic (c) Measured (d) VMF (e) Mie [Osnabrugge] Our simulation Fig. 12. The tilt-shift memory effect. (a) Setup: two beams with spatial displacement ∆a and angular displacement θ enter the volume. We compute the correlation between the speckle patterns measured at the b plane as a function of both tilt and shift, assuming that the input displacement ∆a is selected to maximize Eq. (50). The bottom images visualize the results. (b,c) Analytic and measured correlation, reproduced from Osnabrugge et al. [2017]. (d-e) Correlation computed by our algorithm for two different phase functions with the same average cosine д = 0.98. of the speckle fields due to the two beams if we additionally tilt the beams at angles −θ/2, θ/2 respectively. Intuitively, appropriately selecting the tilting angle as a function of the displacement ∆a helps increase the overlap between the defocused beams inside the medium, and thus increases the correlation of the resulting speckle fields.i This observation motivates evaluating how speckle field correla- tion varies as a function of tilt angle and shift displacements at the input and output planes. Concretely, we can write this as a function: (a) Setup θ[rad] -5 0 5 -0.0319 -0.0158 0 0.0158 0.0319 -5 0 5 -0.0319 -0.0160 0 0.0160 0.0319 -5 0 5 -0.0319 -0.0158 0 0.0158 0.0319 -5 0 5 -0.0319 -0.0158 0 0.0158 0.0319 ∆[µm] ∆[µm] ∆[µm] ∆[µm] (b) Analytic (c) Measured (d) VMF (e) Mie [Osnabrugge] Our simulationf θ[rad] C(∆a, ∆b,θ) ≡E "Õ τ u1(τ −∆b/2) · u2(τ + ∆b/2)∗eikτθ # , (49)i (49) where the expectation is taken over all fields with the same material parameters (e.g., fields generated by different scatterer instantiations O sampled from the same density). Osnabrugge et al. [2017] derived an analytic approximation for this function that takes the form: ∆[µm] (e) Mie ∆[µm] (b) Analytic [Osn ∆[µm] (c) Measured [Osnabrugge] Fig. 12. The tilt-shift memory effect. (a) Setup: two beams with spatial Fig. 12. The tilt-shift memory effect. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 9 THE TILT-SHIFT MEMORY EFFECTi In this section, we use our near-field rendering technique to study the tilt-shift memory effect property introduced by Osnabrugge et al. [2017]. We first briefly review this property, using Fig. 12(a) as a reference: A scattering volume of thickness L is illuminated by two input beams from its top surface, denoted in Fig. 12(a) as the a plane. The scattered fieldsu1,u2 due to the two beams are imaged by a cam- era focused at the lower plane of the volume, denoted in Fig. 12(a) as the b plane. The two illumination beams have a displacement relative to each other equal to i2 −i1 = ∆a. The key observation of Osnabrugge et al. [2017] is that we can increase the correlation To evaluate the accuracy of our near-field covariance rendering algorithm, we use it to estimate the function C(∆a, ∆b,θ) for the 187:16 • Chen Bar, Ioannis Gkioulekas, and Anat Levin Shift Tilt-Shift In Out In Out ∆= 0λ ∆= 1.5λ ∆= 3λ Analytic S Analytic S+T MC S MC S+T -20 -10 0 10 20 0 0.2 0.4 0.6 0.8 1 -20 -10 0 10 20 0 0.2 0.4 0.6 0.8 1 -20 -10 0 10 20 0 0.2 0.4 0.6 0.8 1 ∆[µm] ∆[µm] ∆[µm] (a) OD = 1, g = 0.98 (b) OD = 3, g = 0.98 (c) OD = 6, g=0.99 Fig. 13. Adaptive optics focusing. Illuminating a scattering slab with the complex wave visualized at the first and third columns of the top row leads to a sharp focused point at the other edge of the random media slab. Due to the ME, a small shift of the same pattern can focus at a nearby point, but focusing power degrades with displacement (note the weak power at the third row). Applying both shift and tilt to the input pattern (rightmost columns) leads to a stronger power at the same displacement (compare the power for the non-zero displacements at rows 2 and 3). Lower panel: Using our approach to evaluate the expected power one can achieve with the shit only and shift+tilt approaches, as a function of displacement (that is, the average power of the yellow dot at the three top rows). The scan range predicted by our accurate simulator is wider than the analytical prediction. 9.1 Focusing through turbid media Illuminating a scattering slab with the complex wave visualized at the first and third columns of the top row leads to a sharp focused point at the other edge of the random media slab. Due to the ME, a small shift of the same pattern can focus at a nearby point, but focusing power degrades with displacement (note the weak power at the third row). Applying both shift and tilt to the input pattern (rightmost columns) leads to a stronger power at the same displacement (compare the power for the non-zero displacements at rows 2 and 3). Lower panel: Using our approach to evaluate the expected power one can achieve with the shit only and shift+tilt approaches, as a function of displacement (that is, the average power of the yellow dot at the three top rows). The scan range predicted by our accurate simulator is wider than the analytical prediction. Note that configurations (b) and (c) have the same transport mean free path and should be equivalent according to the simplified analytic model of Eq. (50), yet they are very different according to an accurate MC simulator. The memory effect property of scattered fields provides a way to achieve this refocusing task. The way this works is that we first use a guiding star to measure the scattered field that is created due to emission from a single point ix,y at the a plane (Fig. 12(a)). Measuring this fieldu(vx,y) at all points vx,y at theb plane provides us with exactly the wavefront shape we need to focus at point ix,y. Then, the memory effect property suggests that a shifted wave refocusing simulations using only shifting (Eq. (51)) versus using both shifting and tilting (Eq. (52)). We observe that using the tilted- shifted wave improves refocusing, making the yellow focused spots of the second column in Fig. 13 stronger than the fourth one, and thus validating the observation of Osnabrugge et al. [2017]. We refer to App. A.8 for more details regarding this simulation. u(vx,y + ∆), (51) (51) can be used to roughly focus at ix,y + ∆. Osnabrugge et al. [2017] improve upon this idea by recommending to refocus using a wave that is both shifted and tilted; that is, a wave of the form We can additionally use our rendering algorithms to more ac- curately evaluate the scanning range over which this refocusing technique remains effective. 9 THE TILT-SHIFT MEMORY EFFECTi Note that configurations (b) and (c) have the same transport mean free path and should be equivalent according to the simplified analytic model of Eq. (50), yet they are very different according to an accurate MC simulator. Shift Tilt-Shift In Out In Out ∆= 0λ ∆= 1.5λ ∆= 3λ same tissue phantom. We refer to App. A.7 for details on how to sim- ulate this with our framework. We perform simulations using both the exact Mie-theory phase function describing the phantom, as well as a von Mises-Fisher phase function with the same д = 0.98 av- erage cosine. Comparing with the measured data and the analytical approximation in Fig. 12(b,c), we make the following observations: Both our renderings and the analytical approximation produce a correlation function with a dominant lobe that has the same orien- tation as the one in the measured data. However, our simulations match the dimensions of this lobe more closely than the analytical model. Overall, our simulations reproduce the important qualitative features of the measuremenets, confirming the accuracy of our algo- rithms. Differences between our renderings and the measurements are likely due to inaccurate modeling of the true material properties of the phantom—as seen in Fig. 12(d-e), replacing the exact reported phase function with an approximation results in a better match to the measurements—and due to aberrations in the imaging optics. We also note that Osnabrugge et al. [2017] measured correlation with an interferometric setup, which typically produces very noisy estimates of small signals such as weak speckle correlations. In Analytic S Analytic S+T MC S MC S+T -20 -10 0 10 20 0 0.2 0.4 0.6 0.8 1 -20 -10 0 10 20 0 0.2 0.4 0.6 0.8 1 -20 -10 0 10 20 0 0.2 0.4 0.6 0.8 1 ∆[µm] ∆[µm] ∆[µm] ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 9.1 Focusing through turbid media Having shown that our rendering algorithms can accurately repro- duce the tilt-shift memory effect, we now use them to analyze this effect in the context of a specific biomedical imaging application: we simulate focusing through turbid media with adaptive optics. This involves using, e.g., a spatial light modulator to produce a coherent wavefront whose shape is specific to the tissue sample being imaged. Finding the exact shape of this wavefront is challenging and usually requires having external information or a guiding star [Horstmeyer et al. 2015]. Once we know the wavefront needed to focus at a spe- cific point inside the volume, an important practical consideration is whether we can use this information to refocus at other points in a neighborhood around the original point [Judkewitz et al. 2014].fi 0 ∆[µm] 0 ∆[µm] ∆[µm] ∆[µm] ∆[µm] (a) OD = 1, g = 0.98 (b) OD = 3, g = 0.98 (c) OD = 6, g=0.99 (a) OD 1, g 0.98 (b) OD 3, g 0.98 (c) OD 6, g 0.99 Fig. 13. Adaptive optics focusing. Illuminating a scattering slab with the complex wave visualized at the first and third columns of the top row leads to a sharp focused point at the other edge of the random media slab. Due to the ME, a small shift of the same pattern can focus at a nearby point, but focusing power degrades with displacement (note the weak power at the third row). Applying both shift and tilt to the input pattern (rightmost columns) leads to a stronger power at the same displacement (compare the power for the non-zero displacements at rows 2 and 3). Lower panel: Using our approach to evaluate the expected power one can achieve with the shit only and shift+tilt approaches, as a function of displacement (that is, the average power of the yellow dot at the three top rows). The scan range predicted by our accurate simulator is wider than the analytical prediction. Note that configurations (b) and (c) have the same transport mean free path and should be equivalent according to the simplified analytic model of Eq. (50), yet they are very different according to an accurate MC simulator. ( ) , g ( ) , g ( ) , g Fig. 13. Adaptive optics focusing. 10 CONCLUSION Brian A. Barsky and Todd J. Kosloff. 2008. Algorithms for Rendering Depth of Field Effects in Computer Graphics. ICCOMP (2008). We presented computationally-efficient algorithms for simulating physically-accurate speckle fields and statistics under focused cam- era and light sources. 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Physical Review E (2016). Laurent Belcour, Kavita Bala, and Cyril Soler. 2014. A local frequency analysis of light scattering and absorption. ACM TOG (2014). The closed-form expressions are made possible through the use of von Mises-Fisher functions to approximate all spherical functions appearing in the path integral expression of near-field speckle co- variance. We make four such approximations: (i) We use apodization in the aperture plane to convert the aperture mask into a spheri- cal von Mises-Fisher function. (ii) We assume the exponential at- tenuation is constant through all aperture directions. (iii) We ap- proximate the phase function as a mixture of von Mises-Fisher functions. (iv) We approximate the analytical convolution of von Mises-Fisher functions, which is not in general a von Mises-Fisher function, as such a function. We have shown through simulations that: (i) Even though apodization cannot express an exactly binary aperture, if one matches its variance to the desired NA, a similar depth of field and similar speckle statistics are produced. (ii) The assumption of constant directional attenuation introduces negligible bias. (iii) Real-word and common parametric phase functions can be well-approximated using a small number of von Mises-Fisher functions. (iv) The convolution of von Mises-Fisher functions can be closely approximated as a von Mises-Fisher function. 10 CONCLUSION For narrow phase functions emitting little energy in the backward direction, nu- merical problems arise in back directions. As a result, our approach mostly applies for transmission mode imaging, and further research is required for reflection mode imaging.f Stephan Bergmann, Mahsa Mohammadikaji, Stephan Irgenfried, Heinz Worn, Jürgen Beyerer, and Carsten Dachsbacher. 2016. 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As an application of our algorithms, we evaluated different per- formance metrics for memory-effect-based adaptive optics scan- ning, and showed that in practice performance can be better than what predicted by previous approximate analytical models. Expeditions award 1730147, DARPA REVEAL grant HR0011-16-C- 0028. Expeditions award 1730147, DARPA REVEAL grant HR0011-16-C- 0028. range is larger than the range predicted by the analytical model of Eq. (50). 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These results demonstrate that our rendering algorithms can be used to evaluate how the performance of existing imaging tech- niques depend on exact material parameters, in ways that cannot be predicted using existing analytical models. Eric Akkermans and Gilles Montambaux. 2007. Mesoscopic Physics of Electrons and Photons. Cambridge University Press. Arindam Banerjee, Inderjit S. Dhillon, Joydeep Ghosh, and Suvrit Sra. 2005. Clustering ( ) Arindam Banerjee, Inderjit S. Dhillon, Joydeep Ghosh, and Suvrit Sra. 2005. Clustering on the unit hypersphere using von Mises-Fisher distributions. JMLR (2005). j , j , J y p , g on the unit hypersphere using von Mises-Fisher distributions. JMLR (2005). Ch B M i Alt I i Gki l k d A t L i 2019 A M t C l on the unit hypersphere using von Mises-Fisher distributions. JMLR Chen Bar, Marina Alterman, Ioannis Gkioulekas, and Anat Levin. 2019. A Monte Carlo Chen Bar, Marina Alterman, Ioannis Gkioulekas, and Anat Levin. 2019 Framework for Rendering Speckle Statistics in Scattering Media. ACM TOG (2019). h k l k d b h h b Framework for Rendering Speckle Statistics in Scattering Media. ACM TOG (2019). Chen Bar, Ioannis Gkioulekas, and Anat Levin. 2020. Project Website. https://github. / h b / i B fi ld Framework for Rendering Speckle Statistics in Scattering Media. ACM TOG (2019). Chen Bar, Ioannis Gkioulekas, and Anat Levin. 2020. Project Website. https://github. com/chabner/gaussianBeam-field.f 9.1 Focusing through turbid media In the lower panel of Fig. 13 we plot the expected power we can measure at focus points for different displacements ∆, comparing the prediction by our model with the analytical prediction of Osnabrugge et al. [2017]. We do simulations for a material with a Henyey-Greenstein phase function of д = 0.98 and mean free path of MFP = 75 µm at wavelength λ = 0.5 µm. We test two material thicknesses L = 75 µm and L = 225 µm, cor- responding to optical depths of OD = 1 and OD = 3. We observe that the Monte Carlo simulations predict that the effective scanning u(vx,y + ∆)eikθ(∆)vx,y, (52) (52) where θ(∆) is selected according to Eq. (50). For example, the strength of the yellow spot at the fourth column of Fig. 13 is slightly higher than at the second column. Using Eq. (50), Osnabrugge et al. [2017] also predict the range of shifts for which this refocusing is effective; that is, they estimate the scanning range for which sufficient memory effect correlations exists. We use our rendering algorithms to evaluate these refocusing techniques, as shown in Fig. 13. The top part of the figure compares Rendering Near-Field Speckle Statistics in Scattering Media • 187:17 ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. ACKNOWLEDGMENTS Pierre Jacquot and Pramod K. Rastogi. 1979. 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METODOLOGIAS ATIVAS NAS AULAS DE LÍNGUA PORTUGUESA DO ENSINO FUNDAMENTAL I: UM RELATO DE EXPERIÊNCIA SOBRE O USO DO SMARTPHONE NO PROCESSO DE ENSINO E APRENDIZAGEM
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Resumo Este estudo tem como objetivo relatar uma experiência, com a utilização de metodologias ativas, vivenciada no transcorrer das aulas de Língua Portuguesa, do ensino fundamental I, a partir do uso do smartphone. Na contemporaneidade, observamos o quão as tecnologias digitais de informação e comunicação (TDIC) articulam-se com a realidade que, há tempos, transforma-se em virtude dos avanços tecnológicos. Sendo assim, a escola como agência de fomento cognitivo não pode estar à parte de todo esse processo social, envolvendo, sobretudo, os smartphones que já fazem parte da rotina da população, principalmente, crianças e adolescentes. Dessa forma, amparamo-nos na pesquisa-ação como metodologia, partindo de uma breve revisão de literatura para, na sequência, descrevermos sucintamente o desenvolvimento do trabalho, envolvendo as metodologias ativas e o uso do smartphone. Partimos da hipótese de que esse recurso atrelado à escolha teórico-metodológica pode elevar o nível de engajamento dos estudantes, bem como os resultados de aproveitamento. Palavras-chave: Metodologias Ativas. Smartphone. Ensino fundamental. Língua portuguesa. CÉLIA APARECIDA REGINATO1 PAULO ALEXANDRE FILHO2 DOI: 10.29327/23860.14.25-8 2 Possui graduação em Letras - Alemão pela Universidade Estadual Paulista Júlio de Mesquita Filho - UNESP - Câmpus de Assis. Mestrado em Letras, na área de concentração de Linguagens e Letramentos, pela UNESP - Câmpus de Assis. Atualmente, professor efetivo do Quadro de Magistério, na Secretaria Estadual de Educação - SP, para o componente curricular de Língua Portuguesa. Doutorando em Educação pela Faculdade de Filosofia e Ciências, UNESP - Câmpus de Marília. Desenvolve seu doutorado na linha de pesquisa Teoria e Práticas Pedagógicas em que a tese focaliza os estudos de natureza teórico - metodológica, relacionando-se com as diversas áreas do currículo da Educação Básica e Superior, em especial, com a Didática, a Metodologia, a Psicologia, a Linguística e as políticas educacionais no Brasil. Participa do grupo de pesquisa TECLIN - Tecnologias, Culturas e Linguagens, coordenado pelo professor Dr. Fábio Marques de Souza (UEPE). Tem experiência na área de Linguagens e Letramentos. Keywords: Active Methodologies. Smartphone. Elementary School. Portuguese language. Keywords: Active Methodologies. Smartphone. Elementary School. Portuguese language. Keywords: Active Methodologies. Smartphone. Elementary School. Portuguese language. ACTIVE METHODOLOGIES IN PORTUGUESE LANGUAGE CLASSES OF ELEMENTARY EDUCATION I: AN EXPERIENCE REPORT ON THE USE OF SMARTPHONE IN THE TEACHING AND LEARNING PROCESS 1 Mestranda em Educação pela Faculdade de Filosofia e Ciências - Campus de Marília/SP. Possui Licenciatura Plena em Pedagogia pela Faculdade de Filosofia e Ciências - Campus de Marília/SP (2001). Possui Curso de Pós- Graduação Lato Sensu - Especialização em Coordenação Pedagógica pela Universidade Federal de São Carlos em parceria com o Ministério da Educação - Programa Escola de Gestores da Educação Básica (2016). Especialização Pós-Graduação Lato Sensu em Gestão e Docência na Educação Básica pela Universidade Santa Cecília em parceria com o IFGE - Instituto de Formação e Gestão Educacional (2013); Habilitação Específica de 2º Grau para o Magistério pelo Centro Específico de Formação e Aperfeiçoamento para o Magistério - CEFAM (1994). 2 Possui graduação em Letras - Alemão pela Universidade Estadual Paulista Júlio de Mesquita Filho - UNESP - Câmpus de Assis. Mestrado em Letras, na área de concentração de Linguagens e Letramentos, pela UNESP - Câmpus de Assis. Atualmente, professor efetivo do Quadro de Magistério, na Secretaria Estadual de Educação - SP, para o componente curricular de Língua Portuguesa. Doutorando em Educação pela Faculdade de Filosofia e Ciências, UNESP - Câmpus de Marília. Desenvolve seu doutorado na linha de pesquisa Teoria e Práticas Pedagógicas em que a tese focaliza os estudos de natureza teórico - metodológica, relacionando-se com as diversas áreas do currículo da Educação Básica e Superior, em especial, com a Didática, a Metodologia, a Psicologia, a Linguística e as políticas educacionais no Brasil. Participa do grupo de pesquisa TECLIN - Tecnologias, Culturas e Linguagens, coordenado pelo professor Dr. Fábio Marques de Souza (UEPE). Tem experiência na área de Linguagens e Letramentos. 137 Abstract This study aims to report an experience, with the use of active methodologies, experienced in the course of Portuguese language classes of elementary school I from the use of smartphones. Nowadays, we observe how digital information and communication technologies (TDIC) are articulated with the reality that, for some time, has been transformed by virtue of technological advances. Thus, the school as a cognitive development agency cannot be apart from this entire social process, involving, above all, smartphones that are already part of the population's routine, especially children and adolescents. Thus, we rely on action research as a methodology, starting from a brief literature review and then briefly describing the development of the work, involving active methodologies and the use of smartphones. We start from the hypothesis that this resource linked to the theoretical-methodological choice can raise the level of engagement of students, as well as the results. 1 INTRODUÇÃO Este relato é fruto de uma experiência vivenciada durante as aulas de Língua Portuguesa, no Ensino Fundamental I, tendo as metodologias ativas como suporte teórico-metodológico e o smartphone como recurso pedagógico, no ano de 2019, portanto, anterior ao contexto de pandemia. De início, faz-se necessário descrever brevemente a escola onde se deu o desenvolvimento deste trabalho, uma vez que acreditamos que cada realidade traz consigo suas peculiaridades que devem ser tomadas como base para reflexão de cada sujeito quando confrontado com realidades específicas de diferentes lugares. A unidade escolar localiza-se no interior de São Paulo e faz parte da rede municipal de educação. Os alunos são do 5º ano do ensino fundamental I, e grande parte deles apresenta dificuldade de leitura e escrita, demonstrando pouco engajamento com as atividades propostas. Quanto à infraestrutura, a escola não possuía número suficiente de computadores e internet com sinal estável a ponto de promovermos atividades mais longas e com toda a classe que era composta por 28 alunos. No transcorrer do primeiro bimestre, pudemos comprovar as dificuldades dos alunos por meio de avaliação diagnóstica, estudo das fichas de acompanhamento individual de cada um e compartilhamento de experiências com a professora deles no 4º ano. Durante essas reuniões de formação, a professora do ano anterior sempre me relatava que apesar das defasagens apresentadas pela turma, o que mais lhe preocupava era a apatia desses estudantes que, na maioria das vezes, não se mostravam interessados ou desafiados durante a realização das atividades propostas. Apesar de uma comunidade carente e com grandes vulnerabilidades socioemocionais, observei no transcorrer do bimestre que a maioria dos alunos possuía smartphone e, não raro, era preciso chamar a atenção deles para que não se dispersassem mexendo no aparelho. Dessa forma, notei que grande parte deles se interessava pelo uso desse recurso, porém apenas para fins de 138 comunicação simultânea por meio do aplicativo WhatsApp e acesso às redes sociais. Assim, vi nesse aparelho uma possibilidade de implementar seu uso efetivo para construção do conhecimento e promover o engajamento dos estudantes a partir das metodologias ativas. comunicação simultânea por meio do aplicativo WhatsApp e acesso às redes sociais. 1 INTRODUÇÃO Assim, vi nesse aparelho uma possibilidade de implementar seu uso efetivo para construção do conhecimento e Ao levar a proposta para a coordenadora da escola, fui muito bem acolhida e, após detalhar os acontecimentos e observações de sala de aula, ela me solicitou que compartilhasse com os demais colegas minhas ideias para que todos pudessem opinar. Para minha surpresa, grande parte dos professores, até os mais tradicionais, receberam muito bem a ideia, mas não deixaram de colocar os “contras” como, por exemplo, a possibilidade dos alunos filmarem/gravarem as aulas sem consentimento do professor, uso de internet móvel, uma vez que a escola não possuía wi-fi de qualidade a ponto de fornecer acesso para todos os alunos, entre outras questões. Eu e a coordenadora tomamos nota de todas as observações das demais colegas. Na sequência, marcamos uma reunião para que juntas pensássemos em ações que pudessem solucionar ou minimizar esses entraves, pois o uso de metodologias ativas articulado às TDIC poderia oferecer bons resultados. Chegamos à conclusão de que deveríamos mandar comunicado para os pais ou responsável, esclarecendo nossos objetivos, bem como suas necessidades para a formação dos alunos e, por fim, colocando-nos à disposição para quaisquer dúvidas. Amparamo-nos na metodologia qualitativa em que a pesquisa-ação ofereceu o suporte necessário para o desenvolvimento da prática conforme supracitado. Nesse sentido, Severo (2020, p. 2) ressalta que “dessa forma, não há mais espaço para o modelo de ensino centrado em práticas tradicionais, baseadas fortemente na pura transmissão de conteúdos sem alguma relação com a realidade e necessidades do sujeito aprendente.” A seguir, faremos uma breve revisão de literatura em que buscamos na teoria dos multiletramentos e das metodologias ativas o aporte teórico indispensável para a elaboração de estratégias pedagógicas, bem como promover a reflexão necessária sobre a experiência aqui descrita na forma de relato de experiência. Assim, Rojo e Moura (2019) e Moran (2018) nos ofertaram os estudos acerca dos temas tratados, neste trabalho, que em nenhum momento pretendeu ser recebido de maneira a-crítica. Reiteramos que cada local tem suas especificidades e, assim, este relato além do objetivo, busca também estender as possibilidades de aplicação desse recurso e dessa metodologia. 2 FUNDAMENTAÇÃO TEÓRICA Para esta seção, procuramos subsídios teóricos nas metodologias ativas e na Pedagogia dos multiletramentos para fins de respaldar nossa prática, envolvendo o uso do smartphone, bem como as intencionalidades das ações e estratégias implementadas no transcorrer do processo de ensino e aprendizagem. . 139 Partimos da premissa de que os estudos aqui apresentados nos serviram de base para a compreensão da nossa realidade como forma de estabelecer entre teoria e prática as pontes necessárias para ressignificar nosso lugar de fala enquanto educadores e, ao mesmo tempo, promover alternativas que pudessem viabilizar nosso trabalho, à medida que os desafios iam surgindo. Partimos da premissa de que os estudos aqui apresentados nos serviram de base para a compreensão da nossa realidade como forma de estabelecer entre teoria e prática as pontes necessárias para ressignificar nosso lugar de fala enquanto educadores e, ao mesmo tempo, promover alternativas que pudessem viabilizar nosso trabalho, à medida que os desafios iam surgindo. 2.1 A PEDAGOGIA DOS MULTILETRAMENTOS NAS PRÁTICAS DE ENSINO E APRENDIZAGEM Concebemos a realidade atual como um cenário repleto de tecnologias em que as pessoas passaram a depender sistematicamente de algum desses recursos para realizar suas ações do cotidiano. Se pensarmos na vivência de nossos alunos, comparando-as com aquilo que ofertamos em nossas aulas, constatamos que ainda estamos longe de atingir uma aprendizagem mais significativa e mais próxima daquilo que nos circunda. Nesse sentido, concebemos a Pedagogia dos multiletramentos como possibilidade teórico- metodológica viável no contexto atual de educação, em que as demandas passaram a exigir das pessoas competências, não apenas instrumentais, mas também interpessoais quando se trata das TDIC. Para Rojo e Moura (2019, p. 20), “multiletramentos [...] aponta, a um só tempo, para a diversidade cultural das populações em êxodo e para a diversidade de linguagens dos textos contemporâneos, o que vai implicar, é claro, uma explosão multiplicativa dos letramentos, que se tornam multiletramentos.” Dessa forma, notamos a necessidade da escola, enquanto agenciadora de letramento, incorporar em suas práticas a perspectiva dos multiletramentos, sobretudo, no intuito de oferecer uma educação voltada para as transformações de mundo assistidas nos últimos tempos. Trata-se, portanto, de legitimar as diferentes formas de construir sentido e valorizar as práticas sociais que agora passam a ocorrer em sintonia com a digitalidade. Não podemos deixar de considerar todas essas transformações e, ao mesmo tempo, buscar em nossos alunos a parceria necessária para a construção do conhecimento, uma vez que eles têm muito a nos oferecer de suas experiências com os recursos digitais. Logo, o professor se torna mediador, orientando sobre as potencialidades desses artefatos e também esclarecendo o quão importante é a tecnologia para a aprendizagem quando utilizada adequadamente. Rojo e Moura (2019, p. 24) observam que “[...] o conceito de multiletramentos abre cada vez mais espaço aos conceitos de mídia e de modalidade de linguagem [...].” Trazer para o espaço escolar uma teoria que visa à valorização das linguagens multissemióticas significa, neste momento, reformular o conceito de aprendizagem de que o conhecimento só se constrói a partir de textos canônicos ou expositivos presentes nos livros didáticos e apostilas. Ao articularmos nossas práticas levando em consideração a teoria dos multiletramentos, 140 estamos tornando a educação menos verticalizada e ofertando aos alunos a possibilidade de construir, tamos tornando a educação menos verticalizada e ofertando aos alunos a possibilidade de construi por meio do cooperativismo, seu próprio conhecimento. por meio do cooperativismo, seu próprio conhecimento. 2.2 AS METODOLOGIAS ATIVAS COMO ESTRATÉGIAS TEÓRICO- METODOLÓGICAS NA EDUCAÇÃO BÁSICA 141 Dessa maneira, concebemos a ideia de que, em um primeiro momento, precisamos realocar Dessa maneira, concebemos a ideia de que, em um primeiro momento, precisamos realoc nossa posição de educadores e estarmos sempre abertos a novas experimentações, sem perder de vista que as crianças e jovens têm muito a nos oferecer e, portanto, colaborar com nossas visões sobre nossa profissão. Não se trata de descaracterizar o papel do professor e do aluno, mas sim promover uma pedagogia em que todos possam ser valorizados e protagonistas de suas próprias ações. nossa posição de educadores e estarmos sempre abertos a novas experimentações, sem perder de vista que as crianças e jovens têm muito a nos oferecer e, portanto, colaborar com nossas visões sobre nossa profissão. Não se trata de descaracterizar o papel do professor e do aluno, mas sim promover uma pedagogia em que todos possam ser valorizados e protagonistas de suas próprias ações. 2.2 AS METODOLOGIAS ATIVAS COMO ESTRATÉGIAS TEÓRICO- METODOLÓGICAS NA EDUCAÇÃO BÁSICA A concepção de aprendizagem ativa extrapola a ideia de uma educação pautada em aulas expositivas. Tornar o processo de ensino e aprendizagem ativo requer do educador novas posturas e maneiras de conceber a construção do conhecimento. Sabemos das dificuldades em nos desvencilharmos de modelos pedagógicos tradicionais em que cumprimos com a função de ensinar e o aluno de aprender como se os papéis de um e de outro fossem bem definidos e obedecessem a um roteiro com começo, meio e fim. Para Moran (2018, p. 04), “metodologias ativas são estratégias de ensino centradas na participação efetiva dos estudantes na construção do processo de aprendizagem, de forma flexível, interligada e híbrida.” Assim, podemos compreender que a educação por esse modelo de aprendizagem exige uma concepção mais reflexiva daquilo que objetivamos ao final de cada aula, bimestre ou ano letivo, uma vez que precisamos estar atentos ao percurso formativo de nossos alunos, bem como às estratégias que empregamos. Ou seja, trazer as metodologias ativas para o nosso campo de atuação exige novas atitudes frente aos inúmeros desafios que nossa profissão de educadores nos lançam no cotidiano. As ações que implementamos em sala de aula nem sempre oportunizam aos alunos espaço para o diálogo como forma de escuta reflexiva em que eles podem inclusive externar seus pontos de vista sobre aquilo que acham relevante para sua formação. Na maioria das vezes, cumprimos com o currículo determinado pelas autoridades competentes, deixando de lado a valorização do diálogo, tornando esses estudantes pessoas silenciadas pelo sistema que busca massificar as inteligências, as competências e as relações afetivas. Moran (2018, p. 2) observa que “as metodologias predominantes no ensino são dedutivas: o professor transmite primeiro a teoria e depois o aluno deve aplicá-la a situações mais específicas.” Não estamos aqui colocando em xeque a aula expositiva, tampouco desvalorizando o papel do professor. Pelo contrário, neste momento, buscamos estender as reflexões de modo que as crianças e jovens da atualidade possam aprender de forma mais significativa, engajando-se mais em seu percurso formativo. Desse modo, torna-se imprescindível mobilizar outras competências, tão importantes quanto aquelas de tempos em que a tecnologia ainda não havia tomado proporções como na contemporaneidade, uma vez que “os processos de aprendizagem são múltiplos, contínuos, híbridos, formais e informais, organizados e abertos, intencionais e não intencionais (MORAN, 2018, p. 3). 3 O USO DO SMARTPHONE NO PROCESSO DE ENSINO E APRENDIZAGEM: RELATANDO A EXPERIÊNCIA Para tal, também recorremos à estratégia do agrupamento produtivo, para 142 que o nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido que o nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido que o nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido ou pouco importante. Inicialmente, trabalhamos a ferramenta de busca de maior acesso na internet, ou seja, como pesquisar o sentido das palavras no Google. A priori, pode parecer uma atividade muito simples para o 5º ano, contudo pudemos constatar o contrário, uma vez que nem todos sabiam explorar essa plataforma e suas facilidades. Então, solicitamos que aqueles alunos que já sabiam sobre esse recurso, compartilhassem com os outros colegas, inclusive, relatando em quais momentos faziam uso. Assim, durante as aulas os alunos passaram a contar com o Google como ferramenta indispensável para pesquisa. Com o passar do tempo, um deles me disse que em vez de pesquisar no Google, poderíamos baixar aplicativos de dicionário. Foi então que lhes apresentei o aplicativo de busca Play Store3 e por meio dele pudemos selecionar alguns dicionários que poderiam nos servir de material de apoio. Logo, notamos que muito da capacidade em operar com as tecnologias quando se trata de crianças e jovens não condiz com a realidade, pois sem orientação e os devidos encaminhamentos, nem todos conseguiriam chegar até aquele ponto da aprendizagem. Ao retornarmos das férias de julho para o terceiro bimestre, fui chamada pela coordenadora para descrever como foi o uso do smartphone em sala de aula, expondo se a prática adotada havia se refletido na aprendizagem e se valia a pena continuar. Após relatar a experiência, a coordenadora disse que seria muito positivo continuar, porém promovendo outros desafios como forma de demonstrar às crianças o potencial dessa ferramenta quando utilizada adequadamente. Desse modo, avançamos nos objetivos de uso do smartphone, ampliando as possibilidades de utilização desse artefato. Em um determinado momento, um dos alunos perguntou se eu conhecia um determinado filme ao qual havia assistido e gostaria de me recomendar, mas não se lembrava do título. 3 A Google Play Store — ou simplesmente Play Store, como é popularmente conhecida — é a loja de aplicativos oficial do Google. É por ela que os usuários Android podem fazer download de apps de forma gratuita ou paga — ou, ainda, baixar um programa gratuitamente, mas usá-lo mediante pagamento de assinatura. Disponível em: https://www.techtudo.com.br/listas/2020/10/o-que-e-google-play-loja-de-apps-do- android-oferece-filmes-e-livros.ghtml. Acesso em: 22 set. 2021. 3 O USO DO SMARTPHONE NO PROCESSO DE ENSINO E APRENDIZAGEM: RELATANDO A EXPERIÊNCIA Conforme relatamos, a classe era composta por 28 alunos do ensino fundamental I e grande parte deles apresentava defasagens de leitura e escrita sem contar o pouco envolvimento com a resolução das atividades propostas. Muitos deles perdiam o foco durante a realização das tarefas e isso quando não pegavam o aparelho celular para se comunicarem com os colegas ou até mesmo com os próprios familiares. Após afinar as ideias coletadas durante a reunião com os demais colegas, eu e a coordenadora realinhamos e redefinimos nossas ações para que o trabalho acontecesse a partir do segundo bimestre. Assim, todos os pais e/ou responsável foram comunicados e, nesse sentido, ficamos muito surpresos, pois ninguém se opôs à proposta de utilização do smartphone como recurso pedagógico. Pelo contrário, houve um responsável que, inclusive, elogiou para a coordenadora, em conversa telefônica, a estratégia de usar essa ferramenta com a finalidade de promover conhecimento. Assim, definimos que, logo de início, os estudantes precisariam conhecer os objetivos do uso do smartphone e, ao mesmo tempo, estabelecer com eles algumas regras de conduta para que nossos objetivos fossem alcançados ao final do bimestre. Logo, a coordenadora foi convidada para apresentar junto comigo a proposta de trabalho que seria desenvolvido nesta classe. Por fim, reiteramos as regras de comportamento e a responsabilidade durante a utilização do smartphone. Ao final, fomos questionadas por um dos alunos acerca de como participaria das aulas, sendo que ele não tinha esse recurso. Então, respondemos que todo o trabalho seria desenvolvido em grupo. Reiteramos que se trata de uma prática ocorrida em 2019, momento anterior à pandemia, portanto sem necessidade de distanciamento. Quanto à metodologia empregada, optamos pelas metodologias ativas, pois “[essas] são caminhos para avançar mais no conhecimento profundo nas competências socioemocionais e em novas práticas” (MORAN, 2015, p. 01). Acreditamos que promover uma aprendizagem baseada nos princípios de autonomia, pesquisa e cooperativismo seja o propósito de todo educador e, nesse sentido, reconhecemos a potencialidade dos artefatos tecnológicos, no caso o smartphone. Dessa forma, estabelecemos que aqueles que dispunham de internet iriam se sentar com aqueles que não possuíam. 3 O USO DO SMARTPHONE NO PROCESSO DE ENSINO E APRENDIZAGEM: RELATANDO A EXPERIÊNCIA Nesse momento, aproveitei para trabalhar com eles a pesquisa por meio de palavras-chave com o objetivo de evidenciar que o Google trata-se de um compêndio virtual onde praticamente podemos encontrar tudo. Na sequência, pedi que todos se reunissem em grupos e solicitei para que o estudante nos falasse um pouco sobre a história/enredo do filme. Em seguida, perguntei à classe quais trechos descritos pelo aluno seriam os mais importantes para a compreensão da obra. Desse modo, elenquei na lousa três excertos que poderiam ser fundamentais para o processo de busca na plataforma Google. 143 Logo depois, solicitei que aqueles que estivessem com internet entrassem no Google e no campo de busca digitassem um dos trechos Logo depois, solicitei que aqueles que estivessem com internet entrassem no Google e no campo de busca digitassem um dos trechos. Eles se surpreenderam com as inúmeras possibilidades de filmes que apareceram. O aluno responsável por desencadear toda a atividade nos disse que não era nenhum daqueles filmes que apareceu. Então, falei que precisaríamos consultar cada uma das páginas mostradas, mas logo as crianças perceberam que seria um trabalho muito penoso. Nessa etapa, falei da necessidade de refinarmos a pesquisa, ou seja, deveríamos ser mais objetivos com as palavras no campo de busca. Novamente, voltamo-nos para a lousa e escolhemos apenas palavras que pudessem nos ajudar a encontrar o título do filme. Por fim, após a discussão, retomamos a busca e, nesse momento, o aluno achou o filme o qual havia assistido e que gostaria de me recomendar. Pedi para ele que lesse as informações sobre a obra de modo a compartilhar conosco o enredo da trama. Nesse dia, a aula foi extremamente produtiva, todos participaram e tiveram seu espaço de fala, inclusive, sugerindo novos filmes para os demais colegas. até aquele momento. A nosso ver, a prática foi extremamente exitosa, tendo em vista que esses alunos tiveram a oportunidade de se tornarem sujeitos ativos e protagonistas de sua aprendizagem, desvencilhando-se do papel passivo e receptor de informação que, infelizmente, ainda persiste na educação. Não descartamos ao longo deste relato de experiência os obstáculos, principalmente, aqueles de infraestrutura, que mesmo em meio a uma cultura altamente tecnológica e digital ainda, ainda dificultam muito a implementação de uma aprendizagem mais coerente com as transformações de mundo vigentes. 4 REFLEXÕES ACERCA DO USO DO SMARTPHONE A PARTIR DO USO DE METODOLOGIAS ATIVAS Utilizar as tecnologias digitais de informação e comunicação (TDIC) é de suma importância na atualidade. Todavia, sabemos dos inúmeros desafios acerca da infraestrutura das escolas de nível básico, sobretudo, as públicas. Muito se fala a respeito dos recursos tecnodigitais no processo de ensino e aprendizagem, mas nem sempre encontramos as instituições devidamente paramentadas e prontas para a efetivação de ações mais arrojadas com a realidade de nossos alunos. Durante a implementação da prática de uso do smartphone, alguns colegas me questionavam sobre a monitoração dos aparelhos e se eu não me sentia insegura quanto ao acesso dos alunos a outros sites ou possíveis dispersões. Sempre respondia amparada em estudos e pesquisadores como Rojo e Moura (2019) e Moran (2018) que falam da necessidade dos educadores tornarem as ferramentas tecnológicas suas aliadas diante das demandas educacionais do século XXI. Sabemos que ainda se trata de inovações e, portanto, muitas incertezas ainda nos assolam, tornando o processo de ensino e aprendizagem totalmente desafiador. Por outro lado, houve professores que sentiram a necessidade de relatar durante nossas reuniões o avanço da classe que agora parecia mais engajada, mais responsável e ciente da função do smartphone durante as aulas. A coordenadora também descreveu algumas falas de alguns pais, dizendo que os filhos passaram a utilizar o aparelho não apenas para diversão, mas também para estudar e aprofundar os conhecimentos. As avaliações internas puderam comprovar esse avanço e os 144 próprios estudantes comentavam sobre seus êxitos, reconhecendo a sua evolução do início do ano próprios estudantes comentavam sobre seus êxitos, reconhecendo a sua evolução do início CONSIDERAÇÕES FINAIS Promover uma educação de qualidade requer do educador, não apenas competências teórico- metodológicas, mas também um olhar crítico sobre o contexto em que atua. As crianças de hoje vivem em um mundo onde a virtualidade é a principal característica e as relações passam pela mediação da tela. Desse modo, não podemos concentrar nossos esforços em manter práticas pedagógicas que pouco podem auxiliar os estudantes de hoje em suas vivências e inserção em práticas sociais. As transformações sociais, dos últimos tempos, trouxeram à tona muitos aspectos que precisam ser (re)pensados pelas instituições de ensino de modo que os muros da escola não se mostrem impermeáveis aos avanços de uma sociedade totalmente digital e dependente de recursos móveis como, por exemplo, o smartphone. Concebemos, neste relato, a utilização de metodologias ativas aliada aos recursos digitais como uma das muitas possibilidades de se construir conhecimento. Não tivemos a intenção, em nenhum momento, de transmitir uma receita ou passo a passo efetivo para o processo de aprendizagem, pois acreditamos que por se tratar de um tema muito complexo cada local exige dos profissionais novas estratégias e ações, respeitando sempre a realidade de cada escola. Não podemos deixar de citar, neste trabalho, a atual conjuntura da educação em que grande parte das instituições vivencia a implementação do ensino emergencial remoto em virtude do distanciamento sanitário imposto pelas autoridades públicas. Diante disso, temos observado inúmeros debates e formações acerca das possibilidades ofertadas pelas metodologias ativas e, principalmente, no vasto potencial das TDIC. Nesse sentido, podemos dizer que muitos profissionais têm buscado ressignificar suas práticas, buscando nos recursos tecnológicos instrumentos capazes de minimizar as perdas, tendo em vista o distanciamento sociossanitário. Por fim, vislumbramos, por meio deste breve relato, suscitar novos diálogos, novas reflexões e novos rumos para a educação em nosso país. 145 , p , p https://www2.ifrn.edu.br/ojs/index.php/RBEPT/article/view/6717. Acesso em: 22 set. 2021. REFERÊNCIAS MORAN, J. M. Educação Híbrida: um conceito-chave para a educação, hoje. In: BACICH, L.; TANZI NETO, A.; TREVISANI, F. M. (Orgs.). Ensino híbrido: personalização e tecnologia na educação. Porto Alegre: Penso, 2015. MORAN, J. M. Metodologias ativas para uma aprendizagem mais profunda. In: BACICH, L.; MORAN, J. (Orgs.). Metodologias ativas para uma educação inovadora. 1. ed. Porto Alegre: Penso, 2018. p. 02-25. CÉLIA APARECIDA REGINATO Mestranda em Educação pela Faculdade de Filosofia e Ciências - Campus de Marília/SP. Possui Licenciatura Plena em Pedagogia pela Faculdade de Filosofia e Ciências - Campus de Marília/SP (2001). Possui Curso de Pós-Graduação Lato Sensu - Especialização em Coordenação Pedagógica pela Universidade Federal de São Carlos em parceria com o Ministério da Educação - Programa Escola de Gestores da Educação Básica (2016). Especialização Pós-Graduação Lato Sensu em Gestão e Docência na Educação Básica pela Universidade Santa Cecília em parceria com o IFGE - Instituto de Formação e Gestão Educacional (2013); Habilitação Específica de 2º Grau para o Magistério pelo Centro Específico de Formação e Aperfeiçoamento para o Magistério - CEFAM (1994). ROJO, R.; MOURA, E. Letramentos, mídias e linguagens. 1. ed. São Paulo: Parábola, 2019. ROJO, R.; MOURA, E. Letramentos, mídias e linguagens. 1. ed. São Paulo: Parábola, 2019. SEVERO, C. E. P. Aprendizagem baseada em projetos: uma experiência educativa na educação profissional e tecnológica. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 19, p. 1-13, 2020. Disponível em: , p , p https://www2.ifrn.edu.br/ojs/index.php/RBEPT/article/view/6717. Acesso em: 22 set. 2021. 146 PAULO ALEXANDRE FILHO Graduação em Letras - Alemão pela Universidade Estadual Paulista Júlio de Mesquita Filho - UNESP - Câmpus de Assis. Mestrado em Letras, na área de concentração de Linguagens e Letramentos, pela UNESP - Câmpus de Assis. Atualmente, professor efetivo do Quadro de Magistério, na Secretaria Estadual de Educação - SP, para o componente curricular de Língua Portuguesa. Doutorando em Educação pela Faculdade de Filosofia e Ciências, UNESP - Câmpus de Marília. Desenvolve seu doutorado na linha de pesquisa Teoria e Práticas Pedagógicas em que a tese focaliza os estudos de natureza teórico - metodológica, relacionando-se com as diversas áreas do currículo da Educação Básica e Superior, em especial, com a Didática, a Metodologia, a Psicologia, a Linguística e as políticas educacionais no Brasil. Participa do grupo de pesquisa TECLIN - Tecnologias, Culturas e Linguagens, coordenado pelo professor Dr. Fábio Marques de Souza (UEPE). Tem experiência na área de Linguagens e Letramentos. Artigo Recebido em 25/09/2021 Aceito para Publicação em 30/01/2022 Artigo Recebido em 25/09/2021 Aceito para Publicação em 30/01/2022 REGINATO, Célia Aparecida; FILHO, Paulo Alexandre. METODOLOGIAS ATIVAS NAS AULAS DE LÍNGUA PORTUGUESA DO ENSINO FUNDAMENTAL I: UM RELATO DE EXPERIÊNCIA SOBRE O USO DO SMARTPHONE NO PROCESSO DE ENSINO E APRENDIZAGEM Revista Paidéi@, UNIMES VIRTUAL, Volume 14, Número 25, Jan.2022. Disponível em: https://periodicos.unimesvirtual.com.br/index.php/paideia/index 147
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A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting
Materials
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cc-by
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Review A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting wska 1 , Karolina Cierluk 2, Agnieszka M. Jankowska 1 , Julita Kulbacka 3 , Jerzy Detyna ak 4 Magdalena B. Łabowska 1 , Karolina Cierluk 2, Agnieszka M. Jankowska 1 , Julita Kulbacka 3 , Jerzy Detyna 1,* and Izabela Michalak 4 Magdalena B. Łabowska 1 and Izabela Michalak 4 1 Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering, Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland; 1 Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering, Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland; magdalena.labowska@pwr.edu.pl (M.B.Ł); agnieszka.jankowska@pwr.edu.pl (A.M.J.) 2 1 Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering, Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland; magdalena.labowska@pwr.edu.pl (M.B.Ł); agnieszka.jankowska@pwr.edu.pl (A.M.J.) y gy, g , , ; magdalena.labowska@pwr.edu.pl (M.B.Ł); agnieszka.jankowska@pwr.edu.pl (A.M.J.) 2 Faculty of Chemistry, Wroclaw University of Science and Technology, Norwida 4/6, 50-373 Wroclaw, Pol karolinacierluk@gmail.com 3 Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland; julita.kulbacka@umed.wroc.pl 4 Department of Advanced Material Technologies, Faculty of Chemistry, Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland; izabela.michalak@pwr.edu.pl * Correspondence: jerzy.detyna@pwr.edu.pl Abstract: Sustaining the vital functions of cells outside the organism requires strictly defined pa- rameters. In order to ensure their optimal growth and development, it is necessary to provide a range of nutrients and regulators. Hydrogels are excellent materials for 3D in vitro cell cultures. Their ability to retain large amounts of liquid, as well as their biocompatibility, soft structures, and mechanical properties similar to these of living tissues, provide appropriate microenvironments that mimic extracellular matrix functions. The wide range of natural and synthetic polymeric materials, as well as the simplicity of their physico-chemical modification, allow the mechanical properties to be adjusted for different requirements. Sodium alginate-based hydrogel is a frequently used material for cell culture. The lack of cell-interactive properties makes this polysaccharide the most often applied in combination with other materials, including gelatin. The combination of both materials increases their biological activity and improves their material properties, making this combination a frequently used material in 3D printing technology. The use of hydrogels as inks in 3D printing allows the accurate manufacturing of scaffolds with complex shapes and geometries. materials materials   Keywords: alginate; gelatin; hydrogels; cell cultures; bioprinting Academic Editor: Maurice Collins Received: 8 December 2020 Accepted: 2 February 2021 Published: 10 February 2021 Citation: Łabowska, M.B.; Cierluk, K.; Jankowska, A.M.; Kulbacka, J.; Detyna, J.; Michalak, I. A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting. Materials 2021, 14, 858. https://doi.org/10.3390/ma14040858 Review A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting The aim of this paper is to provide an overview of the materials used for 3D cell cultures, which are mainly alginate–gelatin hydrogels, including their properties and potential applications. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Hydrogels are three-dimensionally cross-linked networks of hydrophilic polymeric materials capable of absorbing large amounts of liquid between their polymeric chains. These materials are created through the reaction of one or more monomers or by association bonds (hydrogen bonds and strong van der Waals forces between chains). The ability of these materials to attract water molecules and their tendency to dissolve in water result from the presence of hydrophilic functional groups connected with the polymeric backbone (−OH, −COOH, −COO, > C = O, −CHNH2, −CONH, −CONH2, −NH2, and −SO3H) [1–6]. The amount of water that polymer networks of hydrogels are capable of absorbing is between 10% and 1000 times greater than dry weight [7]. However, the content—and thus the volume—of a hydrogel can change over time, depending on the external parameters (e.g., the nature of the solvent, temperature, pH), as well as on the drying process [5]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). There are numerous literature reports on the classification of hydrogels. They can be grouped according to their source, configuration (amorphous, semi-crystalline, crys- https://www.mdpi.com/journal/materials Materials 2021, 14, 858. https://doi.org/10.3390/ma14040858 Materials 2021, 14, 858 2 of 24 talline) [2,3,8,9], type of cross-linking (chemically cross-linked, physically cross-linked, en- zymatically cross-linked) [2,10], and polymeric composition (homopolymeric, copolymeric, multipolymer interpenetrating polymeric—which consists of at least two independent cross-linked polymers (synthetic or natural), however they are not covalently bonded to each other) [2,9,11]. Hydrogels can also be divided according to their durability. Despite their high endurance and stability in swelling environments, durable and degradable hydrogels can be distinguished. In comparison to natural sources of hydrogels, synthetic sources have better mechanical strength but a slower degradation rate. However, they are not biocompatible in vivo, unlike natural hydrogels [9,12,13]. Hydrogels can also be divided depending on the presence or absence of electrical charge in the polymeric chain. Hydrogels that have no charge in their backbone are non-ionic or neutral. Conversely, ionic hydrogels are cationic, which are positive-charge-bearing, or anionic, which have a negative charge in their chains. 1. Introduction These states depend on various factors, such as the chemical compositions of the materials, the amounts and types of solvents, the nature of the bonds, the polarity of the polymers, the charges, and the temperature. However, through physico-chemical modification of the structure and cross-linking, it is possible to obtain hydrogels with desired viscous, rheological, and mechanical properties [4,5,18]. p p The universality of hydrogels enables their application as biosensors in the food in- dustry, agriculture, water purification, diagnostics, cosmetics, dentistry, and biomedical fields (e.g., in pharmaceuticals, drug delivery, and wound dressings) [2,10,19]. In bio- engineering, they are mainly utilized due to their biocompatibility, biodegradability, and low immunogenicity. The water content in hydrogels supports their integrity, solubility, and diffusion of their substances, which are useful factors in delivering a therapeutic agent. The properties of hydrogels, such as their soft structures, elasticity, and similarity to living tissues, make them excellent for tissue engineering and regenerative medicine applications, as well as in stem cell cultures and cell implantation. Tissues and organs in the human body are characterized by their unique structures, differing in water content, as well as their mineral and protein contents, therefore it is necessary to use a biopolymer to manufacture a hydrogel with common building blocks [2,5,6,12]. Hydrogels are excellent materials for the production of substrates for cell cultures, for example three-dimensional porous scaffolds that can imitate the extracellular matrix (ECM), ensuring cell proliferation and differentiation in the in vitro microenvironment. Furthermore, their mechanical and structural properties allow imitation of native tissues and provide cell protection against possible damage caused by external factors [6,17,20]. p g y In contrast to 2D cell cultures, 3D structures provide more space for cell proliferation and more closely mimic the natural environment [17]. The use of hydrogel materials for 3D cell cultures involves significant challenges, such as the in vitro behavior of the cells and the influence of external conditions on their appropriate development. Although each cell line has different requirements and the preparation of a universal in vivo environment is not possible, the simplicity with which hydrogel properties can be modified enables them to be customized according to the specific requirements. Furthermore, the utilization in the biomedical field of 3D printing technology makes it possible to achieve more accurate geometries and shapes for the manufactured structures [21,22]. 1. Introduction The presence of a charge in the backbones affects their dissociation in different media environments, i.e., cationic hydrogels swell in media at lower pH (< 7) and anionic hydrogels at higher pH (> 7) [14]. There is also a group of ampholyte hydrogels that contain both cationic and anionic functional groups. These are characterized by their hydrophilicity and good biocompatibility [8,9,15]. The classification of hydrogels is summarized in Figure 1. Figure 1. Classification of hydrogels. The materials used in the hydrogel manufacturing process include both natural and synthetic polymers, as well as hybrid hydrogels, which involve combinations of natural and synthetic polymers [2,12]. These natural polymers can be obtained from natural sources such as plants, bacterial cultures, or animals, including polysaccharides (e.g., agarose, algi- nate, carrageenan, chitosan), glycosaminoglycans (e.g., heparin, hyaluronic acid, keratan sulfate), and polypeptides or proteins (e.g., collagen, fibrin, gelatin, silk). Examples of synthetic polymers used to manufacture hydrogels include poly(ethylene glycol) (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(vinyl alcohol) (PVA) [1,16,17]. Hy- Figure 1. Classification of hydrogels. Figure 1. Classification of hydrogels. Figure 1. Classification of hydrogels. The materials used in the hydrogel manufacturing process include both natural and synthetic polymers, as well as hybrid hydrogels, which involve combinations of natural and synthetic polymers [2,12]. These natural polymers can be obtained from natural sources such as plants, bacterial cultures, or animals, including polysaccharides (e.g., agarose, algi- nate, carrageenan, chitosan), glycosaminoglycans (e.g., heparin, hyaluronic acid, keratan sulfate), and polypeptides or proteins (e.g., collagen, fibrin, gelatin, silk). Examples of synthetic polymers used to manufacture hydrogels include poly(ethylene glycol) (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(vinyl alcohol) (PVA) [1,16,17]. Hy- The materials used in the hydrogel manufacturing process include both natural and synthetic polymers, as well as hybrid hydrogels, which involve combinations of natural and synthetic polymers [2,12]. These natural polymers can be obtained from natural sources such as plants, bacterial cultures, or animals, including polysaccharides (e.g., agarose, algi- nate, carrageenan, chitosan), glycosaminoglycans (e.g., heparin, hyaluronic acid, keratan sulfate), and polypeptides or proteins (e.g., collagen, fibrin, gelatin, silk). Examples of synthetic polymers used to manufacture hydrogels include poly(ethylene glycol) (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(vinyl alcohol) (PVA) [1,16,17]. Hy- Materials 2021, 14, 858 3 of 24 drogels can occur in different forms, such as solid, semi-solid, and liquid states. 1. Introduction Three-dimensional printing is a versatile technique for creating complex biomimetic tissue structures with extraordinary spatial precision [23]. The aim of this review is to present trends in the utilization of alginate–gelatin hydro- gels as substrates in 3D cell cultures, as well as an overview of their material characteristics. Due to the lack of biological activity, sodium alginate cannot be employed alone for the production of hydrogel matrices. It is most often used in combination with other polymers to allow the adhesion of cells and their healthy development. The combination of sodium alginate and gelatin provides an excellent hydrogel for use as a substrate for 3D cell cultures due to its unique biological properties, such as its biocompatibility, biodegradability, and non-toxicity. Some of the recent additions to this field are cellulose nanomaterials, which improve the chemical, physical, and biological properties of the created hydrogels. The eas- ily modifiable mechanical properties of these materials can be adapted to the living tissue, making them ideal environments for cell culture development. Moreover, the rheological properties of alginate-gelatin hydrogels make them excellent candidates for utilization in additive technologies at room temperature. Thus, it is possible to use them as hydrogel inks within the cell content, without risking damage. 2. Materials and Methods for the Preparation and Characterization of Hydrogel Substrates The most important characteristics of a hydrogel are its permeability and water retention capacity. In contact with water, the polar hydrophilic groups are hydrated, which leads to the creation of primary bound water and swelling of the network. This results in the exposure of hydrophobic groups capable of interacting with water molecules and forming Materials 2021, 14, 858 4 of 24 secondary bound water. Often total bound water is also formed by combining primary and secondary bound water. The hydrogel reaches a balanced swelling level by creating an elastic network retracting force by opposing covalent or physical cross-linking [24,25]. g y pp g p y g The biocompatibility of a hydrogel depends on the interaction of the material and its degradation products with living tissue or body fluids—it should not induce toxic or immunological responses. The low interfacial free energy of the hydrophilic surface in contact with body fluids results in a low tendency of proteins and cells to adhere to these surfaces, which makes the hydrogel satisfactorily biocompatible [26,27]. Hydrogels are characterized by their flexibility and soft structures. Therefore, these hydrated polymeric biomaterials are often utilized in biomedical fields as ECM substitutes. The biological, physical, and mechanical properties, as well as the permeability control of hydrogels, provide a biomimetic microenvironment similar to that of living tissue. The physico- chemical viscosity modification of a hydrogel allows control of its mechanical properties, as well as enabling various forms of the material to be achieved for 3D cell cultures, such as beads, injectable gels, moldable gels, and macroporous structures [28,29]. A hydrogel’s porosity is a very important aspect, because large surface areas foster attachment and ingrowth of cells. Moreover, pores connected by channels enable transport of gases, nutrients, and waste [20]. Hydrogel cell culture substrates can be prepared as mono- or multi-component mix- tures of different polymers ratios. Composite substrates are preferred due to their ability to combine the functions of the individual components, as opposed to single-component substrates [30]. Among the natural polymers used for hydrogel fabrication for 3D cell cultures are alginate, collagen, gelatin, and fibrin. One of the most popular materials is based on sodium alginate [29,31]. Alginate hydrogel is an excellent material for imitating the extracellular matrix, which is the outer environment of the cell. 2. Materials and Methods for the Preparation and Characterization of Hydrogel Substrates However, this natu- ral polymer does not provide mammalian cell-adhesive ligands, i.e., short polypeptide sequences in ECM proteins, which mimic ligands used in integrin-mediating cell adhesion formation and facilitate cell attachment [32,33]. In order to form three-dimensional struc- tures in hydrogels for cells, components that are part of the in vivo matrix are required. On the surfaces of cells are receptors, which allow the cells to bind with matrix ligands. Typically, cells have receptors for many different ECM factors, which allow them to simul- taneously bind to various ligands and to form networks. The cells themselves also produce components of the extracellular matrix; nevertheless, supplementation of hydrogels with ligands is required [34]. Thus, to mimic the ECM, this material is utilized in combination with other materials, such as gelatin, chitosan [32], elastin, agarose, or hyaluronic acid [20]. Moreover, additives such as nanocellulose act as reinforcing agents in hydrogel materials, increasing the mechanical properties and improving the printability [35]. In this review, we would like to focus mainly on alginate–gelatin hydrogels for cell cultures. 2.1. Alginate Alginate is commonly used to manufacture hydrogels due to its ability to retain water. This linear polysaccharide is obtained from brown seaweed or bacteria. The quantity and quality of alginate extracted from brown seaweed may depend on the species, as well as the harvest season. Typically, the amount of extracted polysaccharide is up to 40% of the dry matter of brown seaweed and also strongly depends on the extraction method [17,36,37]. Alginate consists of two hexuronic acids—α-L-guluronic (G) and β-D-mannuronic (M), linked by 1–4 glycosidic bonds, as shown in Figure 2 [20,38]. Ionic gel formation depends on the chemical composition and block distribution in the alginate molecule [17,28]. The physical properties are determined by the numbers of MM, GG, and MG blocks. The high viscosity is dependent on the high content of M blocks, while the high content of G gives higher gelling properties [39]. Alginate-based gels can also be evaluated using the M/G ratio. Flexible gels are obtained using a high M/G ratio, while brittle gels are obtained from sodium alginate with a low M/G ratio [40]. Although alginate is biocompatible, biodegradable, and non-toxic, it has several disadvantages, such as low its bioadhesivity Materials 2021, 14, 858 5 of 24 and biological inertness, which limit its applications, particularly in tissue engineering. The main drawback of alginate is the difficulty of obtaining pure alginate from contaminated marine algae. Removal of impurities is possible through further purification steps, which generate additional costs and are time-consuming [41,42]. and biological inertness, which limit its applications, particularly in tissue engineering. The main drawback of alginate is the difficulty of obtaining pure alginate from contaminated marine algae. Removal of impurities is possible through further purification steps, which generate additional costs and are time-consuming [41,42]. Figure 2. Chemical structure of alginate (M and G blocks). Reproduced with permission from [43]. Copyright 2020 IOSR Journal of Pharmacy. Figure 2. Chemical structure of alginate (M and G blocks). Reproduced with permission from [43]. Copyright 2020 IOSR Journal of Pharmacy. In order to increase the mechanical properties of alginate hydrogels, a variety of covalent cross-linking methods have been used. However, the mechanical properties of ionically cross-linked alginate hydrogels decrease over time on account of external streams of cross-linking ions into the surrounding environment [44]. The mechanical properties of alginate-based hydrogels may also depend on the gel homogeneity, which can be controlled by the gelling rate. 2.1. Alginate An important parameter that affects the gelling rate is the temperature of gelation [45]. The combination of alginate hydrogels with other materials can lead to the improvement or changes of their physical and mechanical properties [46]. Alginates are available in various forms, including hydrogels, microcapsules, micro- spheres, fibers, foams, and sponges. However, the most commonly used form, especially in the medical field, is the hydrogel form [46]. The rheological properties of the alginate hydrogel and its ease of gelation, biodegradability, and lack of toxicity make it suitable for a wide range of industrial applications (e.g., in the food industry, agriculture, cosmetics, pack- aging, and textiles). Its biomedical applications include dentistry, pharmaceuticals, wound healing, drug delivery, cell therapy, and as a bone graft substitute for spine fusion [4,47]. Fabrication of this hydrogel in the medical industry requires the use of high-purity sodium alginate to prevent immunogenicity [28,29]. 2.2. Gelatin Gelatin is a type of protein manufactured through hydrolysis of collagen of animal origin (i.e., bovine, porcine, or fish collagen), connective tissues, and bones [32,48]. Its biocompatibility, lack of immune responses in the body, degradability, and absence of toxicity resulted in its approval by the U.S. FDA. Thus, it is used in the food industry, as well as in the biomedical sector (e.g., pharmacy, wound healing, drug delivery, gene therapy, tissue engineering, regenerative medicine). It is one of the most often utilized materials for cell cultures due to its retention of collagen’s bioactive sequences. This enables the creation of a suitable microenvironment for cell adhesion, migration, proliferation, and differentiation [17,32]. Despite its numerous advantages, gelatin also has some drawbacks. One of them is its solubility, because at around 30–40 ◦C it passes from a gel state to a solution, which limits its long-term use in transplantation. In order to prolong the degradation time and increase the water resistance of gelatin, it is necessary to apply cross-linking [49]. In Figure 3, the basic chemical composition of gelatin is shown. Materials 2021, 14, 858 6 of 24 Figure 3. Chemical structure of gelatin. Reproduced with permission from [50]. Copyright 2020 John Wiley & Sons. Figure 3. Chemical structure of gelatin. Reproduced with permission from [50]. Copyright 2020 John Wiley & Sons. Gelatin is exposed to factors such as temperature, pH changes, and the presence of solvents, which modify its flexibility, meaning it takes various forms. Below 40 ◦C it can occur in a gel state (the reaction is thermally reversible), while above this temperature it can become an aqueous solution. Chemical modifications and cross-linking change the structure of gelatin hydrogel, as well as its properties and the forms it acquires [32,51]. Depending on the animal species, gelatin may have different properties. Wang et al. [52] provided a comparison of methacrylate gelatin from cold water fish and pigs and cold-soluble gelatin. Cold-soluble gelatin has a balance between thermal stability and physical and biological properties compared to the other types of gelatin. Cold water fish gelatin also has thermal stability when compared to pork [52]. A comparison of the properties of gelatin from cold water fish and pigs was also made by Yoon’s [53] research team. The research showed the same results as in Wang’s study [53]. 2.3. Alginate Dialdehyde-Gelatin Hydrogels Lately, there has been a lot of interest in hydrogels, which are a combination of natural polysaccharides (e.g., alginate) and proteins (e.g., gelatin). This is due to the adverse properties of alginate itself, such as its inefficient cell attachment, poor alginate– cell interactions, and slow degradability with uncontrolled kinetics [54]. The solution to these limitations may be the use of alginate that has been previously oxidized to form alginate dialdehyde (ADA) and which is subsequently covalently cross-linked with gelatin (GEL). Between aldehyde groups of ADA and free amino groups of gelatin, Schiff’s base is formed [51,55,56]. The chemical structure of ADA-GEL is shown in Figure 4. The obtained alginate-gelatin cross-linked hydrogel (ADA-GEL) can be used to pro- duce microcapsules (for encapsulation of bioactive compounds or cells and for drug delivery) [56–58] and can be applied as a non-cytotoxic biomaterial with good mechanical strength and biocompatibility in regenerative medicine (e.g., bone tissue regeneration) [55], or as a soft tissue adhesive in wound healing [59]. The obtained alginate-gelatin cross-linked hydrogel can differ in its microstructure and physico-chemical properties, depending on the oxidation degree of the ADA and the cross-linking degree and gelation time of the ADA-GEL [51,55,59]. Microcapsules produced from ADA-GEL are characterized by their higher degradability when compared to alginate microcapsules [51], and exhibit good cell adhesion, proliferation, and migration properties [51,57,60]. An interesting application of alginate dialdehyde cross-linked gelatin hydrogel was suggested by Manju et al. [61]. This hydrogel was used as a coating material for polyethylene terephthalate (PET) vascular graft prostheses, which are intended to replace compromised arteries in the body. The ADA-GEL was non-toxic and was charac- terized by its biocompatibility, biodegradability, and reduced water permeability, while it had no effect on the mechanical properties of the graft and improved the adhesion and proliferation of fibroblast cells. Materials 2021, 14, 858 7 of 24 Figure 4. Chemical structure of alginate dialdehyde-gelatin (ADA-GEL). Reproduced with permis- sion from [51]. Copyright 2020 Royal Society of Chemistry. Figure 4. Chemical structure of alginate dialdehyde-gelatin (ADA-GEL). Reproduced with permis- sion from [51]. Copyright 2020 Royal Society of Chemistry. ADA-GEL can be also used as an ink, but this application may be limited due to its viscosity [58]. Nevertheless, there are some studies using ADA-GEL as an ink in 3D printing technology. In the work by Dranseikiene et al. [58], ring-shaped structures containing alginate dialdehyde-gelatin cell-laden hydrogel were fabricated. 2.3. Alginate Dialdehyde-Gelatin Hydrogels The cell viability (MG-63 osteosarcoma cells) in samples depended on the cross-linking agent (CaCl2 or BaCl2), with better results in terms of increased cell viability and cell network formation being observed after cross-linking with Ba2+. Ruther et al. [60] investigated the printing of three- dimensional, cell-containing, vessel-like structures using ADA-GEL ink. This artificial vessel supported the viability, proliferation, and migration of immobilized cells (human dermal fibroblast and human umbilical vein endothelial cells). 3. Material Additives Used in Gel Systems Apart from the basic materials forming the gel systems, additives are also used to increase their efficiency. Material additives are inactive ingredients designed to structurally improve and enhance the hydrogel’s characteristics, as well as influence the degradation profile and interactions between cells and the matrix. Among the additives, materials from natural (e.g., collagen, pectin), synthetic (e.g., polyvinyl alcohol, poloxamers), and semi-synthetic sources (e.g., cellulose derivatives) can be distinguished. The used mate- rials include peptides that increase cell adhesion, nanomaterials that adjust rheological properties to specific applications, as well as growth factors that enable direct stem cell differentiation [62]. Additives added to alginate hydrogel culture media are used to influ- ence cell growth and attachment. Furthermore, they improve resistance to degradation and facilitate the production of the hydrogel, which is a benefit during additive manufacturing and processing in terms of bioprinting [63]. To enhance the strength and increase the mechanical properties of hydrogels when using additive manufacturing technologies in tissue engineering, additives in the form of microfibers, nanofibers, microparticles, and nanoparticles, as well as woven and non- woven scaffolds, are also used. Reinforcement of hydrogels in this manner eliminates the need for dense cross-linking, allowing them to obtain similar mechanical properties, thereby improving cell migration and promoting the formation of neo-tissues [64]. The nanomaterials included in hydrogels, such as cellulose and chitin, can affect the structures of the hydrogels, thus improving their mechanical properties and drug release profiles [65]. Materials 2021, 14, 858 8 of 24 Zhang et al. [66] created hydrogel composites with alginate microcapsules cross-linked with Ca2+ ions and cellulose whiskers. As a result, the mechanical properties of the hydrogels were improved and the release profile of the drug was more stable, without causing cytotoxicity. In Kinneberg’s work [67], the application of a microporous, fibrous collagen scaffold increased the tangent modulus and toughness of the hydrogel. Research carried out by Park et. Al. [68] showed that the addition of nanocellulose to alginate hydrogel enhanced its mechanical strength, but also improved the cells’ ability to proliferate and prolonged their viability. Similar research was carried out by Siquiera et al. [69], whereby the viability of L929 fibroblast cells was examined in alginate hydrogels and compared to nanocellulose–alginate hydrogels containing nanofibers and nanocrystals. The addition of nanocellulose supported the cell bioadhesion and cell growth on the created medium. 3. Material Additives Used in Gel Systems Nanocellulose is one of the materials that improves not only the mechanical properties of the created substrates, but also has a direct effect on cells and their development. 3.1. Hydrogel Fabrication from Materials Useful for 3D Cell Culture Various materials can be used to create different types of hydrogel matrices. The compositions of these media depend on the type of culture and the results expected by the researchers. There is no doubt that 3D cultures and systems are more reliable setups that faithfully recreate cellular responses. The available data show that 3D cells do not have receptors that recognize alginates. Having high purity is of special importance, because some residual endotoxins can interact with receptor CD14 (a receptor for complexes of lipopolysaccharide and lipopolysaccharide binding protein) [28]. Thus, highly purified alginates are crucial in future applications. Natural substrates such as sodium alginate have been used by Zimmermann et al. [70], Dvir-Ginzberg et al. [71], and Bauer et al. [72]. It was also noted that alginates can be functionalized to obtain specific properties. For instance, it was found that arginine–glycine–aspartic acid conjugated to sodium alginate (RGD-alginate) can stimulate cell proliferation and can affect cell adhesion and differentia- tion [73]. De Melo et al. [74] investigated PEG poly(ethylene glycol) and sodium alginate concentrations to optimize a polymer blend for usage as a mechanically resistant support hydrogel. It was also shown that a combination of alginate with gelatin can decrease gelatin leakage over prolonged culture while simultaneously enhancing cell adhesion and the secretion of vascular endothelial growth factor (VEGF) [57]. Chung et al. [75] and Li et al. [76] prepared a sodium alginate-gelatin hydrogel in order to examine cell development. The gelatin strengthened the cell-filled sodium alginate scaffolds. Demirta¸s et al. [77] fabricated sodium alginate and alginate-hydroxyapatite (HA) hydrogels and used them for printing. Schwarz et al. [78] manufactured 3D-printed, grid-like structures for cartilage tissue engineering by using a system composed of an oxidized alginate-gelatin hydrogel. The examples of the applied materials for the 3D cell culture substrates and their concentrations are shown in Table 1. 9 of 24 Materials 2021, 14, 858 Table 1. The examples of hydrogel materials based on alginate and gelatin used for the production of 3D cell culture substrates. 3.1. Hydrogel Fabrication from Materials Useful for 3D Cell Culture Hydrogel Type of Cells Sodium Alginate Concentrations (w/v%) Gelatin/Other Additive Concentrations (w/v%) The Aim of Research Reference sodium alginate C2C12 mouse myoblasts 2.5 testing the influence of substrate stress–relaxation on the regulation of muscle cell (myoblast) proliferation and spreading in vitro [72] sodium alginate-gelatin BL6 primary mouse myoblasts 1, 2, 4 10 optimization of the bioink consistency and investigation of printability with live cells [75] sodium alginate-gelatin rat Schwann cell line RSC96s 4 20 examination of the cell behavior in the created microenvironment [76] sodium alginate-gelatin L929, mouse fibroblast cell line 1 4 evaluation of the cell viability and possibility to develop in the created microenvironment [44] 2–2.5 4–8 analysis of the printability with the cell-filled bioink and optimization of the parameters to obtain structures for cell cultures [79] 2.5 8 sodium alginate-gelatin Non-Small Cell Lung Cancer (NSCLC) PDX (EGFR T790M) cell line 1–6 3–8 research on the cells growth on the manufactured hydrogel; modification of the alginate-gelatin concentration to achieve good printability [80] sodium alginate-gelatin mesenchymal stem cells (MSCs) from adult sheep adipose tissue 1, 3, 5, 7, 9 6 examination of the printing possibilities according to the individual component concentrations [81] 5 2, 4, 6, 8, 10 sodium alginate-gelatin human adipose-derived mesenchymal stem cells (MSCs) 3 10 evaluation of the cell development on produced substrates [82] sodium alginate-gelatin with mouse plantar dermis (PD) mouse mesenchymal stem cell (sMSCs) 1 3 investigation of the chemical and physical properties of the Alg-Gel-PD bioink, and its effect on embedded mouse mesenchymal stem cells (MSCs) [83] sodium alginate-gelatin mixed with hECM HepaRG human bipotent hepatic progenitor cells; A549 human epithelial lung carcinoma cells 2 3 the optimization of a bioink composed of hydrogel and human extracellular matrix (hECM) to print human HepaRG liver cells and testing of its suitability for the study of transduction using an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5) [84] hECM 0; 0.25; 0.5; 1; 2 alginate dialdehyde-gelatin (ADA-GEL) osteosarcoma cells MG-63 5 5 evaluation of the cell encapsulation possibility in prepared hydrogel and monitoring of cell activity [58] alginate dialdehyde–gelatin (ADA-GEL) HUVECs human umbilical vein endothelial cells; rat Schwann cells 2; 3; 6 2 characterization of the printability and cell viability of various concentrations of alginate dialdehyde (ADA)–gelatin (Gel) hydrogels for bioprinting [85] 2 3; 6 sodium alginate with d-gluconic acid rat liver cell 1 0.2 analysis of the cell growth on the created substrate [71] sodium alginate/PEO-bisamine (polyoxyethylene bis(amine)) L929, mouse fibroblast cell line 2 5 and 20 evaluation of the cell viability and possibility of developing in created microenvironment [44] sodium alginate/PEO with gelatin mesenchymal stem cells (MSCs) 2, 4, 5 PEO 1:1 with alginate; gelatin: 10% examination of the cell viability and possibility of proliferating in created microenvironment [86] of hydrogel materials based on alginate and gelatin used for the production of 3D cell culture substrates. 3.1. Hydrogel Fabrication from Materials Useful for 3D Cell Culture PEO 1:1 with alginate; gelatin: 10% Materials 2021, 14, 858 10 of 24 10 of 24 According to the type of cell, different conditions are needed for growth, proliferation, or differentiation, and therefore a surface is required for culture, which will provide the cells with a specific environment. Alginate-gelatin-based hydrogels are mainly found as substrates in laboratory studies, however there are already commercially available cell culture substrates made from other materials, which are intended for specific cell types. One of them is Matrigel, a gelatinous protein mixture obtained from Englebreth– Holm–Swarm tumor cells from mice. It is used as a basement membrane matrix for stem cells and maintains them in an undifferentiated state. However, this substrate is not precisely defined, thus it may not reflect the accuracy of the research results [87]. Similar to Matrigel is Cultrex® BME, which is used as its substitute. Cultrex® BME is also a homogenate of mouse tumor tissue, therefore both substrates differ in composition from human transmembrane proteins [88]. HyStem®-C is a biodegradable and biocompatible material consisting of three blocks: Glycosil® (thiolated hyaluronic acid), Gelin® (tioltated gelatin), and Extralink® (polyethylene glycol diacrylate). It is capable of mimicking ECM, and therefore is used as a tissue model in developing new drugs and cell cultures. It is a suitable material for bioprinting and for designing organ models [89]. Another hydrogel suitable for cell culture is Biogelx™, obtained from synthetic peptide powder functionalized with collagen, fibronectin, and laminin, which was created to mimic the ECM. PuraMatrix™ is a peptide hydrogel consisting of amino acids that is suitable for 3D cell culture [6]. Most of these products are used for research purposes. However, new materials for 3D cell cultivation and bioprinting, such as bacterial cellulose–gelatin hydrogels [90]; synthetic, thermo-responsive polyisocyanopeptide hydrogel [91]; chitosan–fibrin hydrogel for tissue engineering [92]; collagen-based hydrogels [93]; and others are currently being fabricated and tested. 3.2. Hydrogel Cross-Linking Methods Cross-linking is a process that leads to the formation of a three-dimensional polymer network structure by binding one polymer to another. It is a stabilization process—the attached polymers lose their ability to move as a single chain. The consequence of cross- linking is a change in the physical and chemical properties of a substance. Cross-linked polymers are mechanically stronger but less flexible and less heat- or solvent-resistant in comparison to liquid polymers [94]. p q p y Cross-linking of hydrosols (liquid polymers) has positive effects on the physico- chemical properties of hydrogels—including their water holding capacity, hardness, and structural integrity, which affect their cytocompatibility—as well as on their biochemical properties [94,95]. In bioprinting, the cells are mixed in hydrosol and the cross-linking of the hydrogel takes place after extrusion from the bioprinter. Thus, the cross-linker should not be toxic to cells and should have fast cross-linking capability to maintain the highest cell viability [96]. Sodium alginate is most often physically cross-linked through the application of ionic interactions [45,94,95]. Cross-linking with divalent or trivalent ions occurs under mild conditions, at room temperature and neutral pH [94,96]. Such cross-linking conditions are suitable when using cells. Cross-linking of sodium alginate occurs through a bond between G blocks (guluronate blocks) and divalent ions. This is an “egg box” cross-linking model [45,95]. Ca2+, Ba2+, and Zn2+ are divalent ions, which are usually used in such ap- plications [45,95,96]. An example of ionic cross-linking is presented in Figure 5. The choice and concentration of ionic cross-linkers is a critical factor in order to obtain enhanced cell viability, better printability, and mechanical stability of a hydrogel during bioprinting [96]. Other studies have shown that the subsequent addition of cross-linkers in appropriate ratios can improve the cell viability and mechanical stability of hydrogels [96–98]. The sta- bility of hydrogel cross-linking in cell cultures is an important issue. Under physiological conditions, divalent ions can be exchanged with monovalent cations, which contribute to the depletion of the network [45]. 11 of 24 11 of 24 Materials 2021, 14, 858 Figure 5. Graphical illustration of ionic cross-linking of polymer chains (own source). Figure 5. Graphical illustration of ionic cross-linking of polymer chains (own source). Photo cross-linking is another covalent cross-linking approach. Modified sodium alginate with the addition of methacrylate can be cross-linked via exposure to an argon ion laser (514 nm) for 30 s in the presence of eosin and triethanolamine. 3.3. 3D Cell Cultures Cell cultures growing in the three-dimensional form behave quite differently than in a monolayer. Due to the use of 3D culture, the impact of cell interaction and the influence of the external environment on cell development have been investigated. This is why the research on three-dimensional cell cultures is so important. In vitro experiments aim to reflect the in vivo conditions to the greatest extent. Each cell line has individual requirements and conditions that must be met to achieve the most reliable test results. It is not realistic to work out a uniform template of optimized culture conditions for all cell lines. However, for faster optimization of three-dimensional cell culture conditions, it is worth paying attention to certain aspects described below. This review focuses on the use of 3D-printed alginate-gelatin hydrogel scaffolds in three-dimensional cell cultures. Hydrogels obtained from sodium alginate and gelatin imitate the extracellular matrix. Both components of the gel, which are cross-linked under mild conditions, are transparent; decomposition of the network can take place under controlled conditions; and the porosity of the material provides the cells with access to nutrients and metabolite exchange [28,105]. It is worth noting that gelatin as an animal-derived material could be an immunogenic agent. However, the use of sodium alginate alone does not provide optimal conditions for the development of culture [28,106]. Depending on the application of the hydrogel, the selection of the type and con- centration of sodium alginate and gelatin affects the properties of the hydrogel and the cell growth [28,107]. The number and length of G blocks that are involved in ion cross- linking depend on the porosity, stability, and elasticity of the gel [28,108]. The mechanical properties can affect cell proliferation and conditions. Alginate has different affinities for divalent ions, which is why their selection can affect such properties as swelling, stability, and cell growth [28]. Sarker et al. [96] studied the effects of three ionic cross-linkers of alginate—CaCl2, ZnCl2, and BaCl2—in terms of the viability of Schwann cells (the main glial cells of the peripheral nervous system) and the mechanical properties of hydrogels. Research showed that the best of these three cross-linking ions in terms of the impact on mechanical properties was Ba2+, while Ca2+ had the best effect on cell viability. The research team suggested using both cross-linking compounds, namely CaCl2 and BaCl2 [96]. 3.2. Hydrogel Cross-Linking Methods Materials 2021, 14, 858 12 of 24 12 of 24 Gelatin, as a product of collagen hydrolysis, can be cross-linked with transglutaminase, which catalyzes the acyl transfer reaction between the γ-carboxamide group of peptides or protein-bound glutamine residues (acyl donors) and primary amines (acyl acceptor) containing ε-amino groups of lysine. A network of isopeptide bonds is formed [102]. This approach was used in research conducted by Schwarz et al. [78] and Chen et al. [95]. The properties of interpenetrating alginate-gelatin hydrogel have been studied. Sodium alginate has been cross-linked with calcium ions, while gelatin has been cross-linked with transglutaminase [95]. Another gelatin cross-linking agent could be water-soluble 1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide (EDC), which specifically forms amide or peptide bonds between the carboxylic acid and protein amino groups [103]. One way to cross-link gelatin is to modify it with tyramine (TA). TA–gelatin is obtained by carbodiimide coupling of tyramine groups on gelatin. Then, TA–gelatin is covalently cross-linked in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Tyrosine–tyramine, tyrosine–tyrosine, and tyramine–tyramine bonds are formed. The addition of TA–gelatin increases the mechanical stability and gelation rate and slows the enzymatic degradation of silk hydrogels cross-linked in physiological buffers. Ma- nipulation of the amount of TA–gelatin added allows modulation of the silk hydrogel’s bioactivity, in turn affects the cell’s microenviroment [104]. 3.2. Hydrogel Cross-Linking Methods Cross-linking takes place in mild conditions with the proper selection of chemical initiators. This method can be used directly in contact with drugs and cells. To avoid harmful cross-linking conditions (where a light sensitizer or acid release is required as a by-product), polyallylamine partially modified with acetylphenoxyinninamyldiene chloride is used as an alternative, which converts to a dimer under the influence of light at about 330 nm and does not release toxic by-products during the cross-linking reaction [45,99]. Additionally, using light radiation, the resulting hydrogel from sodium alginate and polyallylamine has improved mechanical properties [45]. Thermal gelation is the next cross-linking approach. The use of this method for sodium alginate is not common due to the poor sensitivity of sodium alginate to temperature. How- ever, semi-interpenetrating polymer networks (semi-IPN) can be used. An example is a semi-IPN prepared from copolymerization of N-isopropylacrylamide (NIPAAm) with a poly(ethylene glycol)-co-poly(-caprolactone) (PEG-co-PCL) macromer in the presence of sodium alginate using UV irradiation. The use of sodium alginate in semi-IPNs improves the mechanical strength. The increase in hydrogel swelling with the increase in the con- centration of sodium alginate at a constant temperature and decrease in swelling with the increase in temperature can be used for drug delivery [45,100]. Covalent cross-linking is another method. Covalent networks are formed by poly- merization of end-functionalized monomers or macromers (Figure 6) [94]. In this type of network, cross-linking dissociation and bond transformation do not occur. Water migration causing stress relaxation is also preserved. Chemically modified sodium alginate is used for covalent cross-linking [28]. One of the covalent cross-linkers used in the cross-linking of sodium alginate is poly(ethylene glycol) (PEG), which is used for PEG-diamines. It has been shown that the mechanical properties and swelling can be controlled by using covalent cross-linking agents. Using hydrophilic cross-linkers can compensate for the lack of the hydrophilic nature of the gel due to the cross-linking reaction [45]. Figure 6. Graphical illustration of covalent cross-linking of polymer chains (own source). Figure 6. Graphical illustration of covalent cross-linking of polymer chains (own source). An interesting approach to create cross-linked hydrogels is to use the cells themselves. For example, cells added to adhesive ligand-modified sodium alginate (arginine-glycine- aspartic acid peptide sequence-modified (RGD-modified)) can form networks by binding multiple polymer chains based on the receptor–ligand binding. This method of cross- linking occurs without the addition of cross-linkers [45,101]. 3.3. 3D Cell Cultures When forming hydrogels for cell cultures, an important aspect is sodium alginate modification in order to adapt and control the type of and interaction between cells. Examples of such modifications are the covalent attachment of heparin-binding peptides (HBP) [28,109] or peptides found in extracellular matrix proteins [28,109,110]. In a ligand– Materials 2021, 14, 858 13 of 24 13 of 24 receptor approach, cells can interact with the hydrogel network. For example, attachment of the RGD (arginine–glycine–aspartic acid) peptide sequence has also been shown to affect the initiation of interactions with cells via recognition of an adhesive molecule (RGD sequence) by cell receptors. Some cell lines may show accelerated proliferation when sodium alginate is modified by the RGD sequence [28,111]. As it turns out, the sequence fragments flanking the RGD and the conformation (linear or cyclic) and length of the RGD sequence all impact the integrin receptor affinity and selectivity, depending on the cell type. Additionally, depending on the cell type, the used RGD sequence density may affect the development of the culture [28,112]. An important issue when establishing a 3D cell culture is the printing process. Atten- tion should be paid to parameters such as the rheological properties, printing process, and needle diameter relative to time; the dimensions of the printing thread; and the impact on cell survival. Currently, computational simulation can be used to implement process design strategies, as well as comparisons with other inks [113]. g g p Table 2 shows a comparison of studies on alginate and gelatin-based hydrogels in three- dimensional cell cultures. Depending on the composition of the hydrogel, the modification strategy, and the cross-linking methods used, different concentrations of alginate and gelatin were used. Based on these few examples, it can be seen that studies on three- dimensional cell cultures have been carried out on different cell lines. Table 2. Comparison of the hydrogel compositions depending on the cross-linking methods and modifications used. Cell Line Concentration (w/v%) Cross-Linking Method Modification Reference Sodium Alginate Gelatin Sodium Alginate Gelatin NB SH-SY5Y * 0.50–0.75 2.00–5.00 Ionic (CaCl2) Covalent (transglutaminase) n.a. [95] 1) Hbmsc *; 2) D1 stem cells *; 3) MC3T3-E1 * 2.00 n.a. n.a. n.a. 1) linear RGD 2) cyclic RGD [114] RSC96 * 1.00–2.50 n.a. Ionic (CaCl2) n.a. 1) added poly-L-lysine 2) added fibronectin 3) added RGD 4) covalent binding RGD [115] ACPCs * n.a. 8.00 n.a. 3.3. 3D Cell Cultures 1) Free radical polymerization 2) Free radical polymerization and covalent 1) methacrylic anhydride (GelMA) 2) methacrylic anhydride and tyramine (GelMA-Tyr) [116] 1) HepaRG * 2) A549 * 2.00 3.00 1) Ionic (CaSO4)—before printing 2) Ionic (CaCl2)—after printing 3) Ionic (CaCl2)—during incubation n.a. human extracellular matrix (hECM) [84] 1) Saos-2 * 2) hBMSCs * 4.00 5.00 Ionic (CaCl2) n.a. 1) cellulose nanofiber (CNF) 2) bioactive glass (BaG) [117] 1) HUVECs * 2) MSCs * 1.00; 2.00; 3.00 GelMA:5.00; 7.00 Ionic (CaCl2) Free-radical polymerization 1) 4-arm poly(-ethylene glycol)-tetra-acrylate (PEGTA) 2) methacrylic anhydride [118] Note: NB SH-SY5Y—human neuroblastoma cell line; hBMSC—human bone marrow stem cells; D1—mesenchymal stem cells; MC3T3-E1— osteoblastic cell line; RSC96—neuronal Schwann cell; ACPCs—articular cartilage progenitor cells. Table 2. Comparison of the hydrogel compositions depending on the cross-linking methods and modifi he hydrogel compositions depending on the cross-linking methods and modifications used. Y—human neuroblastoma cell line; hBMSC—human bone marrow stem cells; D1—mesenchymal stem cells; MC3T3-E1— ne; RSC96—neuronal Schwann cell; ACPCs—articular cartilage progenitor cells. Note: NB SH-SY5Y—human neuroblastoma cell line; hBMSC—human bone marrow stem cells; D1—mesenchymal stem cells; MC3T3-E1— osteoblastic cell line; RSC96—neuronal Schwann cell; ACPCs—articular cartilage progenitor cells. Alginate-hydrogel-based cell constructs are good candidates for tissue engineering and regenerative medicine applications (Figure 7). The available studies indicate that stem cells can be combined with biomaterial scaffolds for in vivo and in vitro research. There are various biomaterial formulations for tissue engineering, e.g., hydrogels, electrospun nanofibers, and microspheres. Alginate material is recommended for hydrogels and electrospun nanofibers [119]. This material was efficiently used with adipose-derived adult stem cells (ASCs) and bone marrow-derived MSCs (mesenchymal stem cells) to generate replacement cartilage [120] and was also used with neural tissue [121]. In another study, the authors blended calcium alginate gels for internal and external gelation. The gels were injectable and could be applied as immobilization matrices for human MSCs [122]. Materials 2021, 14, 858 14 of 24 14 of 24 Pandolfiet al. [123] verified that alginate-based microencapsulations can be efficiently applied for transplants in patients that are not eligible for immunosuppressive therapies. Consequently, alginate-based materials seem to be good candidates for tissue engineering and regeneration. Figure 7. Comparison of 2D culture and 3D model based on alginate hydrogel (own source). Figure 7. Comparison of 2D culture and 3D model based on alginate hydrogel (own source). Clinical applications for the materials will be possible in the relatively near future. 3.3. 3D Cell Cultures g y y p y There are reports of clinical trials concerning the utility of gelatin with selected renal cells in patients with type 2 diabetes [131] and chronic kidney disease, and of gelatin with renal autologous cells in patients with chronic kidney disease from congenital anomalies of the kidneys and urinary tract [132]. 3.3. 3D Cell Cultures The application of 3D hydrogels in clinical trials is still in progress, however there are some available data that show that they could be alternatives in regeneration or transplantation treatments. Forte et al. [124] developed a composite hydrogel based on gelatin to form brain tissue phantom . The authors suggested that this newly developed model will be useful in traumatic brain injury (TBI) studies. Gelatin-based hydrogels are used as extracellular matrices (ECMs) in 3D bioprinting technologies [125]. p g g Alginate has been used in wound-healing processes in the form of electrospun hy- drogels and sponges [126,127]. The authors of a previous study observed a significant improvement of the healing process and monocyte stimulation, as well as higher cytokine levels, such as tumor necrosis factor-α and interleukin-6 [128]. It was also proven that alginate might be useful in reconstructive surgery and bone regeneration, e.g., alginate scaffolds helped in bone tissue formation [129]. p [ ] Alginate was also used in cardiac muscle regeneration in the form of a hydrogel and porous 3D scaffold [130]. The most advanced clinical trials involving alginate-based systems for cardiac regeneration have included Algisyl-LVR™sponsored by LoneStar Heart, Inc (Laguna Hills, CA, USA), and PRESERVATION 1 sponsored by Bellerophon BCM LLC (Hampton, NJ, USA). It is also noted that alginate implants are under clinical investigation for intramyocardial delivery in patients with acute myocardial infarct (MI). There are reports of clinical trials concerning the utility of gelatin with selected renal cells in patients with type 2 diabetes [131] and chronic kidney disease, and of gelatin with renal autologous cells in patients with chronic kidney disease from congenital anomalies of the kidneys and urinary tract [132]. Alginate was also used in cardiac muscle regeneration in the form of a hydrogel and porous 3D scaffold [130]. The most advanced clinical trials involving alginate-based systems for cardiac regeneration have included Algisyl-LVR™sponsored by LoneStar Heart, Inc (Laguna Hills, CA, USA), and PRESERVATION 1 sponsored by Bellerophon BCM LLC (Hampton, NJ, USA). It is also noted that alginate implants are under clinical investigation for intramyocardial delivery in patients with acute myocardial infarct (MI). 4. Mechanical Properties of Alginate-Based Hydrogels The mechanical properties of sodium alginate hydrogels depend on the number and sequence of monomer units (M and G), as well as their molecular weight, chain stiffness, and cross-linking density [133,134]. Similarly, the mechanical stiffness of ion-cross-linked sodium alginate hydrogels depends on the molecular weight of the sodium alginate and the concentration of binding cations [135]. There is a relationship between the rheological properties of the pre-gel solution and the mechanical stiffness of the post-hydrogel with the particle concentration and molecular weight distribution of the polymer used to form them [133]. The mechanical properties of biomaterials are an important parameter in regulating the behavior of cell adhesion. Indeed, cells can remodel the mechanical, structural, and chemical composition of the surrounding ECM. It is also known that the mechanical properties of the extracellular microenvironment change the behavior of cells, including Materials 2021, 14, 858 15 of 24 15 of 24 their spreading, proliferation, or differentiation [136]. Most studies have focused on the influence of matrix stiffness on cells using hydrogel substrates, which show purely elastic behavior. Examples of mechanical properties for the most commonly used hydrogels are given in Table 3. Importantly, the matrix stiffness influences the ability of stem cells to differentiate towards specific lineages. Mesenchymal stem cells differentiate towards a neurogenic lineage when cultured on soft 0.1–1 kPa substrates, towards a myogenic lineage when cultured on 8–17 kPa substrates, and towards an osteogenic lineage when cultured on stiffer 25–40 kPa substrates [137]. Besides the stiffness of the matrix, the viscoelasticity and stress relaxation are also important [72]. Commercially available sodium alginate reaches molecular weights in the range of 32,000–400,000 g/mol. A decrease in the pH of the sodium alginate solution results in increased viscosity (reaching a maximum around pH 3–3.5). The shear modulus of the hydrogel depends on the sodium alginate concentration [133]. The physical properties of the obtained gel can be improved by increasing the molecular weight of the sodium alginate. Solutions made from high molecular weight polymers of sodium alginate become more viscous, which is often unsatisfactory during processing [138]. The mechanical strength also depends on the sources from which the alginates are obtained, e.g., Azotobacter alginates have comparatively high stiffness [139]. The viscosity of the pre-gel solution and its stiffness after gelation can be successfully controlled through manipulation of the molecular weight and distribution [133]. Table 3. Mechanical properties of hydrogels. 4. Mechanical Properties of Alginate-Based Hydrogels Material Example Tensile Strength Tensile Modulus Compressive Strength Compressive Modulus Reference Traditional hydrogel (PVA), PEG 1 ~ 100 kPa <100 kPa 10 ~ 100 kPa 1 ~ 100 kPa [140,141] Alginate hydrogel n.a. ~20 kPa ~78 kPa n.a. n.a. [142] Alginate-gelatin hydrogel n.a. ~0.5 MPa ~1 MPa 2 ~ 12 MPa 30 ~ 50 kPa [81,142] Alginate-gelatin- nanocellulose hydrogel n.a. n.a. ~220 kPa ~320 kPa 60 ~ 110 kPa [143,144] Nanocomposite hydrogel PEG/clay 255 kPa 16 kPa 3.7 MPa 38 kPa [145–147] Cartilage n.a. ~3 MPa ~9 MPa ~35 MPa ~15 MPa [148,149] Collagen fiber n.a. ~75 MPa ~1000 MPa n.a. n.a. [140,150] Gelatin hydrogel n.a. n.a. 3–25 kPa n.a. n.a. [151] Table 3. Mechanical properties of hydrogels. Table 3. Mechanical properties of hydrogels. 5. Alginate-Gelatin Hydrogel as an Ink in 3D Bioprinting 5. Alginate-Gelatin Hydrogel as an Ink in 3D Bioprinting 5. Alginate-Gelatin Hydrogel as an Ink in 3D Bioprinting Additive manufacturing is a technology that could be widely applied in biomedicine. Materials and technologies used in medical and biological fields have to fulfill specific requirements. Bioprinting technology is a branch of additive technology, commonly known as 3D printing, which is utilized as an innovative tool in manufacturing living functional tissues from cells and supporting biomaterials as bioinks in computed printing systems. This technology enables a wide range of applications in tissue engineering or regenerative medicine, for example replacing injured or damaged tissue manufactured in an automated way with high repeatability [152]. Moreover, 3D printing, including bioprinting technology, consists of sequential printing with precursor bioink (which consists of a polymer or composite) in a predefined trajectory and within cells. Multilayer ink deposition enables controlled spatial positioning of cells, thus facilitating the precise creation of scalable structures, which cannot be provided by two- and standard three-dimensional cell cultures [153]. Bioink is a specially prepared ink for biological and medical applications that is non-toxic, enabling cell and tissue viability. Bioink is composed mainly of hydrogel due to its biodegradability and ability to mimic the anatomy and mechanical features of the body, however additionally it contains living cells, cell aggregates, or bioactive molecules, creating entirely biologically functional products. The potential application and cell type determine the choice of bioink and bioprinter to be adopted [28,152]. The widely used 3D printing methods for plastics, metals, and ceramics are not ad- equate or appropriate for biomaterials applied in cell culture media. 4. Mechanical Properties of Alginate-Based Hydrogels Conventional 3D Materials 2021, 14, 858 16 of 24 16 of 24 printing strategies described by Bishop [154], such as inkjet printing, microextrusion, and laser printing, have also been developed to generate 3D biological structures directly surrounding cells and bioactive agents, including various biomimetic tissues, e.g., bone tissue, cartilage and osteochondral tissue, heart tissue, blood vessels, liver tissue, and tumors [155,156]. Over the last decade, there has been tremendous progress in the de- velopment of three-dimensional bioprinting. The most commonly used 3D bioprinting techniques include inkjet printing or droplet bioprinting, microextrusion, and laser-assisted bioprinting (LaBP) [152,157] (Figure 8). Figure 8. Schematic representation of the most common methods of bioprinting. (A) Thermal inkjet bioprinting. (B) Piezoelectric inkjet bioprinting. (C) Pneumatic extrusion bioprinting. (D) Mechanical extrusion bioprinting. (E) Laser-assisted bioprinting. Copyright 2017, Chongqing Medical University. Production and hosting by Elsevier B.V [154] Figure 8. Schematic representation of the most common methods of bioprinting. (A) Thermal inkjet bioprinting. (B) Piezoelectric inkjet bioprinting. (C) Pneumatic extrusion bioprinting. (D) Mechanical extrusion bioprinting. (E) Laser-assisted bioprinting. Copyright 2017, Chongqing Medical University. Production and hosting by Elsevier B.V [154] Three-dimensional bioprinting is a versatile technique for creating biomimetic tissue structures [23]. The production of alginate substrates using the additive method is an excellent choice due to the uncomplicated processing procedure and reduction of waste, which affect the material yield and programmable viscosity (ranges from 0.03 Pa/s to 6 × 104 Pa/s). However, in addition to its accuracy, the procedure is limited by its reso- lution (200 µm) [158]. The printability of alginate bioinks is dependent on their viscosity, however 3D biostructures exhibit constant rigidity in relation to certain conditions [153]. k h b bl f f k f h Three-dimensional bioprinting is a versatile technique for creating biomimetic tissue structures [23]. The production of alginate substrates using the additive method is an excellent choice due to the uncomplicated processing procedure and reduction of waste, which affect the material yield and programmable viscosity (ranges from 0.03 Pa/s to 6 × 104 Pa/s). However, in addition to its accuracy, the procedure is limited by its reso- lution (200 µm) [158]. The printability of alginate bioinks is dependent on their viscosity, however 3D biostructures exhibit constant rigidity in relation to certain conditions [153]. Bioinks have to be suitable for a range of key requirements for processing with a 3D bioprinting machine. 4. Mechanical Properties of Alginate-Based Hydrogels It is possible to increase the viscosity by applying higher pressure on the hydrogel extrusion, which eliminates the gel outflow from the nozzle and provides higher accuracy to the printed structures [161]. The efficiency of bioprinting depends on both the bioink printability and the achieved cell viability, as well as the process parameters (e.g., printing speed, resolution, pres- sure) [165,166]. The accuracy of the obtained structure is also affected by the solidification of the bioinks during the bioprinting process, which usually takes place through cross- linking. The gentle solidification of the printout is the main prerequisite for the selected bioinks, as they cannot cause damage or cell death [166]. Certain 3D printing applications (e.g., when building more complex structures for tissue regeneration) do not provide the smart response needed for such an artificially created structure to properly work in the body, creating the opportunity to utilize 4D printing [167]. Sodium alginate hydrogel is widely used in the bioprinting process due to its biocom- patibility, rapid ability for cross-linking, and simple regulation of its mechanical properties through physico-chemical modifications [156,168]. Due to its hydrophilic nature, alginate scaffolds allow the cell viability to be maintained during the culture, because media and cells are pulled into the scaffold via capillary action. One of the benefits of possessing dense cell cultures is the maximization of the contact interactions between cells [165]. Sodium alginate is not suitable for direct printing because the structure obtained is not rigid enough and takes a liquid form after printing. Recently, multi-material bioinks have been used more frequently, as these combinations can benefit from the advantages of different materials [160]. However, it is possible to strengthen the structure of sodium alginate by adding gelatin while maintaining its biocompatibility, because dissolved gelatin forms a solution at body temperature (37 ◦C) and can form a gel after cooling (<29 ◦C). These thermal properties of gelatin guarantee good flow characteristics for bioinks and allow quicker gelation during the bioprinting process compared to alginate combined with Ca2+, which improves the initial stability of the manufactured structure [75,153,169]. One well-known and accessible composite bioink is based on a combination of alginate and gelatin. Alginate allows fast cross-linking, whereas gelatin provides thermoresponsive properties [160]. Alginate-gelatin bioink combines the rapid ionic cross-linking of alginate and the temperature sensitivity of gelatin for multilayer printing and is widely used as bioink in cell bioprinting applications [159]. 4. Mechanical Properties of Alginate-Based Hydrogels Printability should be determined by rheological properties, bioink consistency measurements, and sample dimensions [159–161]. The most important feature of the rheological properties is the bioink viscosity, which can directly affect the printing pressure required for the material dosage, and thus the cell viability in bioink and shape fidelity of the printouts, as well as the elastic recovery, viscoelastic shear moduli, and shear stress. Excessive pressure in the printing nozzle can damage the cells during printing, thus it is important to control shear stress during extrusion because the cells suspended Bioinks have to be suitable for a range of key requirements for processing with a 3D bioprinting machine. Printability should be determined by rheological properties, bioink consistency measurements, and sample dimensions [159–161]. The most important feature of the rheological properties is the bioink viscosity, which can directly affect the printing pressure required for the material dosage, and thus the cell viability in bioink and shape fidelity of the printouts, as well as the elastic recovery, viscoelastic shear moduli, and shear stress. Excessive pressure in the printing nozzle can damage the cells during printing, thus it is important to control shear stress during extrusion because the cells suspended Materials 2021, 14, 858 17 of 24 17 of 24 in the ink have differing sensitivity [162,163]. Gao et al. [162] examined the rheological properties of an alginate-gelatin hydrogel and their influence on the bioink and cells placed in it during the manufacturing process. In a study carried out by Diamantides et al. [164], the effect of the density of cells contained in a hydrogel on its rheological properties was examined. Another factor that may affect the rheological properties of some hydrogels is temperature. The extrusion of bioinks during using 3D bioplotters does not require increased temperature, and therefore does not affect cell destruction either. Such bioinks, which are exist sol form or are gelated after being printed from the nozzle, are not suitable for bioprinting because the mechanical strength of the structure is not sufficient to support the cell culture. However, when bioink is too viscous, the filament is irregular and unstable during printing. The manufactured structure requires the use of a semi-viscous hydrogel to maintain the integrity of the structure consisting of regular fibers, while for the highest cell viability, the viscosity of the bioink should be as low as possible to reduce the induced shear stress [159]. 4. Mechanical Properties of Alginate-Based Hydrogels The substrate properties can also be modified by using different solvents and gelatin concentrations. Their suitability for printing can also be supported by adding an extruder heating system. The rheological properties of alginate-gelatin hydrogels are influenced by temperature, in addition to the concentrations of both substances. Temperature should not lead to cell apoptosis in the culture [161]. Other materials used to support the biological activity of alginate-based bioinks include fibrin, which improves interactions with cells, as well as polyvinyl alcohol and hydroxyapatite (HAp), which enable multicomponent bioinks to be obtained. Cellulose can also be used, which provides a bioink with appropriate shear-thinning action and can accurate reproduce the shapes of the created structures [156]. Materials 2021, 14, 858 18 of 24 18 of 24 Alginate, which has a higher molecular weight and is highly concentrated, is a shear- thinning physical gel. It is important to adjust its concentration in order to obtain the appropriate properties when printing. There are ways to process unprintable alginate compositions using different cross-linking strategies, such as printing on calcium chloride solutions, spraying calcium chloride mist onto the solution coming out of the nozzle, or pre-cross-linking with calcium chloride [160]. p g Traditional 3D structures manufactured using 3D bioprinting technology with static mechanical properties might not be suitable for biomedical applications [170]. A new solu- tion, which can more accurately mimic the dynamics of native tissues, has recently been developed using four-dimensional bioprinting (4D) technology, based on three-dimensional bioprinting with additional shape-shifting capability [171]. It is possible to design biologi- cally active structures that respond to impulses, and after appropriate stimulation, may change their shape to achieve the required functionality. Programmable bioinks capable of being used in 4D bioprinting are being produced to obtain various stimulus-responsive biomaterials, including thermal, humidity, electrical, magnetic, and photo-sensitive materi- als [172]. 6. Conclusions Sodium alginate in the form of hydrogel is a low-cost biomaterial that demonstrates good printability and excellent biocompatibility. This is why it is used in a wide range of applications, such as in wound healing, regenerative medicine, and drug delivery, as well as for cell culture. Alginate-gelatin hydrogel has very beneficial properties; the combination of these two substances provides a substrate with mechanical properties and a structure adapted to free cell growth. While sodium alginate is a less rigid structure, gelatin increases the rigidity and provides a substrate with a precise shape. Bioprinting technology offers promising opportunities for cell cultures. It allows the preparation of media with specific structures and mechanical properties using a variety of substances. The production process providing accurate structures using alginate-gelatin bioink in additive manufacturing technology enables the creation of hydrogel substrates of any shape and high porosity, imitating the ECM. This facilitates the correct ingrowth, proliferation, and differentiation of cells outside the body, which will significantly contribute to the development of tissue engineering and regenerative medicine. In vitro 3D cell cultures will enable the future preparation of tissues or parts of organs and could even be an opportunity for drug testing systems. Author Contributions: Conceptualization, M.B.Ł., K.C., A.M.J., J.K., J.D. and I.M.; writing—original draft preparation, M.B.Ł., K.C. and A.M.J.; writing—review and editing, J.K., J.D. and I.M.; visual- ization, M.B.Ł. and A.M.J.; supervision, J.K., J.D. and I.M.; project administration, M.B.Ł.; funding acquisition, J.D. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.B.Ł., K.C., A.M.J., J.K., J.D. and I.M.; writing—original draft preparation, M.B.Ł., K.C. and A.M.J.; writing—review and editing, J.K., J.D. and I.M.; visual- ization, M.B.Ł. and A.M.J.; supervision, J.K., J.D. and I.M.; project administration, M.B.Ł.; funding acquisition, J.D. All authors have read and agreed to the published version of the manuscript. Funding: This research is a part of Statutory Subsidy No. 8201003902 at the Department of Mechanics, Materials, and Biomedical Engineering (K58), Faculty of Mechanical Engineering, Wrocław University of Science and Technology, Smoluchowskiego 25, 50-372 Wrocław, Poland. Data Availability Statement: No new data were created or analyzed in this study. Conflicts of Interest: Authors declare no conflict of interest. 1. Lapidot, S.A.; Kost, J. Hydrogels. In Encyclopedia of Materials: Science and Technology; Elsevier: Amsterdam, Netherlands, 2001; pp. 3878–3882. 5. Gun’ko, V.; Savina, I.; Mikhalovsky, S. Properties of water bound in hydrogels. Gels 2017, 3, 37. [CrossR Analysis and Applications of Hydrogels; Majee, S.B., Ed.; IntechOpen: London, UK, 2016; pp. 9 38. 4. Aljohani, W.J.; Wenchao, L.; Ullah, M.W.; Zhang, X.; Yang, G. Application of sodium alginate hydrogel. IOSR J. Biotechnol. Biochem. 2017, 3, 19–31. [CrossRef] 2. Ahmed, E.M. 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Evaluation of the Agronomic Performance of Atrazine-Tolerant Transgenic japonica Rice Parental Lines for Utilization in Hybrid Seed Production
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Luhua Zhang1., Haiwei Chen1.¤, Yanlan Li1, Yanan Li1, Shengjun Wang2, Jinping Su2, Xuejun Liu2, Defu Chen1*, Xiwen Chen1* ory of Molecular Genetics, College of Life Sciences, Nankai University, Tianjin, China, 2 Tianjin Crop Research Institute, Tianjin, China Abstract 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 the Key Project of Tianjin Science and Technology Support Program (11ZCGYNC01000), the Key Program of the Natural Science Foundation of Tianjin (12YFJZJC01700, 14JCZDJC34100), the grant of Natural Science Foundation of China (No. 31070273, 31070717) and the 111 Project (No. B08011). 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. * Email: chendefu@nankai.edu.cn (DC); xiwenchen@nankai.edu.cn (XC) * Email: chendefu@nankai.edu.cn (DC); xiwenchen@nankai.edu.cn (XC) * Email: chendefu@nankai.edu.cn (DC); xiwenchen@nankai.edu.cn (XC) . These authors contributed equally to this work. . These authors contributed equally to this work. ¤ Current address: College of Life Sciences, Chifeng College, Chifeng, China ¤ Current address: College of Life Sciences, Chifeng College, Chifeng, China Evaluation of the Agronomic Performance of Atrazine-Tolerant Transgenic japonica Rice Parental Lines for Utilization in Hybrid Seed Production Luhua Zhang1., Haiwei Chen1.¤, Yanlan Li1, Yanan Li1, Shengjun Wang2, Jinping Su2, Xuejun Liu2, Defu Chen1*, Xiwen Chen1* October 2014 | Volume 9 | Issue 10 | e108569 Abstract Currently, the purity of hybrid seed is a crucial limiting factor when developing hybrid japonica rice (Oryza sativa L.). To chemically control hybrid seed purity, we transferred an improved atrazine chlorohydrolase gene (atzA) from Pseudomonas ADP into hybrid japonica parental lines (two maintainers, one restorer), and Nipponbare, by using Agrobacterium-mediated transformation. We subsequently selected several transgenic lines from each genotype by using PCR, RT-PCR, and germination analysis. In the presence of the investigated atrazine concentrations, particularly 150 mM atrazine, almost all of the transgenic lines produced significantly larger seedlings, with similar or higher germination percentages, than did the respective controls. Although the seedlings of transgenic lines were taller and gained more root biomass compared to the respective control plants, their growth was nevertheless inhibited by atrazine treatment compared to that without treatment. When grown in soil containing 2 mg/kg or 5 mg/kg atrazine, the transgenic lines were taller, and had higher total chlorophyll contents than did the respective controls; moreover, three of the strongest transgenic lines completely recovered after 45 days of growth. After treatment with 2 mg/kg or 5 mg/kg of atrazine, the atrazine residue remaining in the soil was 2.9–7.0% or 0.8–8.7% respectively, for transgenic lines, and 44.0–59.2% or 28.1–30.8%, respectively, for control plants. Spraying plants at the vegetative growth stage with 0.15% atrazine effectively killed control plants, but not transgenic lines. Our results indicate that transgenic atzA rice plants show tolerance to atrazine, and may be used as parental lines in future hybrid seed production. Citation: Zhang L, Chen H, Li Y, Li Y, Wang S, et al. (2014) Evaluation of the Agronomic Performance of Atrazine-Tolerant Transgenic japonica Rice Parental Lines for Utilization in Hybrid Seed Production. PLoS ONE 9(10): e108569. doi:10.1371/journal.pone.0108569 Editor: Jin-Song Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China Editor: Jin-Song Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China Received January 27, 2014; Accepted September 1, 2014; Published October 2, 2014 Received January 27, 2014; Accepted September 1, 2014; Published October 2, 2014 pyright:  2014 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ng et al. Atrazine Tolerant Transgenic japonica Rice Parental Lines supplemented with 2 g/L proline, 3 mg/L 2, 4-D and 300 mg/L casein hydrolysate in dark at 28uC. After 2–3 weeks, the scutellum- derived calli were excised and subcultured every four weeks on the same medium but with 0.5 g/L proline, 2 mg/L 2, 4-D in dark at 28uC. The highly embryogenic compact calli (3–5 mm in diameter) that subcultured for less than five generations, were selected and co-cultivated with A. tumefaciens EHA105 harboring p1301C-ubi-22-14 on the co-cultivation medium (subculture medium but with 100 mM acetosyringone) for 3 days in dark at 28uC. Following that, the explants were then transferred into selection medium (subculture medium but with 50 mg/L hygro- mycin and 500 mg/L cefotaxime) in dark at 28uC for selection. After two cycles of selection, hygromycin-resistant calli were transferred onto pre-regeneration medium (NB medium with 0.5 g/L proline, 2 mg/L 6-BA, 1 mg/L NAA, 5 mg/L ABA, 300 mg/L casein hydrolysate and 50 mg/L hygromycin) for 14 to 21 days in dark at 28uC, then to regeneration medium (pre- regeneration medium but without 5 mg/L ABA) for 30 days under 54 mmol/m2/s light at 28uC and finally to the rooting medium (MS medium with 1 mg/L IBA) for 15 days under light at 28uC. production of false hybrids. Furthermore, as the Chinese economy develops, increasing numbers of young men are leaving their home towns to seek work in the cities, leaving the elderly and women to work on the farms. The transformation of heavy and complex farming to light and simple farming is therefore becoming increasingly important. Thus, ensuring hybrid seed purity and reducing labor costs are two key issues in hybrid japonica rice seed production. Genetic engineering, especially herbicide resistance engineering, provides an efficient means of controlling purity in hybrid seed production. Yan first proposed a strategy of utilizing herbicide resistance genes to chemically control purity in hybrid seed production [5]. Since then, two-line hybrid rice production has been extensively investigated [6–12] and progress has recently been reviewed [13]. Additionally, some transgenic hybrid rice combinations have been used in field trials [7,8,10,12]. However, the research has mainly focused on the bar gene isolated from Streptomyces hygroscopicus [6,8,10,12]; other genes, such as the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene from Agrobacterium strain CP4, and the protoporphyrinogen oxidase gene from Bacillus subtilis, have rarely been investigated [11]. Molecular analysis of transgenic lines Genomic DNA was isolated from young leaves using a modified CTAB method [19]. PCR was performed to preliminarily select the transformed plants using primers atzA7/atzA8 [20] and SL- Ubi-C-F/SL-Ubi-C-R (Table 1). RT-PCR was performed to further confirm the expression of atzA in the PCR-positive transformed plants. Total RNA was extracted from young leaves using an RNAultra Extraction Kit (Qiagen). cDNA was synthe- sized using atzA8 and oligo(dT) as primers and SuperScriptTM II RNase H2 (Invitrogen) as reverse transcriptase. RT-PCR was amplified using the cDNA as template and atzA7/atzA2 [20] as primers. b-actin (AB047313) was also amplified as the internal control using actin-R/actin-F as primers [21]. Atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-di- amine) is a triazine herbicide, and is commonly used in maize, sorghum, and sugarcane fields [14]. By inhibiting electron transport to plastoquinone in the photosystem PSII, atrazine terminates photosynthesis and kills weeds [15]. Atrazine was once the most widely used herbicide worldwide, because of its low cost and high effectiveness [14]. We previously isolated an atrazine chlorohydrolase gene (atzA) from a soil bacterium Pseudomonas ADP [16], and modified this gene by using directed evolution, to improve the enzymatic activity [17]. In the present study, we transferred the improved atzA gene into breeding hybrid japonica parental lines. Our results indicate that the transgenic atzA rice lines show tolerance to atrazine, and may be used as parental lines to chemically improve seed purity in hybrid seed production. Germination and seedling growth in the presence of atrazine Fifty seeds from each transgenic line and the respective control plant were directly sown on the surface of filter paper in plates containing 0, 75 or 150 mM atrazine. Seeds were placed in a growth chamber at 28uC with 16 h of 54 mmol/m2/s light per day. Germination (based on radicles .2 mm) was recorded daily and the cumulative values at day 3 and day 7 were calculated to represent as germination potential and germination percentage. The shoot length, root length and their biomass were measured after 7 days. For seedling growth test, seven-day old seedlings germinated in absence of atrazine were placed on filter paper in pots, and incubated in Kimura B nutrition solution [22] containing 0, 75 or 150 mM atrazine in growth chamber described above. During the period, the nutrition solution containing the respective concentration of atrazine was added to keep the filter paper wet. The growth parameters as described above were measured after 10 days. Construction of plant expression vector Construction of plant expression vector Ubiquitin promoter and an improved atrazine chlorohydrolase gene atzA-22-4 [17] were respectively amplified from pSTAR- LING, an RNAi intermediate vector for monocots (a kind gift from the Commonwealth Scientific and Industrial Research Organization, Australia), and AtzA-22-4 using primers SL-Ubi- F/SL-Ubi-R and atzA-TJ-F/atzA-TJ-R (Table 1). After respec- tively inserted into TaKaRa pMD19T-simple vector and con- firmed by sequencing, the pAtzA-22-4-19T-simple was restricted with EcoRV to collect the 1.4 kb fragment, and then lignated with pUbi-19T-simple. The resulting plasmid was restricted with SacI/ SpeI to collect the 3.4 kb fragment, and then ligated with pCAMBIA1301. The recombinant plasmid p1301-ubi-22-14 was then introduced into Agrobacterium tumefaciens EHA105 by the freeze-thaw method [18]. Soil-grown transgenic T2 lines in the presence of atrazine Two-week old seedlings of similar size that germinated in absence of atrazine were transplanted into pots containing 1.12 kg of soil with 0, 2, or 5 mg/kg of atrazine. Nine plants were planted in each pot, and irrigated with the same amount of water every day and with Kimura B nutrition solution [22] twice a week. Plants were incubated in the greenhouse in a 14-h light/10-h dark cycle (28/25uC) at 300 mmol m22s21 light and 75% relative humidity. Plant growth and chlorophyll content were measured at Atrazine Tolerant Transgenic japonica Rice Parental Lines If the strategy is proven to be effective, chemical control of hybrid seed purity will be mainly dependent on an herbicide with a single mode of action, and this will hinder sustainable weed manage- ment. Introduction erably to meet consumer’s demand for high-quality food both in China and globally. Rice (Oryza sativa) is one of the most important staple food crops globally. According to the National Grain and Oil Information Center, the area of China planted with rice in 2012 was 3.06107 hm2, including 9.06106 hm2 of japonica rice [1]. Japonica rice is mainly planted in the northern region of the Qinling Mountains–Huai River, and its planted area has increased in recent years because of its high quality and good taste. Japonica rice production is currently dominated by conventional varieties, with hybrid rice accounting for only 3% of the cultivated area. On the other hand, indica hybrid rice represents 70–80% of the total planted area of indica rice [2]. Therefore, there is considerable potential for the development of japonica hybrid rice. An increase in the annual planted area of japonica hybrid rice from 3% to 50%, i.e., to reach 4.06106 hm2, is estimated to lead to the production of 3.56109 kg of high-quality grain (www.cngrain. com/Publish/qita/200503/207290), thereby contributing consid- The three-line system is a traditional and effective production method for hybrid japonica rice seed [3]. The most widely used male sterile line in the system is BT-type cytoplasmic male sterile (CMS). However, the panicle of this line is loosely enclosed when heading, and this appearance closely resembles that of the maintainer. This makes it difficult for farmers to distinguish the BT-CMS line when eliminating off-type plants [2]. Furthermore, the BT CMS line has good restorability, and may therefore be easily pollinated with exotic pollens that contaminated during mechanical harvesting and storage of seeds, and also with exotic pollens from other plants [4]. The use of contaminated CMS lines in seed production results in decreased hybrid seed purity. Therefore, off-type contamination must be eliminated as early as possible. This is largely a manual process and requires consider- able labor input, particularly in Asia. On the one hand, the need for increased labor will increase the price of hybrid seeds, while on the other hand, the increase in manual procedures may lead to the October 2014 | Volume 9 | Issue 10 | e108569 October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 1 Atrazine Tolerant Transgenic japonica Rice Parental Lines Germination of transgenic atzA lines in the presence of atrazine Atrazine and hydroxyatrazine in the rice leaves and soil were also determined in the soil-grown experiment. One gram of leaves, or five grams of soil samples that collected from the soil layer mixture as thick as possible, were extracted with 5 mL dichloro- methane. After 30 min of incubation at room temperature, the mixture was centrifuged at 15,294 g for 10 min. The supernatant was transferred to a new tube and filtered through a 0.2-mm filter and then subjected to CoM 6000 HPLC system analysis on an analytical C18 column (5 mm, 250 mm64.6 mm) at 30uC with linear gradients phase as follows: 0 to 6 min, 10% to 25% acetonitrile; 6 min to 21 min, 25% to 65% acetonitrile; 21 min to 23 min, 65% to 100% acetonitrile; and 23 min to 25 min, 100% acetonitrile [24] at the wavelength of 228 nm. To investigate the atrazine tolerance of transgenic lines during germination, we germinated seeds of three lines of each genotype and the respective controls in the presence of atrazine (Fig. 1A, Table 2). The germination potential of the wild types decreased as the atrazine concentration increased (58.3–98%, 22.4–64.2%, and 0%, respectively, in 0 mM, 75 mM, and 150 mM atrazine); on the other hand, the germination percentage significantly decreased only in presence of 150 mM atrazine (95.2–100%, 72.1–96.7%, and 0–42.2%, respectively, in 0 mM, 75 mM, and 150 mM atrazine). In the presence of the investigated atrazine concentra- tions, the highest germination potential was determined for Nipponbare, followed by Jinhui1, Jindao8, and Jindao7. In the presence of 0 mM or 75 mM atrazine, the germination potentials and percentages of almost all of the transgenic lines did not differ significantly from those of the respective controls. The exceptions were the germination potential for Jindao8 in the presence of 75 mM atrazine and the germination percentage for Jinhui1 in the presence of 75 mM atrazine. On the other hand, in the presence of 150 mM atrazine, all of the transgenic lines showed significantly higher germination potentials and germination percentages than did the respective controls. Moreover, two of the wild types (Nipponbare and Jinhui1) failed to germinate or only rarely germinated at day 3 or day 7. Interestingly, in the presence of 150 mM atrazine, the highest germination percentage was determined for Jindao7, followed by Jindao8, Jinhui1, and Nipponbare; on the other hand, Jindao7 showed the lowest germination potential. Genetic transformation and plant regeneration Oryza sativa L. Nipponbare and japonica hybrid rice parental lines in the three-line system, Jindao7 (maintainer), Jindao8 (maintainer) and Jinhui3 (restorer) were used for transformation. Mature seeds were dehulled, surface-sterilized and placed on NB medium (N6 macro elements, B5 micro elements and vitamins) October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org 2 Atrazine Tolerant Transgenic japonica Rice Parental Lines Atrazine Tolerant Transgenic japonica Rice Parental Lines Table 1. Primers used in this study. Table 1. Primers used in this study. Primer Sequence (59R39) SL-Ubi-F tgagctcctgcagtgcagcgtgacccggtcgt, with SacI site SL-Ubi-R tgatatcctgcagaagtaacaccaaacaacag, with EcoRV site atzA-TJ-F tgatatcatgcaaacgctcagcatccag, with EcoRV site atzA-TJ-R tgatatcactagtctagaggctgcgccaagctg, with EcoRV and SpeI site SL-Ubi-C-F cccgccgtaataaatagacac SL-Ubi-C-R accacaccacatcatcacaac doi:10.1371/journal.pone.0108569.t001 Table 1. Primers used in this study. Primer Sequence (59R39) SL-Ubi-F tgagctcctgcagtgcagcgtgacccggtcgt, with SacI site SL-Ubi-R tgatatcctgcagaagtaacaccaaacaacag, with EcoRV site atzA-TJ-F tgatatcatgcaaacgctcagcatccag, with EcoRV site atzA-TJ-R tgatatcactagtctagaggctgcgccaagctg, with EcoRV and SpeI site SL-Ubi-C-F cccgccgtaataaatagacac SL-Ubi-C-R accacaccacatcatcacaac doi:10.1371/journal.pone.0108569.t001 doi:10.1371/journal.pone.0108569.t001 15 -day intervals. For chlorophyll content analysis, approximately 10 mg of leaves was extracted with 5 ml acetone and then quantified the absorbance at 663.6 nm and 646.6 nm, as described by Porra et al. [23]. Chlorophyll a = 12.256A663.6– 2.556A646.6, Chlorophyll b = 20.316A646.6–4.916A663.6. lines that showed a segregation pattern of 3:1 resistant/sensitive in the germination test were selected to produce the T2 generations. Individual plants, whose herbicide tolerance did not segregate in the germination test, were considered as homogenous lines and selected. For simplicity, in further research, we used only two or three independent transgenic T2 lines for each genotype. Germination of transgenic atzA lines in the presence of atrazine When germinated in the presence of atrazine, all of the transgenic lines produced larger seedlings (with taller shoots and longer roots) than did the respective control plants (Fig. 1A). However, the presence of atrazine significantly inhibited the growth of transgenic lines and wild types (Fig. 1A). Spraying atrazine to transgenic T2 lines Two-week old seedlings from the germination experiment were also transplanted into pots with soil. Each pot contained 16 plants. After 30 days’ growth, the last second leaves were taken and cut into 2–3 cm section, soaked in 0, 75 and 150 mM atrazine, and incubated at 25uC under light for 2 days. The color of the leaves was observed every day. After 40 days’ growth, each pot of plants was sprayed with 20 ml of 0.15% atrazine solution. Statistical analysis All the experiments were performed for three times. Significant difference between the specific transgenic line and the respective control was performed using independent-samples t-test at 95% or 99% confidence with IBM SPSS Statistics 11.0. Values indicated by * or ** represented significantly difference at P,0.05 or P, 0.01. Tolerance of soil-grown transgenic atzA lines to atrazine To further investigate the tolerance of transgenic lines to atrazine, we transplanted 15-day-old seedlings germinated in the absence of atrazine, to pots of soil containing 0 mg/kg, 2 mg/kg, or 5 mg/kg of atrazine. We measured the plant height (as a non- destructive assay of plant growth) and chlorophyll content at 15- day intervals. In the absence of atrazine, we determined no significant differences in plant height between the transgenic lines and the respective controls (Fig. 2A). In soil containing 2 mg/kg or 5 mg/ kg of atrazine, all the transgenic lines were significantly taller than were the respective control plants. However, the presence of atrazine significantly inhibited the growth of transgenic lines and control plants (by 68.1–111.9% and 46.4–68.2%, respectively). In contrast to the control plants, all of the transgenic lines (except for the Jindao8) were slightly taller in soil containing 2 mg/kg of atrazine than in soil containing 5 mg/kg of atrazine. The growth of transgenic lines gradually recovered with an increase in the growth time. Further, three of the strongest transgenic lines (Jindao7–4, Jinhui1–5, and Jindao8–4) completely recovered after 45 days of growth (data not shown). Figure 1. Germination and seedling growth of transgenic atzA rice lines in the presence of atrazine. (A) Representative images of seeds from the transgenic lines and the respective controls sown on plates containing 0, 75 or 150 mM atrazine for 7 days. (B) Representative images from seven-day old seedlings germinated in absence of atrazine were transplanted into pots containing 0, 75 or 150 mM atrazine for 10 days. For simplicity, only images of transgenic Jindao8 (JD8) and/or Nipponbare (N) were shown here. doi:10.1371/journal.pone.0108569.g001 Atrazine may disrupt the electron flow of photosystem II and destroy photosynthetic pigments in plant leaves [15]. In the absence of atrazine, we observed no difference in chlorophyll content between transgenic lines and control plants (Fig. 2B). However, in soil containing 2 mg/kg or 5 mg/kg of atrazine, the transgenic lines retained more chlorophyll than did the respective control plants. With a few exceptions, the chlorophyll content of the transgenic lines decreased significantly in soil containing 5 mg/kg of atrazine, but not in soil containing 2 mg/kg of atrazine; on the other hand, the chlorophyll content of the control plants decreased significantly under both conditions. For each genotype grown in soil containing the same atrazine concentra- tion, we observed no difference in chlorophyll content at different growth stages. Selection of transgenic atzA plants PCR analysis revealed that 18 (100%) Nipponbare transfor- mants, 15 out of 18 (83%) Jindao7 transformants, 14 out of 16 (88%) Jinhui1 transformants, and 17 (100%) Jindao8 transfor- mants were positive. RT-PCR analysis further confirmed the expression of atzA in the PCR-positive lines (data not shown). Subsequently, the self-pollinated seeds (T1 progenies) from each plant were germinated in the presence of hygromycin. Transgenic To investigate the tolerance of transgenic lines to atrazine, we transplanted 7-day old seedlings germinated in the absence of atrazine, to pots of soil containing different concentration of atrazine (Fig. 1B and Table 3). In the absence of atrazine, we determined no differences in shoot or root growth between any of the transgenic lines and the respective controls. However, in the October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org 3 Atrazine Tolerant Transgenic japonica Rice Parental Lines Atrazine Tolerant Transgenic japonica Rice Parental Lines Figure 1. Germination and seedling growth of transgenic atzA rice lines in the presence of atrazine. (A) Representative images of seeds from the transgenic lines and the respective controls sown on plates containing 0, 75 or 150 mM atrazine for 7 days. (B) Representative images from seven-day old seedlings germinated in absence of atrazine were transplanted into pots containing 0, 75 or 150 mM atrazine for 10 days. For simplicity, only images of transgenic Jindao8 (JD8) and/or Nipponbare (N) were shown here. doi:10.1371/journal.pone.0108569.g001 Plants became yellowish, and subsequently rotted and died after 10 days (Fig. 1B). Note: In each independent treatment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates significant difference (P,0.05) or highly significant difference (P,0.01) between the treatment and the control. aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. For simplicity, only two transgenic lines for each genotype were shown here. doi:10.1371/journal.pone.0108569.t002 Note: In each independent treatment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates highly significant difference (P,0.01) between the treatment and the control. g y g ( ) aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. For simplicity, only two transge shown here. tment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates significant difference (P,0.05) o ,0.01) between the treatment and the control. Tolerance of soil-grown transgenic atzA lines to atrazine After 45 days, the chlorophyll contents of the three presence of 75 mM atrazine, all of the transgenic lines (except for Jindao8) were significantly taller than were the respective controls; further, all of the transgenic lines (except for Jinhui1) produced significantly more root biomass than did the respective controls. On the other hand, in the presence of 150 mM atrazine, shoot and root growth of almost all of the transgenic lines and the respective controls were significantly inhibited. The exceptions were the shoot growth of Nipponbare and the root growth of Jinhui1–5. Table 2. Germination of transgenic atzA rice lines in the presence of atrazine. Line 0 mM 75 mM 150 mM 3 d 7 d 3 d 7 d 3 d 7 d Na-WT 98.062.0 10060.0 54.061.6 94.561.4 0.060.0 0.060.0 N-3 98.061.6 94.260.0 60.062.2 98.261.1 64.361.6** 96.261.8** N-5 96.062.0 10060.0 64.061.3 92.161.5 62.362.6** 92.661.5** JD7-WT 58.361.6 10060.0 24.362.1 90.262.3 0.060.0 42.261.1 JD7-2 56.061.1 98.161.4 22.361.7 88.262.1 2.360.1** 88.561.6** JD7-4 62.362.2 98.661.6 26.361.3 90.260.8 2.460.2** 86.561.3** JH3-WT 86.362.0 95.262.8 64.262.2 72.161.1 0.060.0 4.560.0 JH-3 82.361.2 94.261.8 58.761.6 90.261.2* 32.360.9** 78.661.2** JH3-5 88.363.2 92.261.3 66.362.7 94.161.0* 38.761.1** 80.561.2** JD8-WT 82.064.2 98.760.8 22.461.6 96.761.0 0.060.0 26.561.3 JD8-1 78.362.5 99.260.8 48.361.1** 97.260.2 34.360.7** 94.562.8** JD8-4 78.062.6 10060.0 54.062.6** 98.361.0 35.261.6** 96.861.3** Note: In each independent treatment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates significant difference (P,0.05) or highly significant difference (P,0.01) between the treatment and the control. aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. For simplicity, only two transgenic lines for each genotype were shown here. doi:10.1371/journal.pone.0108569.t002 Table 2. Germination of transgenic atzA rice lines in the presence of atrazine. October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 4 Atrazine Tolerant Transgenic japonica Rice Parental Lines Table 3. Seedling growth performance of transgenic atzA rice lines in the presence of atrazine. Table 3. Seedling growth performance of transgenic atzA rice lines in the presence of atrazine. showed tolerance to the atrazine concentration used in weed control. showed tolerance to the atrazine concentration used in weed control. strongest lines (Jindao7–4, Jinhui1–5, and Jindao8–4) were almost identical when grown in soil containing 5 mg/kg of atrazine and when grown in the absence of atrazine (data not shown). Ability of soil-grown T2 transgenic lines to degrade atrazine Current research on the utilization of herbicide resistance genes in hybrid rice is focused on the two-line hybrid production system. Sterile lines are not stable in the two-line system, and therefore most japonica hybrid combinations are still produced by using three-line system. In the present study, we have developed atrazine-tolerant transgenic japonica rice parental lines with the potential to be used in future hybrid seed production. To investigate the ability of the soil-grown transgenic plants to degrade atrazine, we determined the atrazine residue in the soil at 15-day intervals, after growth of seedlings in pots of soil containing 0 mg/kg, 2 mg/kg, or 5 mg/kg of atrazine. We observed that the atrazine residue in the soil decreased with an increase in growth time, for transgenic and control plants (Fig. 2C). However, the decrease was greater for transgenic lines than for control plants. After treatment with 2 mg/kg of atrazine, the atrazine residue remaining in the soil was 2.9–7.0% for transgenic lines, and 44.0– 59.2% for control plants; after treatment with 5 mg/kg of atrazine, the atrazine residue remaining in the soil was 0.8–8.7% for transgenic lines and 28.1–30.8% for control plant. For each genotype at the same growth stage, the atrazine residue in the soil was higher after growth in 2 mg/kg atrazine than after growth in 5 mg/kg atrazine. We also observed that hydroxyatrazine, not atrazine (only trace) appeared in the leaves of the transgenic plants (Fig. S1), indicating that atrazine was metabolized rather than accumulated in the transgenic plants. Herbicide tolerance during germination is very important when utilizing transgenic atzA lines in hybrid seed production, because off-type plants should be eliminated as early as possible. Atrazine is a slightly water-soluble herbicide, with a saturated concentration of 153 mM. Kawahigashi [25] previously showed that an atrazine concentration of 100 mM did not affect the germination of rice plants. Therefore, we used two atrazine concentrations (75 mM and 150 mM) in the germination test, to evaluate the tolerance of transgenic rice lines. In contrast to Kawahigashi [25], we observed that the transgenic lines germinated well in the presence of atrazine whereas the respective controls did not. However, as the seedling growth of transgenic lines were also inhibited by atrazine, chemical control of contamination during germination may not be appropriate. Tolerance of soil-grown transgenic atzA lines to atrazine Line 0 mM 75 mM 150 mM Shoot length (cm) Root biomass (mg) Shoot length (cm) Root biomass (mg) Shoot length (cm) Root biomass (mg) N a-WT 9.9061.37 32.0063.16 6.5460.86 (66.0)a 21.0062.55 (65.6) 5.0761.17 (51.2) 21.7563.77 (68.0) N-3 10.5561.49 30.2563.50 9.5662.08* (90.7) 29.5063.14** (97.5) 7.5561.20* (71.6) 21.5066.40 (71.1) N-5 8.7862.42 32.7564.50 8.9461.33* (101.8) 29.8561.83** (91.2) 9.3960.51** (106.9) 21.0062.71 (64.1) JD7-WT 8.4760.37 28.5062.56 5.2860.57 (62.4) 20.0063.10 (70.2) 6.5460.61 (77.2) 17.7565.19 (62.3) JD7-2 9.4062.61 31.5063.00 7.0860.78* (75.3) 32.5062.65** (103.2) 5.8160.56 (61.8) 17.0063.46 (54.0) JD7-4 7.9961.65 27.5063.35 8.1661.27* (102.2) 27.5062.65** (100.0) 6.7860.53 (84.8) 17.7564.19 (64.5) JH3-WT 9.8361.25 24.0063.07 5.8060.43 (59.0) 21.7563.36 (90.6) 4.6260.42 (47.0) 13.2566.24 (55.2) JH-3 10.4461.47 26.7562.75 8.2760.59* (79.2) 24.2562.22* (90.7) 5.1260.61 (49.0) 15.2562.63 (57.0) JH3-5 8.8961.58 25.5063.57 8.2560.78* (92.8) 23.2562.87* (83.8) 6.0460.19* (67.9) 22.0063.37** (86.3) JD8-WT 7.8860.93 26.7563.55 7.2061.85 (91.4) 21.7562.87 (81.3) 6.4360.35 (81.5) 12.2563.46 (45.8) JD8-1 8.1161.51 27.5062.15 7.1860.40 (88.6) 27.5061.52** (100.0) 5.6561.12 (69.6) 11.5061.91 (41.8) JD8-4 7.9060.72 27.7562.53 7.6460.69 (96.7) 27.7561.79** (100.0) 5.6460.78 (71.3) 11.7562.99 (42.3) Note: The parameters were recorded after 10 days. Values are shown as mean 6 SEM (n = 3). * or ** indicates significant difference (P,0.05) or highly significant difference (P,0.01) between the treatment and the control. aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. bData in the bracket are the percentage of the treated values to the mock values. doi:10.1371/journal.pone.0108569.t003 Note: The parameters were recorded after 10 days. Values are shown as mean 6 SEM (n = 3). * or ** indicates significant difference (P,0.05) or highly significant difference (P,0.01) between the treatment and the control. aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. bData in the bracket are the percentage of the treated values to the mock values. doi:10.1371/journal.pone.0108569.t003 October 2014 | Volume 9 | Issue 10 | e108569 Utilization of transgenic plants in hybrid seed production Utilization of transgenic plants in hybrid seed production To investigate the tolerance of transgenic plants to the atrazine concentration used in weed control, we first examined the tolerance of leaf sections to atrazine solution. In the presence of 75 mM atrazine, the leaf sections of transgenic lines remained green, whereas those of control plants became bleached (Fig. 3A). In the presence of 150 mM atrazine, the leaf sections of transgenic lines became slightly bleached, but remained greener than those of control plants. We subsequently sprayed plants with 0.15% atrazine, and observed that wild-type plants became curled and withered after 6 days, whereas transgenic lines continued to grow well (Fig. 3B). Our results indicate that the transgenic rice plants To evaluate the tolerance of seedlings to atrazine, we used 7- day-old seedlings germinated in the absence of atrazine as a starting material, to avoid cumulative effects derived from germination. We observed that almost all of the transgenic lines grew well in the presence of 75 mM atrazine with no difference from that in absence of atrazine. Therefore, 75 mM atrazine may be used for effective control of off-type plants during the early seedling growth stage. Our data also suggested spraying plants with 0.15% atrazine (equivalent to the standard dosage used in the field for weed control, i.e., 200–250 g 40% suspension concentrate in 30,50 kg of water) at the subsequently vegetative growth stage could serve as an alternative means of chemically controlling October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org October 2014 | Volume 9 | Issue 10 | e108569 5 Atrazine Tolerant Transgenic japonica Rice Parental Lines h and physiology of transgenic atzA rice lines in the presence of atrazine. Fifteen-day old seedlings germinated in absence transplanted into pots with 1.12 kg soil containing 0, 2 or 5 mg/kg of atrazine. (A) plant height; (B) chlorophyll content; and (C) were measured at 15-day intervals. Values are shown as mean 6SEM (n = 3). * or ** over the bar indicates significant difference (P, gnificant difference (P,0.01) between the treatment and the control. For simplicity, only data of transgenic Nipponbare (N) and re shown here. al.pone.0108569.g002 Figure 2. Growth and physiology of transgenic atzA rice lines in the presence of atrazine. Fifteen-day old seedlings germinated in absence of atrazine were transplanted into pots with 1.12 kg soil containing 0, 2 or 5 mg/kg of atrazine. October 2014 | Volume 9 | Issue 10 | e108569 Utilization of transgenic plants in hybrid seed production (A) plant height; (B) chlorophyll content; and (C) atrazine residue were measured at 15-day intervals. Values are shown as mean 6SEM (n = 3). * or ** over the bar indicates significant difference (P, 0.05) or highly significant difference (P,0.01) between the treatment and the control. For simplicity, only data of transgenic Nipponbare (N) and Jindao8 (JD8) were shown here. doi:10.1371/journal.pone.0108569.g002 off-type plants. However, we did not test the tolerance of transgenic atzA lines at the reproductive growth stage, which is important for the utilization in mechanical harvesting of hybrid seed production. Further studies are required to investigate whether the lethal dosage for untransformed parental lines is tolerated at the reproductive stage. growth of transgenic lines. Our data also suggested that almost all of the atrazine was degraded after growth of the transgenic rice plants for 45 days. This was also verified by the appearance of hydroxyatrazine in the leaves of transgenic plants. Besides, atrazine metabolite hydroxyatrazine could be directly determined in transgenic plants. However, as the leave, stem and root of the plants all possibly metabolize atrazine [28], it would be more complicated if we want to quantitatively evaluate the atrazine metabolized by plants. That is why we chose to determine the atrazine residue in the soil after growth of the transgenic plants in this study. Atrazine degrades relatively slowly, with an average half-life of 4–57 weeks [26]. Therefore, application of this herbicide will inevitably lead to accumulation in the field, which may affect current crop growth or damage sensitive succession crops [27]. The transgenic rice lines were taller, and had higher chlorophyll contents than did the respective control plants when growing in the soil of 2 mg/kg or 5 mg/kg atrazine. Three of the transgenic lines (Jindao7–4, Jinhui1–5, and Jindao8–4) completely recovered after 45 days of growth in the presence of atrazine, suggesting that an atrazine concentration up to 5 mg/kg does not affect sustained Utilization of herbicide resistance genes in hybrid seed production may be achieved by introducing the genes into a maintainer or restorer line in the three-line system. When an herbicide-resistant maintainer is obtained, its corresponding CMS line, which possesses herbicide resistance, may be created by using October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org 6 Atrazine Tolerant Transgenic japonica Rice Parental Lines Figure 3. Tolerance of mature transgenic atzA plants to atrazine. Supporting Information Figure S1 HPLC analysis for determination of atrazine and its metabolite hydroxyatrazine in leaves of trans- genic and WT rice plants after grown in 5 mg/kg atrazine soil for 45 days. (A) Standard of hydroxyatrazine (I) and atrazine (II). (B) Wild type (WT). (C–E) Transgenic Nipponbare (N-6), Jindao7 (JD7-4) or Jindao8 (JD8-4), respective- ly. (TIF) Figure S1 HPLC analysis for determination of atrazine and its metabolite hydroxyatrazine in leaves of trans- genic and WT rice plants after grown in 5 mg/kg atrazine soil for 45 days. (A) Standard of hydroxyatrazine (I) and atrazine (II). (B) Wild type (WT). (C–E) Transgenic Nipponbare (N-6), Jindao7 (JD7-4) or Jindao8 (JD8-4), respective- ly. (TIF) Author Contributions Field trials with genetically modified rice are strictly controlled in China, and therefore we were unable to evaluate our herbicide- resistant hybrid rice combinations on a field scale. Further studies are required to verify the validity of utilizing atrazine-tolerant transgenic lines in the field, and to determine whether herbicide- Conceived and designed the experiments: DFC XWC. Performed the experiments: LHZ HWC YLL YNL SJW JPS. Analyzed the data: LHZ DFC XWC. Contributed reagents/materials/analysis tools: SJW XJL DFC XWC. Wrote the paper: DFC XWC. Utilization of transgenic plants in hybrid seed production (A) Representative images of leave sections in the presence of atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 30 days’ growth, the last second leaves were cut into 2–3 cm section, and soaked in 0, 75 and 150 mM atrazine, and incubated at 25uC under light for 2 days. (B) Representative images of mature plants after sprayed with atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 40 days’ growth, each pot of transgenic lines (9 plants) was sprayed with 20 mL of 0.15% atrazine solution. For simplicity, only data of transgenic Jindao8 (JD8) was shown here. doi:10.1371/journal.pone.0108569.g003 Figure 3. Tolerance of mature transgenic atzA plants to atrazine. (A) Representative images of leave sections in the presence of atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 30 days’ growth, the last second leaves were cut into 2–3 cm section, and soaked in 0, 75 and 150 mM atrazine, and incubated at 25uC under light for 2 days. (B) Representative images of mature plants after sprayed with atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 40 days’ growth, each pot of transgenic lines (9 plants) was sprayed with 20 mL of 0.15% atrazine solution. For simplicity, only data of transgenic Jindao8 (JD8) was shown here. doi:10.1371/journal.pone.0108569.g003 a backcross. The resulting herbicide-resistant CMS breeder seeds may be used to cross with the original maintainer (herbicide sensitive), to multiply the herbicide-resistant foundation seeds. During this process, the selfed maintainer and off-type plants may easily be eliminated by herbicide spraying, thus ensuring the purity of CMS seeds. The herbicide-resistant CMS line may also be used in mechanical harvesting, by spraying herbicide to kill the pollen plants after pollination, and harvesting seeds from the surviving CMS plants. A similar result may be achieved when crossing the herbicide-resistant CMS line with the herbicide-sensitive restorer line. Alternatively, the herbicide-resistant restorer line may be crossed with the herbicide-sensitive sterile line during the hybrid seed production process, by spraying herbicide to kill the off-type plants. Further, the herbicide-resistant maintainer and restorer lines may be useful for eliminating off-type plants, and for weed management when growing hybrid rice. resistant hybrid rice combinations retain their original superiority in terms of yield. Nevertheless, the development of atrazine- tolerant transgenic japonica rice parental lines represents a valuable tool for future application in hybrid seed production. References 10. Xiong X, Tang L, Deng X, Xiao G (2004) A preliminary report on the experiments of herbicide-resistant two-line hybrid rice Xiang 125S/Bar 68-1. Hybrid Rice 19(5): 41–43. 1. Peng C, Zhang H (2013) Analysis of paddy and rice markets at home and abroad in the first quarter of 2013 and its prospect. Agric Outlook (4): 4–9 (in Chinese, with English abstract). 1. 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Oral Fluorescein angiography in the diagnosis of paediatric optic nerve abnormalities.
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Oral Fluorescein angiography in the diagnosis of paediatric optic nerve abnormalities. Brinda Muthusamy  (  brinda.muthusmamy@nhs.net ) Addenbrooke's Hospital Robert Thomas Brady  Addenbrooke's Hospital, Cambridge, UK https://orcid.org/0000-0001-5839-8566 Hrishikesh Kaza  Nikhil Jain  Article Keywords: Posted Date: November 14th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3417412/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International Licen Read Full License Additional Declarations: There is no conflict of interest Oral Fluorescein angiography in the diagnosis of paediatric optic nerve abnormalities. Brinda Muthusamy  (  brinda.muthusmamy@nhs.net ) Addenbrooke's Hospital Robert Thomas Brady  Addenbrooke's Hospital, Cambridge, UK https://orcid.org/0000-0001-5839-8566 Hrishikesh Kaza  Nikhil Jain  Article Keywords: Posted Date: November 14th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3417412/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International Licen Read Full License Additional Declarations: There is no conflict of interest Brinda Muthusamy  (  brinda.muthusmamy@nhs.net ) Brinda Muthusamy  (  brinda.muthusmamy@nhs.net ) OBJECTIVE FFA is a well-established investigation for the diagnosis of optic nerve abnormalities, requiring an intravenous cannula and extended imaging acquisition time. Cannulation can present a challenge in paediatric patients and whilst oral FFA has been used for decades, it has been limited by imaging technology and unconfirmed image acquisition timings. For years, we have used a modern ultra-widefield retinal camera, and established imaging time-points to demonstrate dynamic optic nerve head changes upon ingestion of fluorescein and collected a database of oFFA images for various presentations. RESULTS Our study presents a series of images and descriptions for common optic nerve abnormalities in paediatric populations. In the interpretation part of the study, overall sensitivity of 76.8% in the consultant group vs 63.3% in the combined consultant + trainees and specificity of 87.5% vs 68.4% in the combined group. CONCLUSIONS This is the first study that describes characteristic features of several common, and serious, optic nerve abnormalities specifically for oFFA interpretation in a paediatric population. It also highlights the rapid accumulation of oFFA interpretation skills in non-specialist consultant and trainee ophthalmologists such as to obtain a high diagnostic accuracy with high sensitivity and specificity. METHODS Using an established protocol, optic nerve colour images were obtained, followed by oral administration of fluorescein dye. The optic nerves are then imaged at established intervals. An interpretation of oFFA tutorial was delivered to consultant ophthalmologists and trainees. Subsequently, these groups were assessed using a series of fifteen cases with sensitivity and specificity of the test determined. Keywords: License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: There is no conflict of interest Additional Declarations: There is no conflict of interest Version of Record: A version of this preprint was published at Eye on February 10th, 2024. See the published version at https://doi.org/10.1038/s41433-024-02956-y. Page 1/11 Page 1/11 Page 1/11 INTRODUCTION Fundus Fluorescein angiography (FFA) is a well-established investigatory tool for the diagnosis of optic nerve abnormalities. The test typically requires the intravenous administration of a fluorescein solution followed by the sequential imaging of ocular structures as the dye is perfused through the vasculature (1). Filling defects and leakage are two commonly observed features which can be observed and used to determine a diagnosis. Characteristic findings of paediatric optic nerve abnormalities have been described in paediatric patients to discern between optic nerve head drusen and papilloedema (2). A Page 2/11 study by Chang et al described fluorescein angiography as the single most important imaging modality to distinguish between these presentations (3). Intravenous FFA requires the placement of an intravenous cannula and an extended imaging acquisition time to capture the different sequences of dye perfusion through the vasculature. Whilst this is not typically difficult to complete in adults, it can be a challenge in paediatric patients. In particular, those who are very young, are needle phobic, and those with learning difficulties (4). Therefore, an alternative oral administration technique could overcome these barriers. Oral FFA (oFFA) has been used for decades but was limited by the quality of imaging technology and unconfirmed optimised timings of photograph acquisition (5). In our unit we utilise a retinal camera with a rapid, high-quality, image capture time with established timing protocol to obtain images at different phases of fluorescein dye perfusion. This technique has the benefit of avoiding cannulation and obtaining quality images to aid diagnosis. Having utilised this protocol for several years, we have collected a database of oFFA images for various optic nerve presentations including optic nerve head drusen, papilloedema, inflammatory papillitis and normal optic nerves. A selection of images were used to construct an oFFA interpretation tutorial for consultant ophthalmologists and trainee ophthalmologists. This study outlines the clinical findings of oFFA in optic nerve abnormalities and the results of an assessment upon interpretation which found it to have a high diagnostic accuracy with high sensitivity and specificity. METHODS Completing an oFFA investigation. Completing an oFFA investigation. Protocol outlined in Fig. 1. Pre-procedure, informed written consent is obtained. We advise parents that appointment will take approximately two hours and there are risks due to exposure to fluorescein. This includes pruritus, nausea, vomiting and allergy. Children will typically have discolouration of their urine. A patient information leaflet is provided which details post-procedure care. Staffing required: medical photographer, a paediatric nurse, and a doctor to cross-check prescription and supervise the child during the one-hour period post-ingestion of the fluorescein. The oral dose of fluorescein is calculated and prescribed based on the child’s current weight. Pupils are dilated on arrival using gutt cyclopentolate 1% & gutt phenylephrine 2.5%. Initial optic nerve colour images are captured using a Silverstone SS OCT - Optos ultra-widefield (UWF™) retinal imaging device (Nikon Co. LTD. Japan), followed by oral administration of fluorescein dye diluted in 20ml fruit juice with a straw to prevent staining of teeth and mouth. Post-ingestion the optic nerves are imaged at 15-, 30-, 45-, and 60-minute intervals or as requested. The paediatric nurse records vital signs at 15-minute intervals during imaging. The nurse will also monitor the child for one-hour post-procedure prior to discharge. Impact of interpretation tutorial. Page 3/11 An interpretation of oFFA tutorial describing the features of normal and 3 optic nerve pathologies was prepared and delivered over an electronic meeting platform (Zoom video communications, Qumu corporation, USA). This format facilitated the inclusion of consultant and trainee ophthalmologists from across a wide geographical area to participate. Following the tutorial, participants were invited to complete an assessment of magnified coloured optic disc photos and oFFA images of 15 optic nerves. Each nerve had five associated questions. Importantly, participants were given no clinical context to the patient’s presenting signs, symptoms, or other investigations. The diagnosis around the optic nerve images were confirmed by a consultant paediatric neuro- ophthalmologist who had access to all the clinical data for these patients. Images were used from patients who had a full neuro-ophthalmic examination, investigations, and duration of follow-up in order to be certain of the accuracy of the diagnosis. Answers were collated and analysed using a Chi-squared statistical test to compare results between consultant and trainee ophthalmologist groups. Test sensitivity and specificity was determined. The final analysis was done with the use of Statistical Package for Social Sciences (SPSS) software, IBM(manufacturer), Chicago, USA, version 25.0. Completing an oFFA investigation. For statistical significance, p value of less than 0.05 was considered significant. RESULTS Interpreting images from an oFFA investigation. Normal optic nerve: Figure 2 illustrates the common oFFA features observed in the normal optic nerve. There is a gradual increase in hyperfluorescence through the three phases. There should not be any expansion of the dye beyond the optic nerve margin. The vasculature on and surrounding the optic nerve should be clearly visible throughout all phases. Papilloedema: In the case of papilloedema, Fig. 3, hyperfluorescence observed at the optic nerve increases over time as the series progresses. There is leakage beyond the margins of the optic nerve. There is obscuration of the vessels on the optic nerve head. Maximal hyperfluorescence occurs in the late phase. Optic nerve head drusen (ONHD): Impact of interpretation tutorial. There were a total of 37 participants in the study – who were divided into two groups – consultants (group A, n = 8) and trainees who ranged from year 2 to year 7 + of their ophthalmology training (group B, n = 29). Total number of optic discs evaluated by each participant was 15, with 5 photograph-based questions linked with each optic disc and FFA. These questions included comments on all aspects of the optic disc and oFFA as described above. The total responses evaluated were 2769 (group A + group B). The total number of correct and incorrect responses received were 2020 (72.95%) and 749 (27.94%) respectively. The diagnosis given matched the actual diagnosis, in 81.7% of the responses amongst the consultant group and 58.8% in the trainee group. The responses to the first question, to comment on whether the optic discs were normal versus abnormal, revealed the following – nearly 4/5ths of the trainees gave the correct response. This compares to more than 90% with the correct response in the consultant group, and this was statistically significant (p =  0.004). The proportion of correct responses on disc vessels (clear at the disc margins or not clear) were significantly higher in consultant group as compared to the trainees. (85% vs 71.59% respectively (p value = 0.003). The correct responses received on the presence or absence of optic disc swelling was again higher in consultant as compared to ophthalmology trainees, and this was statistically significant. (76.67% vs 66.74% respectively, p value = 0.038). The two groups were also asked to comment on disc leak and a possible diagnosis on the basis of disc and oFFA photos. Correct responses to both these questions were higher in the consultant group than trainees and this was statistically significant (p = 0.002 and p < 0.0001 respectively). Sensitivity and specificity for detection of disc leak and correct diagnosis were calculated from the data and both were higher in the consultant group. Sensitivity of 76.8% in the consultant group vs 63.3% in the combined consultant + trainees and Specificity of 87.5% vs 68.4% in the combined group. Subsequently, diagnostic accuracy was calculated in the consultant’s group (82.5%) and combined consultants + trainees (65.95%). The positive and negative predictive value of both groups are shown in Table:7 (supplementary information). Inflammatory papillitis: With inflammatory optic neuropathy, there is early phase hyperfluorescence of the optic nerve head. There is leakage of fluorescein beyond the margins of the optic nerve. The hyperfluorescence increases moderately as the series progresses. Significantly, there is obscuration of the blood vessels overlying the optic nerve head. Optic nerve head drusen (ONHD): There is uptake of fluorescein by the optic nerve drusen in the early phase of the series. This forms a well demarcated area of hyperfluorescent drusen that gradually increases in hyperfluorescence as the series Page 4/11 Page 4/11 progresses, Fig. 4. The overlying vasculature should be clearly visible and not obscured even in the late images. progresses, Fig. 4. The overlying vasculature should be clearly visible and not obscured even in the late images. Impact of interpretation tutorial. The area under curve (AUC) was calculated from Sensitivity with True negatives and presented as a probability for the consultant and combined groups, which was 0.82 and 0.66 respectively. DISCUSSION Page 5/11 This study discusses the clinical findings of a range of optic nerve pathologies as revealed by oral fundus fluorescein angiography (oFFA). We describe a protocol for administering oral fluorescein and acquiring angiographic images in a population where intravenous cannulation may be too difficult or too distressing. In addition to this, we found that the images we generated can be interpreted as for images obtained by intravenous (IV) FFA. Specifically, these clinical observations support those previously published by Pineles and Arnold (2) who described that early and late leakage on IV FFA can be used to reliably distinguish papilloedema from pseudo-papilloedema (mainly optic disc drusen). The same authors produced a subsequent study years later that compared various imaging modalities to differentiate papilloedema from pseudo-papilloedema and found FFA to be the most accurate in the paediatric population (3), highlighting the benefit and need of accurate interpretation of these images in day-to-day clinical work. Moreover, this manuscript highlights the rapid accumulation of oFFA interpretation skills in non-specialist consultant and trainee ophthalmologists. We can highlight a significant difference in accuracy, sensitivity and specificity between consultant interpretation and trainee interpretation. This inter-observer variability could have ramifications in clinical practice as patients present to a variety of clinics and, should an ophthalmologist not feel comfortable in interpreting oFFA images, they may not be able to benefit from their high sensitivity and specificity of oFFA. There have been multiple studies since the 1960s in which FFA has been used to identify papilloedema in an adult population (6) and how to differentiate the findings from those in pseudo-papilloedema (7). The clear consensus between these studies is that leakage around the disc is suggestive for papilloedema and directs away from pseudo-papilloedema. Our study corroborates these findings and expands on them to represent the paediatric population. It is clear from our observations, and others, that timing plays a critical role is discerning signs such as leakage which are fundamental to an accurate diagnosis. Our findings support the dynamic nature of changes occurring over time, that further aid in diagnosis when compared to investigations using a single image at a single time point. This is particularly relevant when comparing studies that quote a lower diagnostic accuracy of this investigation but do not capture earlier time points (8). In addition, our study presents a series of images and description of pertinent features for inflammatory papillitis. DISCUSSION The presence of disc leakage in inflammatory optic nerve head pathology has been well described in patients with an underlying diagnosis of sarcoidosis (9) and Vogt-Koyangai-Harada Syndrome (10). Our findings in paediatric patients with inflammatory papillitis secondary to panuveitis are very similar in description to the adult patients with the above conditions. It is therefore apparent that paediatric inflammatory optic nerve pathology looks very similar on oFFA to adult IV FFA. Differentiating between underlying conditions may be very limited using FFA but occult granulomas that may be not visible on clinical examination can be become apparent on FFA (9). In addition, our study presents a series of images and description of pertinent features for inflammatory papillitis. The presence of disc leakage in inflammatory optic nerve head pathology has been well described in patients with an underlying diagnosis of sarcoidosis (9) and Vogt-Koyangai-Harada Syndrome (10). Our findings in paediatric patients with inflammatory papillitis secondary to panuveitis are very similar in description to the adult patients with the above conditions. It is therefore apparent that paediatric inflammatory optic nerve pathology looks very similar on oFFA to adult IV FFA. Differentiating between underlying conditions may be very limited using FFA but occult granulomas that may be not visible on clinical examination can be become apparent on FFA (9). Inter observer variability has been well demonstrated before and in addition to their other findings Chang et al (3) demonstrated a range of accuracy between neuro-ophthalmologists of 83%-97% in interpreting Page 6/11 FFA images of paediatric optic nerve pathology. Our study further extends the finding of variability to the interpretation of ophthalmologists fully trained in other subspecialties as well as a wide range of training grades. This suggests a need for a training model for ophthalmologists of all grades to standardise interpretation acumen. A limitation of this study was the presentation of clinical images without any clinical context. The signs and symptoms upon presentation, complete with investigations and additional imaging modalities is fundamental to reaching a correct diagnosis. We would expect the diagnostic accuracy to be higher if the participants were provided with this information. An additional limitation was the imposition of an arbitrary time limit of one minute to answer all questions on each image. Finally, the presentation of images did not allow the interpreter to magnify optic nerve features which may have limited their ability to observe key findings such as vessel obscuration. DISCUSSION In reality, such time limits do not exist and clinicians can magnify areas of interest to further evaluate image features. Future work will focus on validating the investigation by assessing additional images presented to the same body of interpreters. An additional goal would be to optimise a teaching strategy and delivery of an oFFA tutorial to maximise participants engagement, understanding, and application of new skills. FUNDING The authors did not receive any additional financial resources for completion of this work. CONCLUSION This is the first study that describes the characteristic features of several common, and serious, optic nerve abnormalities specifically for oFFA interpretation. We found that our methodology achieved comparable clinical features to those previously published. Moreover, we found that interpretation skills could be rapidly developed such as to obtain a high diagnostic accuracy with high sensitivity and specificity. CONFLICT OF INTEREST The authors declare no confilicts of interests . References Int Ophthalmol. 2007;27(2–3):155–61. ACKNOWLEDGMENTS The authors would like to acknowledge the paediatric nursing team and imaging technician team at Addenbrooke’s hospital for their support in completing the oFFA investigations described above. We Page 7/11 would also like to acknowledge the participation of consultant and trainees colleagues who participated in the study. would also like to acknowledge the participation of consultant and trainees colleagues who participated in the study. References 1. Cavallerano AA. Ophthalmic fluorescein angiography. Optom Clin. 1996;5(1):1–23. 1. Cavallerano AA. Ophthalmic fluorescein angiography. Optom Clin. 1996; 2. Pineles SL, Arnold AC. Fluorescein angiographic identification of optic disc drusen with and without optic disc edema. J Neuroophthalmol. 2012;32(1):17–22. 3. Chang MY, Velez FG, Demer JL, Bonelli L, Quiros PA, Arnold AC, et al. Accuracy of Diagnostic Imaging Modalities for Classifying Pediatric Eyes as Papilledema Versus Pseudopapilledema. Ophthalmology. 2017;124(12):1839–48. 3. Chang MY, Velez FG, Demer JL, Bonelli L, Quiros PA, Arnold AC, et al. Accuracy of Diagnostic Imaging Modalities for Classifying Pediatric Eyes as Papilledema Versus Pseudopapilledema. Ophthalmology. 2017;124(12):1839–48. 4. Mohan S, Raman R. Recommended protocol and technique for doing oral fundus fluorescein angiography for adults and children. Indian J Ophthalmol. 2022;70(12):4430–3. 4. Mohan S, Raman R. Recommended protocol and technique for doing oral fundus fluorescein angiography for adults and children. Indian J Ophthalmol. 2022;70(12):4430–3. 5. Kelley JS, Kincaid M. Retinal Fluorography Using Oral Fluorescein. Archives of Ophthalmology. 1979;97(12):2331–2. 5. Kelley JS, Kincaid M. Retinal Fluorography Using Oral Fluorescein. Archives of Ophthalmology. 1979;97(12):2331–2. 6. Hayreh SS. Pathogenesis of optic disc edema in raised intracranial pressure. Progress in retinal and eye research. 2016;50:108–44. 6. Hayreh SS. Pathogenesis of optic disc edema in raised intracranial pressure. Progress in retinal and eye research. 2016;50:108–44. 7. Miller SJ, Sanders MD, Ffytche T. Fluorescein fundus photography in the detection of early papilloedema and its differentiation from pseudo-papilloedema. Lancet. 1965;2(7414):651–4. 7. Miller SJ, Sanders MD, Ffytche T. Fluorescein fundus photography in the detection of early papilloedema and its differentiation from pseudo-papilloedema. Lancet. 1965;2(7414):651–4. 8. Elhusseiny AM, Fong JW, Hsu C, Grigorian F, Grigorian AP, Soliman MK, et al. Oral Fluorescein Angiography for the Diagnosis of Papilledema Versus Pseudopapilledema in Children. American Journal of Ophthalmology. 2023;245:8–13. 8. Elhusseiny AM, Fong JW, Hsu C, Grigorian F, Grigorian AP, Soliman MK, et al. Oral Fluorescein Angiography for the Diagnosis of Papilledema Versus Pseudopapilledema in Children. American Journal of Ophthalmology. 2023;245:8–13. 9. Moschos MM, Guex-Crosier Y. Anterior segment granuloma and optic nerve involvement as the presenting signs of systemic sarcoidosis. Clin Ophthalmol. 2008;2(4):951–3. 9. Moschos MM, Guex-Crosier Y. Anterior segment granuloma and optic nerve involvement as the presenting signs of systemic sarcoidosis. Clin Ophthalmol. 2008;2(4):951–3. 10. Arellanes-García L, Hernández-Barrios M, Fromow-Guerra J, Cervantes-Fanning P. Fluorescein fundus angiographic findings in Vogt-Koyanagi-Harada syndrome. Int Ophthalmol. 2007;27(2–3):155–61. 10. Arellanes-García L, Hernández-Barrios M, Fromow-Guerra J, Cervantes-Fanning P. Fluorescein fundus angiographic findings in Vogt-Koyanagi-Harada syndrome. Figures Page 8/11 gure 1 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating papilloedema. Figure 1 Pathway to completing an oFFA investigation. This begins with the decision to undertake the investigation, the informed consent of the paediatric patient and guardians. The fluorescein is then prescribed, and the dose is adjusted by weight of the patient. The dosing phase involves baseline observations of blood pressure, heart rate, temperature, and baseline colour photographs. This is followed by fluorescein ingestion, dilated in apple juice, using a straw. Finally, the patient is imaged at Page 9/11 time points of 15-, 30-, 45-, and 60-minutes post ingestion. The patient is monitored for any adverse effects and the sequence of images can be reviewed by the ordering physician once completed. Figure 2 Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating the normal optic nerve. Figure 5 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating inflammatory papillitis. Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating inflammatory papillitis. Figure 4 Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating optic nerve head drusen. Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating optic nerve head drusen. Figure 5 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating inflammatory papillitis. Figure 2 Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating the normal optic nerve. Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating the normal optic nerve. Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating the normal optic nerve. Figure 3 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating papilloedema. Figure 3 Figure 3 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating papilloedema. Page 10/11 Figure 4 Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes illustrating optic nerve head drusen. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SUPPLEMENTARYINFORMATION.docx Page 11/11
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Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap Kinerja Guru SMK Swasta Se-Kecamatan Bangil
Kelola/Kelola: Jurnal Manajemen Pendidikan
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Magister Manajemen Pendidikan FKIP Universitas Kristen Satya Wacana jurnalkelola@gmail.com Magister Manajemen Pendidikan FKIP Universitas Kristen Satya Wacana jurnalkelola@gmail.com e-ISSN 2549-9661 Volume: 6, No. 2, Juli-Desember 2019 Halaman: 188-192 Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap Kinerja Guru SMK Swasta Se-Kecamatan Bangil Windasari Manajemen Pendidikan Universitas Negeri Surabaya windasari@unesa.ac.id Muchammad Zulham Yahya SMK Darut Tauhid Bangil zulwamyahya27@gmail.com ABSTRACT This research objective to find out the impact of motivation and compensation through teachers performance Vocational High School in Bangil district. Collecting data technique used questionnaire and distributed to respondent. Sampling technique is cluster random sampling. Sample for every school are 3 teachers from each major. Totally sample 42 respondents from 5 schools. Validity test used pearson product moment. Analisis technique to answer the research questions are inferential statistic multiple linier regression. Research results showed motivation and compensation have an impact through teachers performance Vocational High School in Bangil District. Keywords: Motivation, Compensation, Performance, Teachers Article Info Received date: 3 Oktober 2019 Revised date: 28 November 2019 Accepted date: 20 Desember 2019 Keywords: Motivation, Compensation, Performance, Teachers Article Info Revised date: 28 November 2019 Accepted date: 20 Desember 2019 Received date: 3 Oktober 2019 Received date: 3 Oktober 2019 PENDAHULUAN besar dalam mengembangkan potensi peserta didik di sekolah. Pendidikan memiliki peranan penting dalam mencerdaskan manusia. Berdasarkan Undang-Undang Nomor 20 tahun 2003 tentang Sistem Pendidikan Nasional, Pasal 3 dijelaskan bahwa tujuan pendidikan nasional adalah mengembangkan potensi peserta didik agar menjadi manusia yang beriman dan bertakwa kepada Tuhan Yang Maha Esa, berakhlak mulia, sehat, berilmu, cakap, kreatif, mandiri, dan menjadi warga negara yang demokratis serta bertanggung jawab. Untuk mencapai tujuan pendidikan tersebut, kualitas guru menjadi salah satu faktor penentu keberhasilan peserta didik dalam proses belajar mengajar. Kinerja guru memiliki kontribusi yang cukup Motivasi guru memiliki dampak/ implikasi terhadap pemimpin dan pengelola sekolah karena motivasi guru juga berpengaruh terhadap motivasi peserta didik. Guru dengan tingkat motivasi yang tinggi akan mampu menciptakan lingkungan sosial dan psikologi yang lebih baik untuk peserta didik. Oleh karena itu, motivasi guru juga merupakan kunci keberhasilan kinerja organisasi atau sekolah. Bukti empiris menunjukkan terdapat hubungan yang kuat diantara kompensasi dan kualitas guru. Penelitian yang ada mengindikasikan bahwa kompensasi berpotensi untuk meningkatkan kinerja guru. Kompensasi 188 Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap Kinerja Guru … | Windasari & Muchammad Z. Yahya masih memiliki kinerja yang kurang. Penelitian sebelumnya yang sudah pernah dilakukan terkait motivasi merupakan penelitian yang dilakukan untuk mengetahui kinerja guru di sekolah negeri. Kompensasi yang diberikan kepada guru negeri memiliki system yang berbeda dengan sistem kompensasi yang diberikan kepada guru swasta. Oleh karena itu peneliti ingin mengetahui pengaruh motivasi dan kompensasi terhadap kinerja guru SMK swasta di kecamatan bangil. Adapun tujuan dari penelitian ini adalah untuk mengukur seberapa besar pengaruh motivasi kerja dan kompensasi terhadap kinerja guru SMK Swasta se- Kecamatan Bangil? yang diterima oleh guru baik berupa financial ataupun non finanasial juga akan berdampak kepada kepuasan kerja. Guru yang memiliki kepuasan kerja bagus maka akan terus menunjukkan kinerja yang maksimal. Motivasi dan kompensasi merupakan faktor yang berpengaruh terhadap kinerja guru. Motivasi menjadi poin yang penting mengingat tanggung jawab guru adalah menyampaikan ilmu pengetahuan dan keterampilan kepada peserta didik. Sedangkan tujuan dari kompensasi yang baik adalah untuk menarik, memotivasi dan mempertahankan orang-orang yang berkinerja baik untuk pencapaian tujuan organisasi. Kompensasi yang diberikan kepada individu merupakan suatu bentuk apresiasi jasa dan upaya dalam menyelesaikan tugas dan pekerjaan mereka. Saat ini negara maju dan berkembang mengalami kekurangan guru berkualitas di bidang pendidikan. Sebagai factor penting dalam keberhasilan dalam pembelajaran, guru seharunya memiliki kinerja yang bagus. PENDAHULUAN Sekolah Menengah Kejuruan (SMK) adalah salah satu lembaga pendidikan di Indonesia yang berperan dalam menghasilkan lulusan yang memiliki kompetensi siap kerja. Sehingga, kualitas SMK sangat menentukan mutu Sumber Daya Manusia (SDM) bangsa Indonesia. Data (Dikbud, 2016) menunjukkan bahwa kinerja guru SMK masih dalam kategori kurang yaitu dnegan nilai masih kurang yaitu 77.60%. Kelola: Jurnal Manajemen Pendidikan, Vol. 6, No. 2, Juli-Desember 2019 Desian penelitian ini akan mengukur variable motivasi dan kompensasi secara bersama-sama terhadap kinerja. Keterangan: X1 : Motivasi Kerja X2 : Kompensasi Guru Y : Kinerja Guru erhadap kinerja. Gambar 1.1 Pengaruh Variabel X1 dan X2 terhadap Y X1 X2 Y Hasil dari analisis data menggunakan spps diperoleh hasil regresi ganda seperti tabel 1.1 dibawah ini. Gambar 1.1 Pengaruh Variabel X1 dan X2 terhadap Y Gambar 1.1 Pengaruh Variabel X1 dan X2 Tabel 1.1 Analisis Persamaan Linier Regresi Ganda Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 Constant 3.684 2.826 1.304 .200 Tot Mot .359 .079 .367 4.530 .000 Tot Komp 1.096 .138 .645 7.972 .000 Karena untuk motivasi kerja t hit = 4,530 >1.68 = t 0,05;39, maka motivasi berpengaruh signifikan terhadap kinerja guru. Begitu juga untuk kompensasi guru, t hit = 7,972 >1.68= t 0,05;39, maka kompensasi guru berpengaruh signifikan terhadap kinerja guru. Dari tabel diatas, persamaan linier regresi ganda Kinerja Guru (Y) dalam Motivasi Kerja (X1) dan Kompensasi Guru (X2) adalah sebagai berikut : Y = 3,684 + 0,0356 X1 + 1,096 X2 ANOVAa Model Sum of Squares df Mean Square F Sig. 1 Regression 621.882 2 310.941 102.253 .000b Residual 118.595 39 3.041 Total 740.476 41 Dari tabel diatas terlihat F hit = 102,253 > 3,23 = F 0,05(2;39), maka regresi linier ganda signifikan. mengacu kepada penguasaan konsep dan keterampilan mengajar di dalam kelas. Semakin tinggi motivasi guru untuk mengajar, guru akan menunjukkan kemampuan optimal di dalam kelas sehingga kinerjanya akan terus meningkat. METODE PENELITIAN Penelitian ini merupakan penelitian kuantitatif deskriptif dengan metode survey. Penelitian ini dilaksanakan di 5 SMK di Bangil dengan subyek penelitian ini adalah 3 orang guru dari tiap jurusan pada setiap sekolah. Setiap guru akan mengisi dan menjawab kuesioner yang telah disebarkan. Metode penarikan sampel yang digunakan adalah menggunakan Central Limit Teorema (sebuah teorema yang menyatakan bahwa kurva distribusi sampling untuk ukuran sampel 30 atau lebih akan berpusat pada nilai parameter populasi dan akan memiliki semua sifat-sifat distribusi normal). Agar tidak berpusat pada satu titik maka digunakan metode cluster random sampling dengan mengambil 3 guru dari tiap jurusan yang ada di sekolah masing- masing sehingga jumlah sample yang diambil dari populasi yang ada sebanyak 42 guru dari 5 sekolah yang terbagi 14 jurusan. Berdasarkan penelitian sebelumnya yang dilakukan oleh (Nowsu, 2016) kompensasi memiliki pengaruh terhadap motivasi guru di sekolah negeri. Penelitian ini juga menyatakan bahwa motivasi juga berpengaruh terhadap kinerja guru di sekolah negeri di Kenya.Selain itu, berdasarkan hasil penelitian (Sukrapi, 2014) menunjukkan bahwa motivasi kerja guru berpengaruh positif dan signifikan terhadap kinerja guru. Dalam penelitian ini akan digunakan uji validitas product moment dan uji reliabilitas croanbach’s alpha dengan bantuan software SPSS for windows versi 21. Teknik analisis data yang digunakan untuk menguji dalam penelitian ini yaitu Regresi Linier Berganda untuk menguji apakah ada pengaruh variabel motivasi dan kompensasi terhadap kinerja guru. Dari data rendahnya kinerja guru SMK yang dikeluarkan oleh Kemendikbud dapat dilihat bahwa secara umum kualitas guru SMK 189 Kelola: Jurnal Manajemen Pendidikan, Vol. 6, No. 2, Juli-Desember 2019 Simpulan Motivasi dan kompensasi merupakan dua variabel penting dalam mempengaruhi tinggi rendahnya kinerja guru. Keinginan guru untuk mengajar dengan baik di kelas serta didukung pemberian kompensasi pelatihan peningkatan kompetensi dari sekolah akan memberikan dampak yang sangat positif terhadap kinerja guru di sekolah. Hasil penelitian ini menunjukkan hasil yang signifikan korelasi antara motivasi dan kompensasi terhadap kinerja guru. Pengaruh motivasi kerja dan kompensasi terhadap kinerja guru SMK Swasta se-Kecamatan Bangil positif dan signifikan sebesar 83,98%. Jika semakin tinggi motivasi kerja yang dimiliki dan kompensasi yang diterima oleh guru maka kinerja guru SMK swasta se-Kecamatan Bangil semakin tinggi. Selain itu, penelitian ini juga membuktikan bahwa kompensasi guru berpengaruh positif juga terhadap kinerja guru SMK Swasta se-Kecamatan Bangil. Semakin besar atau tinggi kompensasi yang diberikan oleh sekolah terhadap guru maka akan semakin tinggi dan baik pula kualitas kinerja guru. Hal ini sejalan dengan pendapat Handoko (2008:157) kompensasi hendaknya mendorong perilaku-perilaku yang diinginkan. Prestasi kerja yang baik, pengalaman, kesetiaan, tanggung jawab baru dan perilaku-perilaku lain dapat dihargai melalui rencana kompensasi yang efektif. Pembahasan Berdasarkan data analisis, diperoleh hasil bahwa motivasi kerja dan kompensasi guru secara bersama-sama berpengaruh terhadap kinerja guru SMK Swasta se- kecamatan Bangil. Motivasi guru sangat penting untuk menumbuhkan dan mengembangkan proses pembelajaran yang baik di dalam kelas. Motivasi guru di sekolah Kompensasi dan insentif finansial yang diberikan kepada guru merupakan factor penting dalam persaingan yang kompetitif di antara SMK swasta yang ada, bentuk lain pemberian kompensasi berupa kesempatan guru untuk mengikuti pelatihan dan 190 Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap Kinerja Guru … | Windasari & Muchammad Z. Yahya swasta di Bangil merupakan sekolah SMK dalam pengelolaan SDM nya sudah terstruktur. Beberapa guru yang direkrut merupakan guru- guru yang memiliki kompetensi sesuai dengan bidang yang diampunya dan juga merupakan lulusan dari universitas yang bereputasi. pengembangan diri juga sangat berpengaruh terhadap kinerja guru. Guru yang memiliki kinerja bagus, melaksanakan tugas pembelajaran di kelas dengan baik didukung dengan teknikal skills atau kompetensi yang bagus. Artinya pemberian kompensasi baik dalam bentuk finasial ataupun pelatihan keduanya sangat berpengaruh terhadap kinerja guru di sekolah. Hasil tersebut sesuai dengan pendapat (Hasibuan,2008) yang mendefinisikan motivasi sebagai suatu perangsang (want) dan daya penggerak kemauan bekerja seseorang. Selain input SDM yang cukup memiliki kualifikasi dan motivasi yang bagus, sistem kompensasi di beberapa sekolah SMK memang sudah baik. Saran Adapun saran untuk peneliti kedepannya adalah bahwa ruang lingkup studi ini difokuskan pada guru SMK Swasta se- Kecamatan Bangil yang berdampak pada generalisasi studi yang terbatas. Keterbatasan ini mengisyaratkan perlunya studi studi lanjutan untuk menggeneralisasi hasil-hasil yang diperoleh pada konteks yang berbeda dan lebih luas, sehingga konsep-konsep yang diuji dalam model dapat ditingkatkan validitas eksternalnya. Seorang guru yang memiliki tingkat motivasi bagus akang sangat membantu dan memudahkan organisasi dalam meraih tujuan organisasi. Guru dengan tingkat motivasi yang tinggi akan melaksanakan tugas pengajaran dengan sebaik mungkin kepada peserta didik. Begitu pula sebaliknya, guru dengan tingkat motivasi yang rendah akan berdampak kepada kualitas pengajaran yang diberikan kepada peserta didik. Hampir sebagian besar SMK DAFTAR PUSTAKA DAFTAR PUSTAKA Afifah, Marnesya. (2011). Pengaruh Motivasi, Kompensasi, dan Pengembangan Karir terhadap Kinerja Guru (Studi Kasus Pada SMK Nusantara Jl. Tarumanegaran dalam No.1 Ciputat). Jakarta : UIN Syarif Hidayatullah. 191 Kelola: Jurnal Manajemen Pendidikan, Vol. 6, No. 2, Juli-Desember 2019 Mulyasa. (2013). Uji Kompetensi dan Penilaian Kerja Guru. Bandung : PT. Remaja Rosdakarya. Arikunto, Suharsimi. (1999). Prosedur Penelitian: Suatu Pendekatan Praktek. Yogyakarta: Rineka Cipta. Fauzie, Ahmad. (2009). Pengaruh Motivasi dan Kompensasi Terhadap Kinerja Pegawai Perpustakaan UIN Syarif Hidayatullah Jakarta (Tesis). Jakarta : UIN Syarif Hidayatullah. Nowsu, J (2016). Motivation And Teachers’ Performance In Selected Public Secondary Schools In Ikenne Local Government Area Of Ogun State.. British Journal of Psychology Research Vol.5, No.3, pp 40-50. Ghozali, Imam. (2005). Aplikasi Analisis Multivariate dengan Program SPSS. Semarang: Universitas Diponegoro. Robbins, Stephen P. (2002). Prinsip-prinsip Perilaku Organisasi. Jakarta : Erlangga. Handoko, T. Hani. (2008). Manajemen Personalia & Sumberdaya Manusia. Yogyakarta: BPFE. Robbins, Stephen P dan Timothy A. Judge. (2009). Perilaku Organisasi Organizational Behavior Edisi 12. Jakarta : Salemba Empat. Hasibuan, Malayu S.P. (2008). Manajemen Sumber Daya Manusia. Jakarta : Penerbit Bumi Aksara. Sarimaya, Farida. (2008). Sertifikasi Guru. Jakarta : Yrama Widya. Kadarisman, M (2012). Manajemen Kompensasi. Jakarta : Rajagrafindo Persada. Wekesa, Jane N, dan Silas Nyaroo. (2013). “Effect of Compensation on Performance of Public Secondary School Teachers in Eldort Municipality Kenya”. International Journal of Scientific and Research Publications (ISSN 2250-3153) Vol. 03 - Issue 6, June 2013. Keran, Kristina N. (2012). Pengaruh Motivasi Kerja, Kompetensi, Kompensas dan Kinerja Karyawan di Yayasan Bintang Timur Tangerang (Tesis). Jakarta : Universitas Esa Unggul. 192
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Presence of a Haloarchaeal Halorhodopsin-Like Cl&lt;sup&gt;−&lt;/sup&gt; Pump in Marine Bacteria
Microbes and environments
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Presence of a Haloarchaeal Halorhodopsin-Like Cl– Pump in Marine Bacteria Yu Nakajima1,2*, Takashi Tsukamoto3†, Yohei Kumagai1,2, Yoshitoshi Ogura4, Tetsuya Hayashi4, Jaeho Song5, Takashi Kikukawa6,7, Makoto Demura6,7, Kazuhiro Kogure1,2, Yuki Sudo3, and Susumu Yoshizawa1,2 Yu Nakajima1,2*, Takashi Tsukamoto3†, Yohei Kumagai1,2, Yoshitoshi Ogura4, Tetsuya Hayashi4, Jaeho Song5, Takashi Kikukawa6,7, Makoto Demura6,7, Kazuhiro Kogure1,2, Yuki Sudo3, and Susumu Yoshizawa1,2 1Atmosphere and Ocean research Institute (AORI), The University of Tokyo, 5–1–5, Kashiwanoha, Kashiwa, Chiba 277–8564, Japan; 2Department of Natural Environmental Studies, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277–8563, Japan; 3Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700–8530, Japan; 4Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka-shi, Fukuoka 812–8582, Japan; 5Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea; 6Faculty of Advanced Life Science, Hokkaido University, Sapporo 060–0810, Japan; and 7Global Station for Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo 060–0810, Japan Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl–-pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl– pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl–-transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl–-pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR. Key words: family Rhodothermaceae, rhodopsin, genome analysis, chloride ion pump Rhodopsins are seven-transmembrane proteins composed of a protein moiety (opsin) and light-absorbing chromophore (retinal). * Corresponding author. E-mail: yunakajima@aori.u-tokyo.ac.jp; Tel: +81–4–7136–6168; Fax: +81–4–7136–6169. † Present address: Faculty of Advanced Life Science, and Global Station for Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo 060–0810, Japan Microbes Environ. Vol. 33, No. 1, 89-97, 2018 Microbes Environ. Vol. 33, No. 1, 89-97, 2018 https://www.jstage.jst.go.jp/browse/jsme2  doi:10.1264/jsme2.ME17197 Key words: family Rhodothermaceae, rhodopsin, genome analysis, chloride ion pump Materials and Methods Strain information and genomic sequencing of R. marinus SG-29T, R. marina SAORIC-28T, and R. profundi SAORIC-476T. R. marinus SG-29T was isolated from surface seawater (30° 40' N, 138° 00' E; depth 50 m), and additional information has been reported previously (27). R. marina SAORIC-28T was isolated from a deep seawater sample obtained from the western North Pacific Ocean (32° 00' N, 138° 13' E; depth 3000 m) by the R/V Tansei maru (Atmosphere and Ocean Research Institute, The University of Tokyo and Japan Agency for Marine-Earth Science and Technology [JAMSTEC]) on 3 July 2010 (KT-10-12 cruise). R. profundi SAORIC- 476T was isolated from a deep seawater sample obtained from the western North Pacific Ocean (32° 00' N, 140° 00' E; depth 3000 m) during the research cruise (MR-11-05) of R/V Mirai (JAMSTEC) in May 2011. Details of the culture conditions and phenotypic traits of these strains have been described previously (28, 30). The genomic DNA of strains SAORIC-28T and SAORIC-476T was extracted using phenol-chloroform and ethanol precipitation (21). Library preparation was performed for strain SAORIC-28T, as previously reported for strain SG-29T (24). A KAPA HyperPlus Kit (Kapa Biosystems, Boston, MA, USA) was used for library preparation for strain SAORIC-476T. Each end of the libraries (300 bp) of SAORIC- 28T and SAORIC-476T were sequenced on a MiSeq instrument with MiSeq Reagent kit version 3 (Illumina, San Diego, CA, USA). ( pp μ ) Retinal isomer compositions were analyzed using normal-phase high-performance liquid chromatography (HPLC). The procedures for the retinal oxime extraction and HPLC analysis were the same as those described in our previous study (35). Regarding measurements under dark conditions, the sample was maintained in the dark for more than 1 week. In measurements under light conditions, the sample was illuminated with 540±10 nm light for 5 min before retinal oxime extraction. The retinal oxime extracted from the purple membrane (PM) containing Halobacterium salinarum bacteriorho- dopsin (HsBR) was used as a reference. The eluting retinal oxime was monitored by its absorption at 360 nm. The flow rate was 1 mL/ min. All measurements were performed at 25°C. p All UV-Vis spectra were recorded at 25°C using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Presence of a Haloarchaeal Halorhodopsin-Like Cl– Pump in Marine Bacteria pH was measured using a pH meter (LAQUA F-72; HORIBA, Kyoto, Japan). Measurements were repeated under the same conditions after the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP, final concen- tration, 30 μM). All measurements were performed at 4°C. Different solutes (100 mM NaCl, KCl, MgCl2, NaBr, NaI, NaNO3, and Na2SO4) were used in the ion selectivity analysis of RmHR. (XeR) cluster, and the other is a unique cluster consisting of rhodopsin containing a TSA motif (amino acid residues 85, 89, and 96 in BR numbering). A phylogenetic analysis showed that this unique rhodopsin cluster is closely related to the HR of halophilic archaea. Therefore, we named these HR-like rhodopsins RmHR (R. marinus SG-29T halorhodopsin), R28HR1, and R28HR2 (R. marina SAORIC-28T halorhodopsin 1 and 2). A functional analysis using heterologously expressed RmHR was subsequently conducted to identify its ion specificity. We further performed a spectroscopic analysis using purified RmHR to clarify its spectroscopic characteristics because even rhodopsins with the same ion transport specificity have intermediates with different ion affinities and structural changes, and the properties are important factors that define rhodopsins. In the present study, we performed genomic and phylogenetic analyses, and also characterized RmHR spec- troscopically, and these properties were compared with those of known microbial rhodopsins. Spectroscopic analysis of RmHR. E. coli BL21 (DE3) cells were used for the functional expression of RmHR as a recombinant protein. The procedures for protein expression and purification were the same as those described in our previous study (35). In spectroscopic measurements, the buffer was sufficiently exchanged by centrifugation (AmiconUltra centrifuge unit; 30,000 molecular weight cut-off, Merck Millipore, Bedford, MA, USA) and the PD-10 column (GE Healthcare, Waukesha, WI, USA) with 10 mM 3-(N-morpholino)-propanesulfonic acid (MOPS) buffer (Dojindo Laboratories, Kumamoto, Japan) containing 0.1% (w/v) n-dodecyl-β-D-maltoside (DDM, Dojindo Laboratories) and NaCl at the desired concentrations (1 mM to 4 M). In order to maintain ionic strength at 4 M, Na2SO4 was added to the samples at the desired concentrations (0–1.333 M). Neither Na+ nor SO42– was a substrate ion for RmHR (see the Results and Discussion section). The optical density of DDM-solubilized RmHR was adjusted to approximately 0.5 optical density (OD) at its absorption maximum (approx. 10 μM). Presence of a Haloarchaeal Halorhodopsin-Like Cl– Pump in Marine Bacteria They are widely distributed among animals and prokaryotes and have been classified into two groups: type-1 (microbial origin) and type-2 (eukaryotic origin). Microbial type-1 rhodopsins were initially discovered in extremely halophilic archaea: a light-driven outward H+-pumping rhodopsin (bacteriorhodopsin [BR] [26]), inward Cl–-pumping rhodopsin (halorhodopsin [HR] [22]), and light-sensing rhodopsins (sensory rhodopsin I and II [SRI and SRII]) (31). In 2000, an H+-pumping rhodopsin was also found in a marine bacterial genomic fragment using a metagenomic approach and was named proteorhodopsin (PR) (4). Recent studies have shown that PR genes are widely present among marine bacteria and archaea inhabiting the euphotic layer of the ocean (7). Furthermore, outward Na+-pumping rhodopsin (NaR) (15) and inward Cl–-pumping rhodopsin (ClR) (43) were discovered in marine bacteria. Of note, BR and HR of haloarchaea and PR and ClR of marine bacteria belong to distinct phylogenetic lineages, suggesting that these ion-pumping rhodopsins evolved independently in their respective habitats. The family Rhodothermaceae belongs to the class Cytophagia in the phylum Bacteroidetes. This family includes 7 genera (Longimonas [40], Longibacter [41], Rhodothermus [1], Rubricoccus [27], Rubrivirga [28], Salinibacter [3], and Salisaeta [36]) and has been found in diverse environments, e.g., hydrothermal vents, hypersaline environments, and oceans. The genera Longimonas, Longibacter, Salisaeta, and Salinibacter include halophilic bacteria, and members of the genus Rhodothermus are thermophiles. In contrast, members of the genera Rubricoccus and Rubrivirga have been isolated from surface or deep seawater and are not extremophiles. Salinibacter, a halophilic bacterium, dominates with halophilic archaea in hypersaline environments (5–25% according to fluorescence in situ hybridization [2] and 7–9% according to a metagenomic analysis [8]). Previous studies reported that Salinibacter ruber M31T carries multiple rhodopsin genes: xanthorhodopsin (XR), HR, and SRI (23). In addition, a genomic analysis suggested that S. ruber acquired not only the HR and SRI genes, but also many other genes that are necessary for salt adaptation from halophilic archaea by lateral gene transfer (LGT) because both of these halophilic prokaryotes inhabit and dominate the same environment (23). Following the genome information of Rubricoccus marinus SG-29T (24), we herein report two genome sequences of Rubrivirga marina SAORIC-28T and R. profundi SAORIC- 476T. A genomic analysis revealed that R. marinus SG-29T and R. marina SAORIC-28T have five rhodopsin genes belonging to two different clusters: one is included in the xenorhodopsin 90 Nakajima et al. then irradiated through a 520±10 nm bandpass filter (MX0520; Asahi Spectra) for 3 min. Gene preparation and ion transport measurements of RmHR. The DNA fragments encoding RmHR, R28HR1, and R28HR2 were chemically synthesized by Eurofins Genomics (Eurofins Genomics, Tokyo, Japan) with codon optimization for Escherichia coli. These gene fragments were inserted into the NdeI and XhoI sites of the pET21a vectors (Novagen, Darmstadt, Germany); consequently, the plasmids encoded RmHR, R28HR1, and R28HR2 with hexahistidines at the C terminus. The vectors were transformed into E. coli strain C41 (DE3) (Lucigen, Middleton, WI, USA). E. coli cells with the plasmid were incubated at 37°C on an LB medium agar plate supple- mented with ampicillin (final concentration, 100 μg mL–1). Cells carrying the plasmid were then grown at 37°C in 200 mL of 2×YT medium supplemented with ampicillin (final concentration, 100 μg mL–1), and protein expression was induced at an OD 660 nm (OD660) of 0.4–0.6 with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 10 μM all-trans-retinal (Sigma-Aldrich, St Louis, MO, USA). Rhodopsin-expressing cells were collected by centrifugation (8,000×g for 3 min), washed three times with 100 mM NaCl, and then re-suspended in the solvent for measurement. The light source was a 300 W xenon lamp (MAX-303; Asahi Spectra, Tokyo, Japan), and 6 mL of the cell suspension was initially placed in the dark and ΔA=1/(1+10(pKa-pH)) Materials and Methods In the Cl– titration experiment, the absorption maximum wavelength (λmax) of RmHR was plotted against the logarithm of the Cl– concentration ([Cl–]) and analyzed by fitting the data to the following Hill equation: λmax=a+b·[Cl–]n/([Cl–]n+[Kd]n) λmax=a+b·[Cl–]n/([Cl–]n+[Kd]n) where a, b, Kd, and n represent the offset, the amplitude of the λmax change, the dissociation constant for Cl–, and the Hill coefficient, respectively. In the pH titration experiment, the sample was suspended in a 6-mix buffer (0.89 mM citrate, 0.89 mM MES, 1.1 mM TES, 0.78 mM TAPS, 1.1 mM CHES, 0.33 mM CAPS; MES, TES, TAPS, CHES, and CAPS are Good’s buffers and were purchased from Dojindo Laboratories) (34) containing 0.05% DDM, 0–4 M NaCl, and 0–1.333 M Na2SO4. The initial pH was approximately 3.8 and was then adjusted to the desired value by adding a very small amount of 2 M NaOH, followed by measurements of the UV-Vis absorption spectrum. The concentration change in the RmHR sample was almost negligible. The acid dissociation constant, pKa, of the protonated Schiff base of the retinal chromophore was estimated by fitting the data to the Henderson-Hasselbalch equation: Gene preparation and ion transport measurements of RmHR. ΔA=1/(1+10(pKa-pH)) where P0 and P1–P4 represent the unphotolyzed original pigment and the 1st–4th kinetically defined states, respectively. All data on time-dependent changes in absorbance were simultaneously fit with a sum of 4 exponential decay functions. The appropriate number of exponents was selected based on reductions in the standard deviation of the residuals. The P1–P4 states were allowed to contain a few physically defined photointermediates such as L, N, and O with individual absorption maxima when a quasi-equilibrium state existed between them. The time constants τ1–τ4 and the absorbance differences Δε1–Δε4 between the P1–P4 states and the original P0 state were assessed according to the fitting results. Independently, the pure retinal spectrum of the P0 state was extracted from the UV-Vis absorption spectrum of the initial state (unphotolyzed) RmHR by spectral decomposition using the skewed Gaussian function as described previously (10). Finally, the absolute spectra of each P1–P4 state were obtained by adding the P0 spectrum to each absor- bance difference Δε1–Δε4. Results and Discussion Genomic and phylogenetic analysis of R. marinus SG-29T, R. marina SAORIC-28T, and R. profundi SAORIC-476T. A previous study showed that R. marinus SG-29T has a 4.43-Mbp chromosome encoding 3847 CDSs (24). The present genome sequence analysis of R. marina SAORIC-28T and R. profundi SAORIC-476T revealed that these strains have 4.98 Mbp and 4.48 Mbp chromosomes encoding 4267 and 3842 CDSs, respectively (Table S1). The G+C contents of both strains from the genus Rubrivirga were two of the highest (72.5% and 71.3%) among the available genomes of class Cytophagia in the NCBI database. The genera Rubrivirga and Rubricoccus belong to the family Rhodothermaceae, and the 16S rRNA phylogenetic tree is shown in Fig. 1. This family contains members of thermophilic (genus Rhodothermus), halophilic (genera Salinibacter, Salisaeta, Longimonas, and Longibacter), and slightly halophilic (genera Rubricoccus and Rubrivirga, isolated from ocean) strains. Growth ranges for the NaCl concentration of thermophilic strains were similar to those of the genera Rubricoccus and Rubrivirga (Fig. 1). In contrast, halophilic bacteria in the family Rhodothermaceae grew under high-salinity conditions (20–30%). In addition, the lower limit of the growth range for NaCl of the genera Longimonas, Longibacter, and Salisaeta (2–4%), was lower than that of the genus Salinibacter (12–15%). The tempera- ture growth range for the genera Rubricoccus and Rubrivirga was approximately 4–42°C (Fig. 1). In contrast, halophilic Rhodothermaceae strains grew at 20–50°C, and this range was between the temperature growth range for thermophiles (50–85°C) and marine isolates (4–42°C). These physiologi- cal traits clearly showed that the family Rhodothermaceae contained two different types of extremophiles, indicating that the common ancestor of this family was a halophilic or thermophilic bacterium. Therefore, the genera Rubricoccus and Rubrivirga with members that inhabit the ocean may have a higher G+C content in class Cytophagia as a remnant of their ancestors. As described in the Results and Discussion section, the P3 state was obtained as a quasi-equilibrium mixture of N-like and O-like intermediates with relative fractions that were dependent on the Cl– concentration. P3 state spectra contained not only the main absorption bands of the N-like and O-like intermediates, but also the respective β-bands. The procedures for the spectral decomposition of the P3 state were the same as those described in previous studies (10, 29). Assuming both β-bands are identical to that of the P0 state, the spectrum of the P3 state is described as: ΔA=1/(1+10(pKa-pH)) salinarum R1, genes with an e-value less than 1e-10 were counted, and a threshold with a sequence identity >50% was established. We used the genome family Rhodothermaceae, Cytophaga hutchinsonii ATCC 33406T, which is a type species of the class Cytophagia, Indibacter alkaliphilus LW1T, Spirosoma linguale DSM 74T, N. marinus S1-08T, and Flavobacterium aquatile LMG 4008T. Since I. alkaliphilus and S. linguale, belonging to the class Cytophagia, have a rhodopsin gene, these genome data were used for this assay. Furthermore, because N. marinus is a marine flavobacterium with the ClR gene and F. aquatile is a type species of the class Flavobacteriia, these genome data were also used. with a Nd:YAG laser (5 mJ pulse–1, 532 nm, 7 ns) as an actinic light source as described previously (18, 35). Due to the setting for the response time (0.5 μs) of a homemade I-V converter, a large scattering artifact from the laser flash appeared before 10 μs. Therefore, plots were started from 10 μs. Time-dependent changes in absorbance were measured from 400 to 710 nm at 10-nm intervals. Data before the laser pulse were adopted as a baseline. In order to improve the signal-to-noise ratio, the results of 30 flashes were averaged at each wavelength. No further data processing was applied. The temperature was kept at 20°C using a thermostat. We analyzed the data using a sequential model based on our previous studies (10, 18) as follows: P0→P1→P2→P3→P4→P0 where P0 and P1–P4 represent the unphotolyzed original pigment and the 1st–4th kinetically defined states, respectively. All data on time-dependent changes in absorbance were simultaneously fit with a sum of 4 exponential decay functions. The appropriate number of exponents was selected based on reductions in the standard deviation of the residuals. The P1–P4 states were allowed to contain a few physically defined photointermediates such as L, N, and O with individual absorption maxima when a quasi-equilibrium state existed between them. The time constants τ1–τ4 and the absorbance differences Δε1–Δε4 between the P1–P4 states and the original P0 state were assessed according to the fitting results. Independently, the pure retinal spectrum of the P0 state was extracted from the UV-Vis absorption spectrum of the initial state (unphotolyzed) RmHR by spectral decomposition using the skewed Gaussian function as described previously (10). Finally, the absolute spectra of each P1–P4 state were obtained by adding the P0 spectrum to each absor- bance difference Δε1–Δε4. Spectrum of the P3 state=f×Abs(N,λ)+(1–f)×Abs(O,λ)+Abs(β,λ) where f, Abs (N, λ), Abs (O, λ), and Abs (β, λ) represent the fraction of the N-like intermediate, the main absorption band of the N-like intermediate, the main absorption band of the O-like intermediate, and the β-band, respectively. We employed skewed Gaussian functions to describe these three absorption bands and then decomposed the spectrum of the P3 state to estimate f, which we assumed to be the only parameter dependent on the Cl– concentration. The fractions of the N-like and O-like intermediates were plotted against the Cl– concentration, and data were simultaneously analyzed by the Hill equation as follows: ΔA=1/(1+10(pKa-pH)) where pH and ΔA are the adjusted pH value and absorption change at 380 nm, which corresponds to the deprotonated Schiff base, respectively.l where pH and ΔA are the adjusted pH value and absorption change at 380 nm, which corresponds to the deprotonated Schiff base, respectively.l Time-dependent changes in absorbance were measured by flash- photolysis using a homemade computer-controlled apparatus equipped 91 HR-Like Cl– Pump in Marine Bacteria order to compare gene synteny around the Cl–-pumping rhodopsin gene and blh gene, we acquired the genomes of H. salinarum R1, S. ruber M31T, Synechocystis sp. PCC 7506, Mastigocladopsis repens PCC 10914, Salisaeta longa DSM 21114T, and Rhodothermus marinus DSM 4252T from the NCBI RefSeq database. The ortholog clustering of the CDSs of each genome was performed using eggNOG-mapper (13) and the bactNOG dataset in the eggNOG database, version 4.5 (14). We visualized the operon structure and colored each gene based on each eggNOG ortholog cluster using R software. In order to evaluate the potential LGT, a BLAST analysis was performed on the genomes of H. salinarum R1, genes with an e-value less than 1e-10 were counted, and a threshold with a sequence identity >50% was established. We used the genome family Rhodothermaceae, Cytophaga hutchinsonii ATCC 33406T, which is a type species of the class Cytophagia, Indibacter alkaliphilus LW1T, Spirosoma linguale DSM 74T, N. marinus S1-08T, and Flavobacterium aquatile LMG 4008T. Since I. alkaliphilus and S. linguale, belonging to the class Cytophagia, have a rhodopsin gene, these genome data were used for this assay. Furthermore, because N. marinus is a marine flavobacterium with the ClR gene and F. aquatile is a type species of the class Flavobacteriia, these genome data were also used. order to compare gene synteny around the Cl–-pumping rhodopsin gene and blh gene, we acquired the genomes of H. salinarum R1, S. ruber M31T, Synechocystis sp. PCC 7506, Mastigocladopsis repens PCC 10914, Salisaeta longa DSM 21114T, and Rhodothermus marinus DSM 4252T from the NCBI RefSeq database. The ortholog clustering of the CDSs of each genome was performed using eggNOG-mapper (13) and the bactNOG dataset in the eggNOG database, version 4.5 (14). We visualized the operon structure and colored each gene based on each eggNOG ortholog cluster using R software. In order to evaluate the potential LGT, a BLAST analysis was performed on the genomes of H. Fraction of N-like (or O-like)=a+b·[Cl–]n/([Cl–]n+[Kd]n) where a, b, Kd, and n represent the offset, amplitude of the λmax change, dissociation constant for Cl–, and Hill coefficient, respectively. Phylogenetic and comparative genome analyses Multiple alignments of amino acid sequences were performed using the CLUSTALW option in MEGA 6.0 software (32). Phylogenetic trees were constructed using the maximum-likelihood (ML) method (5) with 1,000 bootstrap replications (6). The nucleotide sequences of 16S rRNA and the amino acid sequences of rhodopsins, 15,15'-β-carotene dioxygenase (blh), and lycopene cyclase (crtY and crtYcd) genes were collected from the NCBI database and RAST annotated sequences for the genera Rubricoccus and Rubrivirga. In 92 Nakajima et al. Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences. Phylogenetic tree of 16S rRNA showing the taxonomic positions of strains SG-29T, SAORIC-28T, and SAORIC-476T among related species of the family Rhodothermaceae in the classes Cytophagia and Sphingobacteria. The habitats of each strain are indicated by different-colored squares. The red and open circles indicate strains containing the rhodopsin gene and no rhodopsin gene, respectively. The G+C content and growth range for temperature and NaCl concentrations are also shown on the right side. Black closed circles indicate G+C content of each strain. ND: No genome data. + content Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences. Phylogenetic tree of 16S rRNA showing the taxonomic positions of strains SG-29T, SAORIC-28T, and SAORIC-476T among related species of the family Rhodothermaceae in the classes Cytophagia and Sphingobacteria. The habitats of each strain are indicated by different-colored squares. The red and open circles indicate strains containing the rhodopsin gene and no rhodopsin gene, respectively. The G+C content and growth range for temperature and NaCl concentrations are also shown on the right side. Black closed circles indicate G+C content of each strain. ND: No genome data. The genome sequence analysis showed that the genomes of R. marinus SG-29T and R. marina SAORIC-28T encoded two different types of rhodopsin genes (RmXeR [WP_094549673] and RmHR [WP_094550238]) and three rhodopsin genes (R28XeR [WP_095509440], R28HR1 [WP_095509924], and R28HR2 [WP_095512583]), respectively. The identities of amino acid sequences among RmHR, R28HR1, and R28HR2 were 73% (RmHR-R28HR1), 62% (RmHR-R28HR2), and 57% (R28HR1-R28HR2) respectively. No rhodopsin gene was found in the genome of R. profundi SAORIC-476T. Light-induced pH changes and ion selectivity of RmHR. In order to clarify the function of this unique rhodopsin cluster, codon-optimized RmHR, R28HR1, and R28HR2 genes were chemically synthesized and each gene was heterologously overexpressed in E. coli. Light-induced pH changes were observed in suspensions of E. coli cells expressing each gene in 100 mM NaCl. However, light-induced pH changes in R28HR1 were smaller than those in RmHR, and were not observed in R28HR2. Therefore, RmHR was used in subse- quent analyses. Light-induced alkalization was observed in 100 mM NaCl, and this pH change was not abolished by the addition of CCCP (Fig. 3A). Since CCCP is a protonophore, this alkalization may be explained by passive proton influx due to the negative membrane potential, which was created by outward Na+ or inward Cl– translocation. In order to eluci- date the ion selectivity of RmHR, we performed similar observations in different salt solutions (Fig. 3B). The signal almost disappeared in 100 mM NaI and Na2SO4, suggesting that RmHR cannot transport Na+. In contrast, strong signals were observed in 100 mM KCl, NaBr, and MgCl2, and a slight pH change was noted in 100 mM NaNO3. These anion-dependent transport properties are very similar to those of archaeal HRs in halophilic archaea, suggesting that RmHR functions as a light-driven inward anion (Cl–, Br–, and NO3–) pump. Fraction of N-like (or O-like)=a+b·[Cl–]n/([Cl–]n+[Kd]n) Although R28HR1 and R28HR2 were found from the deep- sea isolate, it currently remains unclear whether these are deep-sea adapted rhodopsins because the habitat of the genus Rubrivirga is not yet understood. In the BLASTP search against the metagenome database (env_nr), no sequence with >50% identity with RmHR was found. This result implies that the RmHR gene is very rare in the marine environment. In the family Rhodothermaceae, four strains (S. ruber M31T, S. longa DSM 21114T, R. marinus SG-29T, and R. marina SAORIC-28T) have rhodopsin genes. In contrast, no rhodopsin gene was found in the two thermophilic strains belonging to the genus Rhodothermus (Fig. 1). A recent study showed that RmXeR of SG-29T functions as a light-driven inward proton pump (17). R28XeR also belongs to the same cluster of RmXeR, suggesting that the function of R28XeR is an inward H+ pump. RmHR, R28HR1, and R28HR2 formed a unique cluster close to HR and SyHR (Fig. 2 and S1). Although HR and SyHR were discovered from halophilic prokaryotes and a freshwater cyanobacterium, RmHR, R28HR1, and R28HR2 formed a unique marine bacterial cluster. RmHR and R28HR1 had the same TSA motif as HR, and R28HR2 has TTD motif sequences (Table 1). In contrast, cyanobacterial HRs had the TSD, TSV, or TSL motif sequence (9). Spectroscopic analysis of RmHR. Spectroscopic analysis of RmHR. In order to investigate the spectroscopic properties of RmHR, we prepared a detergent DDM-solubilized sample. Under this condition, the results described below were able to be precisely compared to those of all the other anion-pumping rhodopsins investigated, such as Natronomonas pharaonis halorhodopsin (NpHR), H. salinarum halorhodopsin (HsHR), Nonlabens marinus S1-08T rhodopsin 3 (NM-R3), Synechocystis 93 HR-Like Cl– Pump in Marine Bacteria Fig. 2. Unrooted maximum-likelihood phylogenetic tree of microbial rhodopsins. (A) Amino acid sequences of microbial rhodopsins were aligned using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. All rhodopsin amino acid sequence data used in this study were obtained from the public database (http://www.ncbi.nlm.nih.gov/). Dark gray indicates the clade consisting of the freshwater cyanobacterial rhodopsin clade, and light gray indicates halorhodopsin. An open circle and closed gray circle indicate the positions of strain SG-29T and SAORIC-28T, respectively. Sodium ion pump rhodopsin (NaR); chloride ion pump rhodopsin (ClR); xanthorhodopsin-like rhodopsin (XLR); proteorhodopsin (PR); xenorhodopsin (XeR); sensory rhodopsin (SR); bacteriorhodopsin (BR); halorhodopsin (HR). The motif sequence (amino acid residues 85, 89, and 96 in BR numbering) is shown under the rhodopsin name. (B) Detailed phylogenetic tree of the SR, BR, HR, cyanobacterial HR, and RmHR homologues. The habitats of each strain are indicated by squares of different colors. Bootstrap values >80% are indicated as a percentage of the replicates tested. Asterisk represents the strain that was isolated from seawater by using medium for freshwater cyanobacteria. Fig. 2. Unrooted maximum-likelihood phylogenetic tree of microbial rhodopsins. (A) Amino acid sequences of microbial rhodopsins were aligned using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. All rhodopsin amino acid sequence data used in this study were obtained from the public database (http://www.ncbi.nlm.nih.gov/). Dark gray indicates the clade consisting of the freshwater cyanobacterial rhodopsin clade, and light gray indicates halorhodopsin. An open circle and closed gray circle indicate the positions of strain SG-29T and SAORIC-28T, respectively. Sodium ion pump rhodopsin (NaR); chloride ion pump rhodopsin (ClR); xanthorhodopsin-like rhodopsin (XLR); proteorhodopsin (PR); xenorhodopsin (XeR); sensory rhodopsin (SR); bacteriorhodopsin (BR); halorhodopsin (HR). Spectroscopic analysis of RmHR. The motif sequence (amino acid residues 85, 89, and 96 in BR numbering) is shown under the rhodopsin name. (B) Detailed phylogenetic tree of the SR, BR, HR, cyanobacterial HR, and RmHR homologues. The habitats of each strain are indicated by squares of different colors. Bootstrap values >80% are indicated as a percentage of the replicates tested. Asterisk represents the strain that was isolated from seawater by using medium for freshwater cyanobacteria. Table  1. Comparison of important residues, particularly motifs, among ion pumps. Residue numbers in BR (above) and PR (below) 85 86 87 88 89 90 91 92 93 94 95 96 Rhodopsin type Taxonomic group Motif 97 98 99 100 101 102 103 104 105 106 107 108 BR Halobacterium salinarum R1 DTD D W L F T L P L L L L D PR Ca. Pelagibacter ubique HTCC 1062 DTE D W L I T V P L L M L E ClR Nonlabens marinus S1-08T NTQ N W M A T I P C L L L Q SyHR Synechocystis sp. PCC 7506 TSD T W F L S T P L L L L D HR Salinibacter ruber M31T TSA T W A F S T P F I L L A HR Halobacterium salinarum R1 TSA T W A L S T P M I L L A RmXeR Rubricoccus marinus SG-29T DTA D W V V T T P L L L T A R28XeR Rubrivirga marina SAORIC-28T DTA D W V V T T P L L L A A RmHR Rubricoccus marinus SG-29T TSA T W F T S T P L L L L A R28HR1 Rubrivirga marina SAORIC-28T TSA T W F L S T P L L L L A R28HR2 Rubrivirga marina SAORIC-28T TTD T W F L T T P L L L L D Table  1. Comparison of important residues, particularly motifs, among ion pumps. sp. PCC 7509 halorhodopsin (SyHR), and Mastigocladopsis repens PCC 10914 halorhodopsin (MrHR) (11, 25, 33, 35, 42). Based on this background, we used DDM-solubilized RmHR in the subsequent spectroscopic analysis. We also focused on the photochemical properties of RmHR in the presence of NaCl because Cl– is considered to be a candidate substrate anion for RmHR in the native habitat of R. marinus SG-29T (Cl– concentration of approximately 500 mM). sp. Spectroscopic analysis of RmHR. coli cell suspensions expressing RmHR in solutions containing (A) 100 mM NaCl without (black solid line) and with (gray broken line) 30 μM CCCP and (B) 100 mM KCl, MgCl2, NaBr, NaI, NaNO3, and Na2SO4 without CCCP. The cell suspension was illuminated with green light (520 nm). Temperature was maintained at 4°C. The word “div.” represents division. dependency, Cl–-binding affinity in the initial state (practically the dissociation constant, Kd,int.) was estimated to be 7.6±1.7 mM by the Hill equation (Fig. S2C). The Hill coefficient was 1.2±0.23, indicating positive cooperativity for Cl– binding. The estimated Kd,int. value of RmHR was in the same order as those of NpHR, HsHR, and MrHR (2–6 mM) (10, 11, 18, 33, 42), but was approximately 100-fold lower than that of SyHR (approximately 0.1 mM) (25) and approximately 3- and 5-fold larger than those of NM-R3 (24 mM) and Fulvimarina pelagi HTCC 2506T rhodopsin (FR) (84 mM), respectively (16, 35). In addition, the acid dissociation constant, pKa, of the Schiff base was dependent on the Cl– concentration (Fig. S2D), which is similar to archaeal HsHR and NpHR (39). At low concentrations of NaCl (0 - several tens of mM), the absorption band of the deprotonated Schiff base appeared in the shorter wavelength region at approximately 380 nm (Fig. S2B). Therefore, subsequent experiments were performed in the presence of more than 100 mM NaCl. section). The P1 and P2 states each contained a blue-shifted photointermediate with a similar absorption maximum at 510 nm. Analogous to NpHR, we tentatively assigned these interme- diates as L1- and L2-like, respectively. The following P3 state contained at least two photointermediates with absorption maxima at 520 and 590 nm, which we tentatively assigned as N- and O-like intermediates, respectively, by referring to the P3 state of NpHR. In the case of NpHR, the molar ratio of N and O changed in a Cl– concentration-dependent manner (10, 18). In order to evaluate this phenomenon, we performed flash photolysis measurements on RmHR with varying Cl– concentrations. It is important to note that the Cl– concentration- dependent absorption change was significant in the P3 state (Fig. S4). Fig. S3D shows the Cl–-dependent change in the molar ratio of N-like and O-like intermediates between 100 mM and 4 M NaCl. The fraction of the N-like intermediate increased with elevations in the Cl– concentration, indicating that this intermediate is the Cl–-binding form. Spectroscopic analysis of RmHR. PCC 7509 halorhodopsin (SyHR), and Mastigocladopsis repens PCC 10914 halorhodopsin (MrHR) (11, 25, 33, 35, 42). Based on this background, we used DDM-solubilized RmHR in the subsequent spectroscopic analysis. We also focused on the photochemical properties of RmHR in the presence of NaCl because Cl– is considered to be a candidate substrate anion for RmHR in the native habitat of R. marinus SG-29T (Cl– concentration of approximately 500 mM). rhodopsin encoded in the R. marinus SG-29T gene (RmXeR) showed a light-dependent retinal composition change in the initial state (75 and 45% of all-trans retinal under dark and light conditions, respectively) (17). The biological significance of RmXeR currently remains unclear. In order to evaluate this feature in RmHR, we measured the UV-Vis absorption spectra under various Cl– concentrations ranging between 1 mM and 4 M (Fig. S2B). Many anion- pumping rhodopsins show a spectral blue-shift upon substrate anion binding. In the experiment, ionic strength was maintained at 4 M by adding Na2SO4 at appropriate concentrations because in many Cl–-pumping rhodopsins, Cl– binding reaches a plateau at 4 M and SO42– is not a substrate ion for RmHR, as shown in Fig. 3B. As shown in Fig. S2B, RmHR exhibited a Cl–- dependent spectral blue-shift from 550 nm in the presence of 1 mM Cl– to 542 nm in the presence of 4 M Cl–. Based on this We initially examined the retinal isomer composition of RmHR under dark and light conditions in the presence of 1 M NaCl. As shown in Fig. S2A, RmHR predominantly possesses all-trans retinal as a chromophore, which is responsible for the light-driven anion pump function, at 99.6 and 96.2% under dark and light conditions, respectively. This property is the same as those of the other anion-pumping rhodopsins reported to date (11, 25, 33, 35). On the other hand, another Nakajima et al. 94 Nakajima et al. 94 Fig. 3. Anion transport activity of RmHR. Light-induced pH changes in E. coli cell suspensions expressing RmHR in solutions containing (A) 100 mM NaCl without (black solid line) and with (gray broken line) 30 μM CCCP and (B) 100 mM KCl, MgCl2, NaBr, NaI, NaNO3, and Na2SO4 without CCCP. The cell suspension was illuminated with green light (520 nm). Temperature was maintained at 4°C. The word “div.” represents division. Fig. 3. Anion transport activity of RmHR. Light-induced pH changes in E. Spectroscopic analysis of RmHR. In contrast, the fraction of the O-like intermediate decreased with the increase in the Cl– concentration, indicating that the O-like intermediate is the Cl–-releasing form. Data were simultaneously fit by the skewed Gaussian function in order to identify the fractions of the N-like and O-like intermediates. As shown in Fig. S3E, we plotted these fractions against NaCl concentrations and then estimated the dissociation constant for Cl– release, Kd,rel, as 308±45.6 mM using the Hill equation. The value was approximately 40-fold larger than that of the initial state (Kd,int.=7.6 [mM]), the behavior of which was the same as NpHR, whereas the magnitude of the change was different (Kd,int.=2 [mM] and Kd,rel=1.2 [M]) (33). In the P4 state (Fig. S3C), a photointermediate with the same absorption maximum at 540 nm as the initial state (P0) was identified, and we named it the RmHR’-intermediate by referring to other anion-pumping rhodopsins, including NpHR (18, 35). Analogous to NpHR, in the O-like-to-RmHR’ transition, Cl– was entered into the initial binding site on the extracellular side of RmHR, which was supported by the blue-shifted absorption from O-like to RmHR’ and the same absorption maximum of RmHR’ as that of the initial state. We summarized the Cl– transporting pho- The Cl–-transporting photoreaction, called a photocycle, of RmHR was analyzed using a time-resolved flash-photolysis technique. Fig. S3A shows the flash-induced light-minus- dark difference absorption spectra in the 10-μs to 388-ms time domain. After flash excitation, photointermediates with absorption bands at approximately 460 nm and 610 nm, respectively, were generated with the concomitant disappear- ance of the initial state absorption band at approximately 550 nm. Time-dependent absorption changes in these three absorption bands are shown in Fig. S3B. Data were simulta- neously fit to the exponential decay function with 4 exponents, which was the same number as NpHR (10, 18), indicating that RmHR passed through 4 kinetically intermediate states, P1–P4, in its photocycle. The time constants between each transition, τ1–τ4, were 0.105, 0.483, 2.14, and 23.5 ms, respectively. According to the sequential model (10, 18), we calculated the absolute spectra of the P1–P4 states of RmHR in the presence of 1,000 mM NaCl, as shown in Fig. S3C. The spectrum of P0 corresponded to the initial state pure absorption spectrum of RmHR, which was extracted from the experimentally measured absorption spectrum (see the Materials and Methods HR-Like Cl– Pump in Marine Bacteria 95 tocycle model of RmHR in Fig. Phylogenetic analyses based on retinal biosynthesis pathway genes. Functional and spectroscopic analyses revealed that RmHR is a light-driven inward Cl–-pumping rhodopsin similar to the HR of halophilic archaea rather than to ClR (e.g., NM-R3 and FR) of marine flavobacteria. In order to further examine the evolutionary history of RmHR, we performed phylogenetic analyses on retinal biosynthesis pathway genes, such as crtY Table  2. Comparison of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. Rhodopsin (Type) Retinal isomer (dark/light) (%) Kd,int/Kd,rel (mM) Schiff base pKa Photo- intermediates Existence of N-like–O-like equilibrium Similarity of the photocycle scheme References RmHR (marine bacterial HR) 99.6/96.2 7.6/308 7.3 (0 M NaCl)– 10 (4 M NaCl) (K), L1, L2, N, O, RmHR’ Yes (This study) This study HsHR (archaeal HR) 50/86 2.6/N.D. Approx. 8 (10 mM NaCl)– Approx. 11.5 (below 1 M NaCl) K, L1, L2, N No Different from RmHR 38, 39, 42 NpHR (archaeal HR) 83/77 2/1200 Approx. 8 (10 mM NaCl)– Approx. 10 (below 1 M NaCl) (K), L1, L2, N, O, NpHR’ Yes Similar to RmHR 10, 18, 33, 37, 39 NM-R3 (marine bacterial ClR) 90.3/91.6 24/N.D. N.D. K, L, N, O1, O2, NM-R3’ No Different from RmHR 35 FR (marine bacterial ClR) 96.4/86.6 84/N.D. N.D. K, L, O, FR’ N.D. Different from RmHR 16 MrHR (cyanobacterial HR) >98/>98 1.99/N.D. N.D. (K), L, N, O, MrHR’ Yes Different from RmHR 9, 11 SyHR (cyanobacterial HR) N.D./97.5 0.1/N.D. 10.6 (1 M NaCl) K, L, N, O, SyHR’ Yes Similar to RmHR 25 N D indicates “Not determined” Table  2. Comparison of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. Rhodopsin (Type) Retinal isomer (dark/light) (%) Kd,int/Kd,rel (mM) Schiff base pKa Photo- intermediates Existence of N-like–O-like equilibrium Similarity of the photocycle scheme References RmHR (marine bacterial HR) 99.6/96.2 7.6/308 7.3 (0 M NaCl)– 10 (4 M NaCl) (K), L1, L2, N, O, RmHR’ Yes (This study) This study HsHR (archaeal HR) 50/86 2.6/N.D. Approx. 8 (10 mM NaCl)– Approx. 11.5 (below 1 M NaCl) K, L1, L2, N No Different from RmHR 38, 39, 42 NpHR (archaeal HR) 83/77 2/1200 Approx. 8 (10 mM NaCl)– Approx. 10 (below 1 M NaCl) (K), L1, L2, N, O, NpHR’ Yes Similar to RmHR 10, 18, 33, 37, 39 NM-R3 (marine bacterial ClR) 90.3/91.6 24/N.D. N.D. K, L, N, O1, O2, NM-R3’ No Different from RmHR 35 FR (marine bacterial ClR) 96.4/86.6 84/N.D. N.D. Spectroscopic analysis of RmHR. S3F. The initially captured Cl– on the extracellular side of RmHR was transferred to the intracellular side in the L2-like-to-N-like transition and was then released into the intracellular bulk space in the N-like- to-O-like transition. The next Cl– was entered into the initial binding site through the intracellular side of the molecule in the O-like to RmHR’ and RmHR’ to initial state transitions. The mechanism was similar to that of NpHR (18), but signifi- cantly different from that of the bacterial anion pumps, NM-R3 and FR, which are evolutionally distant from RmHR (Table 2) (Fig. 2) (16, 35). (lycopene cyclase) and blh (15,15'-β-carotene dioxygenase). The enzymes encoded by the crtY and blh genes catalyze the terminal cyclization reaction from all-trans-lycopene to all- trans-β-carotene and cleave β-carotene to produce two mole- cules of all-trans-retinal as a chromophore of rhodopsin. ML phylogenetic trees based on the blh and crtY genes are shown in Fig. 4 and S5. Regarding SAORIC-476T, since this strain lacks rhodopsin and blh genes, only the crtY gene was used in this analysis. In the phylogenetic tree of the blh gene, although each cluster was mainly formed according to the taxonomic group, the blh genes of SG-29T and SAORIC-28T in the class Cytophagia belonged to the lineage containing halophilic archaea and bacteria (Fig. 4). The phylogenetic tree based on crtY showed that three clusters: Bacteroidetes crtY and Proteobacteria crtY and crtYcd, had formed, and the genes of strains SG-29T, SAORIC-28T, and SAORIC-476T were included in the crtYcd cluster (Fig. S5). Carotenoid cyclases such as crtY are considered to be diversified by LGT or gene duplication, and some Actinobacteria (20), archaea (12), and Bacteroidetes (19) contain crtYcd. Although the Phylogenetic analyses based on retinal biosynthesis pathway genes. K, L, O, FR’ N.D. Different from RmHR 16 MrHR (cyanobacterial HR) >98/>98 1.99/N.D. N.D. (K), L, N, O, MrHR’ Yes Different from RmHR 9, 11 SyHR (cyanobacterial HR) N.D./97.5 0.1/N.D. 10.6 (1 M NaCl) K, L, N, O, SyHR’ Yes Similar to RmHR 25 N.D. indicates “Not determined”. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. (B) Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap values >80% are indicated as a percentage of the replicates tested. Table  2. Comparison of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. Rhodopsin (Type) Retinal isomer (dark/light) (%) Kd,int/Kd,rel (mM) Schiff base pKa Photo- intermediates Existence of N-like–O-like equilibrium Similarity of the photocycle scheme References RmHR (marine bacterial HR) 99.6/96.2 7.6/308 7.3 (0 M NaCl)– 10 (4 M NaCl) (K), L1, L2, N, O, RmHR’ Yes (This study) This study HsHR (archaeal HR) 50/86 2.6/N.D. Approx. 8 (10 mM NaCl)– Approx. 11.5 (below 1 M NaCl) K, L1, L2, N No Different from RmHR 38, 39, 42 NpHR (archaeal HR) 83/77 2/1200 Approx. 8 (10 mM NaCl)– Approx. 10 (below 1 M NaCl) (K), L1, L2, N, O, NpHR’ Yes Similar to RmHR 10, 18, 33, 37, 39 NM-R3 (marine bacterial ClR) 90.3/91.6 24/N.D. N.D. K, L, N, O1, O2, NM-R3’ No Different from RmHR 35 FR (marine bacterial ClR) 96.4/86.6 84/N.D. N.D. K, L, O, FR’ N.D. Different from RmHR 16 MrHR (cyanobacterial HR) >98/>98 1.99/N.D. N.D. (K), L, N, O, MrHR’ Yes Different from RmHR 9, 11 SyHR (cyanobacterial HR) N.D./97.5 0.1/N.D. 10.6 (1 M NaCl) K, L, N, O, SyHR’ Yes Similar to RmHR 25 N.D. indicates “Not determined”. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. Phylogenetic analyses based on retinal biosynthesis pathway genes. similarity between the crtYcd and crtY genes is not high, the crtYcd gene is in a carotenoid biosynthesis gene cluster and has been shown to encode an enzyme that functions as a lycopene cyclase-like crtY gene. Halophilic archaea, the genera Natronomonas, Halobacterium, and Haloarcula, and halophilic bacteria, S. ruber M31T and S. longa DSM 21114T, possess the crtYcd, but not crtY gene. Similar to the phylogenetic tree of the blh gene, the crtYcd genes of Rubricoccus and Rubrivirga were not close to the genes of the Bacteroidetes crtY clade, whereas these crtYcd genes were closely related to the halophilic archaeal ones. Although the family Rhodothermaceae belongs to the class Cytophagia in the phylum Bacteroidetes, blh and lycopene cyclase genes formed a phylogenetic lineage that is distinct from these genes in Bacteroidetes. In conclusion, we herein report a novel light-driven inward Cl–-pumping rhodopsin (RmHR), similar to the HR of halo- philic archaea, in marine isolates of the family Rhodothermaceae in the phylum Bacteroidetes. Spectroscopic assays also showed that the properties of RmHR resembled those of HR from the aspect of the intermediate states and mechanisms of Cl– uptake and release. Phylogenetic analyses revealed that strains of the genera Rubricoccus and Rubrivirga may have acquired some genes by LGT, including the rhodopsin and blh genes from halophilic archaea. These results suggest that these genera and halophilic archaea had once lived in the same environment. Collectively, the present results provide insights into gene sharing between marine bacteria and halophilic archaea and facilitate our understanding of the evolutionary processes occurring in the ecologically diverse environments of this family. Previous genomic and phylogenetic analyses showed that S. ruber M31T acquired several genes from halophilic archaea via LGT (23). Gene transfer beyond a domain may have occurred because Salinibacter and halophilic archaea dominate in the same niche, such as a hypersaline environment. The best example of this gene transfer is the HR gene shared by Salinibacter and halophilic archaea. In addition, not only this case, but also other genes (e.g., blh, crtYcd, the cytochrome c oxidase gene, and nitrous oxide reductase gene) were shared among halophilic bacteria and archaea. Our results based on the phylogenetic analysis suggest that the rhodopsin gene and its related genes were shared not only between Salinibacter and halophilic archaea, but also among those halophiles and marine bacteria (R. marinus SG-29T and R. marina SAORIC- 28T). References 1. Alfredsson, G.A., J.K. Kristjansson, S. Hjörleifsdottir, and K.O. Stetter. 1988. Rhodothermus marinus, gen. nov., sp. nov., a thermophilic, halophilic bacterium from submarine hot springs in Iceland. Microbiology 134:299–306. Visualizing genomic flanking regions of rhodopsin and blh genes. The results showing that several genes were shared among the halophiles, Rubricoccus and Rubrivirga, indicated that a common ancestor of strains SG-29T, SAORIC-28T, and SAORIC-476T inhabited the same environment, such as salt lakes, as halophiles. In order to clarify whether other genes were acquired together with the rhodopsin and blh genes, we compared the genomic flanking regions of these genes among the genera Rubricoccus and Rubrivirga and the strains pos- sessing genes closely related to this rhodopsin or the blh gene (Fig. S6). The results of the analysis showed that there was low similarity around rhodopsin genes (Fig. S6A). This dissimilarity may suggest that only Cl–-pumping rhodopsin genes were acquired by LGT or that a gene transfer event has not recently occurred. The same analysis based on the blh gene also showed no remarkable similarity to the flanking regions of the rhodopsin gene. The flanking regions of R. profundi SAORIC-476T lacking the rhodopsin and blh genes showed the presence of multiple conserved genes between SAORIC-28T and SAORIC-476T (Fig. S6B). This result sug- gested that only the blh gene was deleted in SAORIC-476T or acquired in SAORIC-28T. The results of a BLAST analysis revealed that although the numbers of potential LGT of strains SG-29T, SAORIC-28T, and SAORIC-476T were lower than that of S. ruber M31T, these numbers were approximately 2- to 4-fold higher than other phylum Bacteroidetes strains 2. Antón, J., R. Rosselló-Mora, F. Rodríguez-Valera, and R. Amann. 2000. Extremely halophilic bacteria in crystallizer ponds from solar salterns. Appl. Environ. Microbiol. 66:3052–3057. pp 3. Antón, J., A. Oren, S. Benlloch, F. Rodríguez-Valera, R. Amann, and R. Rosselló-Mora. 2002. Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. Int. J. Syst. Evol. Microbiol. 52:485–491. 4. Beja, O., L. Aravind, E.V. Koonin, et al. 2000. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289:1902– 1906. 5. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum-likelihood approach. J. Mol. Evol. 17:368–376.i 6. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791. g 7. Fuhrman, J.A., M.S. Schwalbach, and U. Stingl. 2008. Phylogenetic analyses based on retinal biosynthesis pathway genes. Furthermore, these results indicate that the common ancestor of the genera Rubricoccus and Rubrivirga acquired rhodopsin and its related genes from halophilic archaea via LGT in a hypersaline environment, as reported previously (23). Acknowledgements We are grateful to the captains and crews of R/V Mirai (JAMSTEC) and R/V Tansei Maru (Atmosphere and Ocean Research Institute, The University of Tokyo and JAMSTEC) for their assis- tance and support with sample collection. This work was supported by the Japan Society for the Promotion of Science (JSPS) to TT (15K18519) and SY (15H02800) and the Interdisciplinary Collaborative Research Program of the Atmosphere and Ocean Research Institute, the University of Tokyo. This work was also supported by JST- CREST and AMED to YS. Phylogenetic analyses based on retinal biosynthesis pathway genes. (B) Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap values >80% are indicated as a percentage of the replicates tested. n of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. N.D. indicates “Not determined”. N.D. indicates “Not determined”. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. (B) Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap values >80% are indicated as a percentage of the replicates tested. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. (B) Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap values >80% are indicated as a percentage of the replicates tested. 96 Nakajima et al. (Fig. S7). This result suggests that genes in strains SG-29T, SAORIC-28T, and SAORIC-476T may be shared with H. salinarum, similar to S. ruber. similarity between the crtYcd and crtY genes is not high, the crtYcd gene is in a carotenoid biosynthesis gene cluster and has been shown to encode an enzyme that functions as a lycopene cyclase-like crtY gene. Halophilic archaea, the genera Natronomonas, Halobacterium, and Haloarcula, and halophilic bacteria, S. ruber M31T and S. longa DSM 21114T, possess the crtYcd, but not crtY gene. Similar to the phylogenetic tree of the blh gene, the crtYcd genes of Rubricoccus and Rubrivirga were not close to the genes of the Bacteroidetes crtY clade, whereas these crtYcd genes were closely related to the halophilic archaeal ones. Although the family Rhodothermaceae belongs to the class Cytophagia in the phylum Bacteroidetes, blh and lycopene cyclase genes formed a phylogenetic lineage that is distinct from these genes in Bacteroidetes. References Kato, R. Abe-Yoshizumi, and H. Kandori. 2014. Spectroscopic study of a light-driven chloride ion pump from marine bacteria. J. Phys. Chem. B. 118:11190–11199. 32. Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Kumar. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30:2725–2729. 17. Inoue, S., S. Yoshizawa, Y. Nakajima, K. Kojima, T. Tsukamoto, T. Kikukawa, and Y. Sudo. 2018. 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Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208–218. g 22. Matsuno-Yagi, A., and Y. Mukohata. 1980. ATP synthesis linked to light-dependent proton uptake in a red mutant strain of Halobacterium lacking bacteriorhodopsin. Arch. Biochem. Biophys. 199:297–303. 38. Váró, G., L. Zimányi, X. Fan, L. Sun, R. References Opinion- proteorhodopsins: an array of physiological roles? Nat. Rev. Microbiol. 6:488–494. 8. Ghai, R., L. Pašić, A.B. Fernández, et al. 2011. New abundant microbial groups in aquatic hypersaline environments. Sci. Rep. 1:135. 9. Harris, A., M. Saita, T. Resler, A. Hughes-Visentin, R. Maia, F. Pranga-Sellnau, A.N. Bondar, J. Heberle, and L.S. Brown. 2018. Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin. Phys. Chem. Chem. Phys. 20:3184–3199. 10. Hasegawa, C., T. Kikukawa, S. Miyauchi, A. Seki, Y. Sudo, M. Kubo, M. Demura, and N. Kamo. 2007. Interaction of the halobacterial transducer to a halorhodopsin mutant engineered so as to bind the transducer: Cl– circulation within the extracellular channel. Photochem. Photobiol. 83:293–302. 11. Hasemi, T., T. Kikukawa, N. Kamo, and M. Demura. 2016. Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump. J. Biol. Chem. 291:355–362. HR-Like Cl– Pump in Marine Bacteria 97 28. Park, S., J. Song, S. Yoshizawa, A. Choi, J.C. Cho, and K. Kogure. 2013. Rubrivirga marina gen. nov., sp. nov., a member of the family Rhodothermaceae isolated from deep seawater. Int. J. Syst. Evol. Microbiol. 63:2229–2233. 12. Hemmi, H., S. Ikejiri, T. Nakayama, and T. Nishino. 2003. Fusion-type lycopene beta-cyclase from a thermoacidophilic archaeon Sulfolobus solfataricus. Biochem. Biophys. Res. Commun. 305:586–591. 13. Huerta-Cepas, J., D. Szklarczyk, K. 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Management of children's urinary tract infections in Dutch family practice: a cohort study
BMC family practice
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BioMed Central BioMed Central Management of children s urinary tract infections in Dutch family practice: a cohort study Mirjam Harmsen*1, Michel Wensing1, Jozé CC Braspenning1, René J Wolters1, Johannes C van der Wouden2 and Richard PTM Grol1 Address: 1Centre for Quality of Care Research (WOK), Radboud University Nijmegen Medical Centre, PO Box 9101 (114 kwazo), 6500 HB Nijmegen, The Netherlands and 2Department of General Practice, Erasmus MC – University Medical Center Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands Email: Mirjam Harmsen* - M.Harmsen@kwazo.umcn.nl; Michel Wensing - M.Wensing@kwazo.umcn.nl; Jozé CC Braspenning - J.Braspenning@kwazo.umcn.nl; René J Wolters - R.Wolters@kwazo.umcn.nl; Johannes C van der Wouden - J.vanderWouden@erasmusmc.nl; Richard PTM Grol - R.Grol@kwazo.umcn.nl * Corresponding author Received: 17 July 2006 Accepted: 13 March 2007 BMC Family Practice 2007, 8:9 doi:10.1186/1471-2296-8-9 This article is available from: http://www.biomedcentral.com/1471-2296/8/9 ; 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. BMC Family Practice Open Access Abstract Background: Optimal clinical management of childhood urinary tract infections (UTI) potentiates long-term positive health effects. Insight into the quality of care in Dutch family practices for UTIs was limited, particularly regarding observation periods of more than a year. Our aim was to describe the clinical management of young children's UTIs in Dutch primary care and to compare this to the national guideline recommendations. Methods: In this cohort study, all 0 to 6-year-old children with a diagnosed UTI in 2001 were identified within the Netherlands Information Network of General Practitioners (LINH), which comprises 120 practices. From the Dutch guideline on urinary tract infections, seven indicators were derived, on prescription, follow-up, and referral. Results: Of the 284 children with UTI who could be followed for three years, 183 (64%) were registered to have had one cystitis episode, 52 (18%) had two episodes, and 43 (15%) had three or more episodes. Another six children were registered to have had one or two episodes of acute pyelonephritis. Overall, antibiotics were prescribed for 66% of the children having had ≤ 3 cystitis episodes, two-thirds of whom received the antibiotics of first choice. About 30% of all episodes were followed up in general practice. Thirty-eight children were referred (14%), mostly to a paediatrician (76%). Less than one-third of the children who should have been referred was actually referred. Conclusion: Treatment of childhood UTIs in Dutch family practice should be improved with respect to prescription, follow-up, and referral. Quality improvement should address the low incidence of urinary tract infections in children in family practice. Page 1 of 7 (page number not for citation purposes) BMC Family Practice 2007, 8:9 http://www.biomedcentral.com/1471-2296/8/9 http://www.biomedcentral.com/1471-2296/8/9 Design and setting A prospective cohort study in Dutch family practice was performed. Study population We identified all children 0–6 years old (born between 1994 and 2001) with UTI diagnosed in 2001 within the Netherlands Information Network of General Practice (LINH). The LINH network contains 120 practices and is representative of the Dutch population of patients, family practitioners, and types of practices [11]. Informed con- sent was arranged within the network (general board of the National Institute for Health Services Research (NIVEL), general board of the Centre for Quality of Care Research (WOK), general board of the Dutch College of General Practitioners (NHG), and the general board of the National Association of General Practitioners (LHV)). According tothe Dutch Central Committee on Research Involving Human Subjects (CCMO) regulations only research in which the study participant has to be physi- cally present during the study is subject to the Medical Research Involving Human Subjects Act (WMO) and therefore ethical approval is not required for studies that use patient databases. The guideline on UTIs of the Dutch College of General Practitioners (DCGP) states that every childhood UTI should be treated with antibiotics because of the risk of renal scarring. Amoxicillin/clavulanic acid or co-trimoxa- zole are the medications of choice in an attempt to reach effective tissue levels and to maximize the chance of the most effective medicine until test results are available. The follow-up recommendation takes into account that young children may not express their complaints clearly, while they have a high risk of renal scarring. The referral recom- mendations are based on the patients' age and sex, which predict the probability of anatomical abnormalities of the urinary tract system [9]. The International Classification of Primary Care (ICPC) [12] defines UTI as acute pyelonephritis (ICPC code U70) or cystitis (ICPC code U71). These definitions imply that UTI was diagnosed by urine testing, not just suspected or assumed. The one-year incidence of UTIs in 0 to 6-year-old children in Dutch family practices averages for girls 25.7 and for boys 4.5 per 1000 life-years [10]. Insight into the quality of care for UTI was limited, particularly regarding observa- tion periods of more than a year. We have acquired the necessary prospective data to provide insight into the pri- mary-care-based management of childhood UTIs in the Netherlands. We aimed to describe the clinical manage- ment of young children's UTIs in Dutch primary care and compare this to the national guideline recommendations. Methods Design and setting g Awareness of the importance of timely diagnosis and treatment of childhood urinary tract infections (UTIs) is growing. Without timely treatment, renal scarring can occur [1], which is likely to affect approximately 5–15% of young children with a UTI [2-5]. Renal scarring is associ- ated with serious health problems in later life, such as hypertension, complications during pregnancy, and renal failure [1]. Optimal clinical management of childhood UTI potentiates long-term positive health effects. There- fore, guidelines across different countries advocate an active approach concerning prescription, follow-up, and referral [6-9]. Follow-up Twenty-eight percent to 37% of all episodes was followed- up (Table 3). The overall follow-up rate was 32%. About 60% of all episodes with follow-up contacts consisted of two contacts. The second contact took place within 14 days of the first for 90% of the episodes. There were no differences for age or sex groups. Student's t-test or chi-square tests, as appropriate, were used to investigate whether more boys or girls were treated as recommended by the guideline, and whether younger children were treated more consistently with the guideline than older children. We also investigated whether recur- rent childhood UTIs were more often treated according to the guideline than single episodes. We considered a prob- ability level of P < 0.05 statistically significant. Prescriptions Table 2 shows that, overall, 66% of the children received antibiotics, varying from 61% to 70%. First choice medi- cation, Amoxicillin/clavulanic acid or co-trimoxazole, was prescribed for 55% to 83% of the children with antibiotics (overall 66%). Another 4% to 25% (overall 13%) of the children with antibiotics received ceftibuten, ofloxacin, or nitrofurantoin. Since childhood UTIs should be treated as complicated UTIs according to the DCGP-guideline, these last three antibiotics should not be prescribed according to the guidelines. Furthermore, 10% to 25% (overall 19%) of the children with antibiotics received amoxicillin without clavulanic acid, which is also not according to the DCGP-guideline. In eight cases, a child who did not receive medication was referred to a medical specialist directly after seeing the GP. No differences in prescription for age or sex were found in any group. Descriptive statistics were applied to patient characteris- tics, and the numbers of children receiving medication, follow-up, or referrals. For each indicator, the number of children (or episodes in the case of follow-up) to whom family practitioners (FPs) offered the appropriate care was divided by the total number of children (or episodes in the case of follow-up) needing the provision of such care (Table 1). We calculated the percentage of children with more than one contact during a UTI episode as an indica- tor of follow-up. Age groups were based on age when hav- ing the first UTI. Measurements Although there is no specific DCGP guideline on UTIs in children, the Dutch UTI guideline does include specific recommendations for children [13]. We derived seven clinical indicators of appropriate performance. Two indi- cators focused on medication, one on follow-up, and four on referring (Table 1). Page 2 of 7 (page number not for citation purposes) Table 1: Indicators urinary tract infections (UTIs) in children in general practice Indicator Measurement Numerator Denominator First-choice antibiotics 1. Antibiotics given All children receiving antibiotics All children 2. Amoxicillin/clavulanic acid OR co- trimoxazole Children receiving first-choice antibiotics All children receiving antibiotics Follow-up 3. Episodes with at least one follow-up contact Number of UTI episodes with >1 contact All UTI episodes Referral For all groups: Total referred within the group Total within the group 4. Children <1 year old 5. Boys <12 years old 6. Girls 1–4 years old with second UTI 7. Girls 5–12 years old with > 1 recurrent UTI Table 1: Indicators urinary tract infections (UTIs) in children in general practice Table 1: Indicators urinary tract infections (UTIs) in children in general practice Numerator Denominator All children All children receiving antibiotics All UTI episodes Total within the group All children All children receiving antibiotics All UTI episodes Total within the group Number of UTI episodes with >1 contact For all groups: Total referred within the group Page 2 of 7 (page number not for citation purposes) BMC Family Practice 2007, 8:9 http://www.biomedcentral.com/1471-2296/8/9 ences between included children and children excluded in step 2 of the flow chart. Analyses The contacts were expressed in units of episodes. Episodes were considered new episodes if the preceding contact for UTI occurred more than 28 days previously. Prescriptions and referrals within 28 days after the last contact for an episode were linked to that episode. The first UTI in 2001 was assumed to be the child's first episode ever. To be able to compare groups, four groups of children were created, based on ICPC code and number of UTI episodes: group 1 (1 episode cystitis), group 2 (2 episodes cystitis), group 3 (≥ 3 episodes cystitis) and group 4 (1 or 2 episodes acute pyelonephritis). Referrals Table 3 shows that overall 38 of 278 (14%) children were referred for specialist treatment (range: 8–16%). Seventy- six percent was referred to a paediatrician; 8% to a urolo- gist; 3% to radiography; and from 13% it is not clear to what specialist they were referred. Data collection In the LINH network, the family practice staff routinely records the encoded patient information in electronic medical records (EMR). For the period 2001–2003 data were extracted from the EMR, concerning contacts with the family practice, prescriptions, referrals, and patient characteristics (age, sex). Of the 284 children included, 278 (98%) were diagnosed with cystitis. Of these children, 66% had one episode (group 1), 19% had two (group 2), and 15% had 3 to 10 (group 3). Six children were diagnosed with acute pyelonephritis (2%; group 4). Practices were excluded from the analyses if they had reg- istered fewer than 46 weeks in 2002 or 2003. Patients who were not on the practice list and patients who had left the practice before 1 January 2004 were also excluded. During the first UTI episode, the mean age varied from 2.8 (SD 2.5) to 4.1 years (SD 1.5). About 80% of the children in group 1, 90% in groups 2 and 3, and 100% in group 4 were girls (figure 1). Because of the small numbers, no fur- ther data are presented for group 4 and for episodes 4–10 in group 3. Page 3 of 7 (page number not for citation purposes) Study population Less than one-third of the children who should have been referred concerning the guideline on referring specific age and/or sex categories, was actually referred. Three of four children younger than one year were referred, and fewer than 25% of the boys were referred during at least one epi- sode. In group 2, 9% of the girls aged 1–4 years were Figure 1 shows the selection of children included in this study. Of 38,408 children in the 120 practices in the year 2001, 461 from 92 practices had a diagnosed UTI (1.2%). Of these 461 children, 284 (62%) in 59 practices could be followed for three years. There were no age or sex differ- Page 3 of 7 (page number not for citation purposes) Page 3 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2296/8/9 BMC Family Practice 2007, 8:9 of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standar TI = urinary tract infection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standar TI = urinary tract infection. Study population All children born between 1994 and 2001 (n=38,408) All children with UTI in 2001 (n=461) Exclusion of children not having had a UTI All children with UTI completely registered for the years 2001- 2003 (n=284) Exclusion of practices with incomplete registration and patients not belonging to the practice or with incomplete registration Group 1: Children with 1 episode cystitis/ UTI (n=183) 78% female mean (sd) age: 3.8 (1.6) years Group 2: Children with 2 episodes cystitis/UTI (n=52) 92% female mean (sd) age: 3.6 (1.4) years Group 3: Children with 3 or more episodes cystitis/UTI (n=43) 93% female mean (sd) age: 4.1 (1.5) years Group 4: Children with acute pyelonephritis (n=6) 100% female mean (sd) age: 2.8 (2.5) years All children born between 1994 and 2001 (n=38,408) Exclusion of children not having had a UTI Exclusion of practices with incomplete registration and patients not belonging to the practice or with incomplete registration All children with UTI completely registered for the years 2001- 2003 (n=284) Group 3: Children with 3 or more episodes cystitis/UTI (n=43) 93% female mean (sd) age: 4.1 (1.5) years Group 3: Children with 3 or more episodes cystitis/UTI (n=43) Group 4: Children with acute pyelonephritis (n=6) Group 2: Children with 2 episodes cystitis/UTI (n=52) Group 1: Children with 1 episode cystitis/ UTI (n=183) 78% female mean (sd) age: 3.8 (1.6) years Group 1: Children with 1 episode cystitis/ UTI Group 1: Children with 1 episode cystitis/ UTI (n=183) 78% female mean (sd) age: 3.8 (1.6) years Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi- ation; UTI = urinary tract infection Figure 1 Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi- ation; UTI = urinary tract infection. Page 4 of 7 (page number not for citation purposes) Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi- ation; UTI = urinary tract infection Figure 1 Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi- ation; UTI = urinary tract infection. Study population Page 4 of 7 (page number not for citation purposes) Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi- ation; UTI = urinary tract infection Figure 1 Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi- ation; UTI = urinary tract infection. http://www.biomedcentral.com/1471-2296/8/9 BMC Family Practice 2007, 8:9 Table 2: Prescription of antibiotics in children with urinary tract infections Group 1 (183 children) Group 2 (52 children) Group 3 (43 children) Total (278 children) Percentage (number) of children/antibiotic prescription Overall children with antibiotics (indicator 1) 66 (121/183) 70 (73/104) 61 (79/129) 66 (273/416) First episode 66 (121/183) 73 (38/52) 67 (29/43) 68 (188/278) Second episode - 67 (35/52) 63 (27/43) 65 (62/95) Third episode - - 53 (23/43) 53 (23/43) Percentage (number) of children with antibiotics/choice of antibiotic Amoxicillin/clavulanic acid or co-trimoxazole Overall first-choice antibiotics (indicator 2) 65 (79/121) 56 (41/73) 76 (60/79) 66 (180/273) First episode 65 (79/121) 55 (21/38) 69 (20/29) 64 (120/188) Second episode - 57 (20/35) 78 (21/27) 66 (41/62) Third episode - - 83 (19/23) 83 (19/23) Ceftibuten, ofloxacin, or nitrofurantoin Overall 7 (9/121) 21 (15/73) 14 (11/79) 13 (35/273) First episode 7 (9/121) 18 (7/38) 14 (4/29) 11 (20/188) Second episode - 23 (8/35) 15 (4/27) 19 (12/62) Third episode - - 13 (3/23) 13 (3/23) Amoxicillin Overall 25 (30/121) 18 (13/73) 10 (8/79) 19 (51/273) First episode 25 (30/121) 18 (7/38) 17 (5/29) 22 (42/188) Second episode - 17 (6/35) 7 (2/27) 13 (8/62) Third episode - - 4 (1/23) 4 (1/23) Table 2: Prescription of antibiotics in children with urinary tract infections increased risk of complications or underlying pathology in boys and younger children. Such unawareness may lead to health complications when the child is older. increased risk of complications or underlying pathology in boys and younger children. Such unawareness may lead to health complications when the child is older. referred during the second episode, and in group 3 33%. Two girls in the 5–9 year olds group (7%) were referred during the third episode. No differences regarding age and sex subgroups were found. Study population The LINH network provided a unique opportunity for col- lecting prospective data regarding clinical management in routine healthcare settings, but one can question FP regis- tration behaviour and whether all childhood UTIs were identified with the ICPC codes 'acute pyelonephritis' and 'cystitis'. However, the incidence we found for 0 to 6-year- olds, which is 12.0 (461*1000/38408), is comparable to those in other Dutch studies: 15.1 [10] and 13.2 [14]. Since direct observation and hand-searching medical records are infeasible, using databases of consultation reg- istrations seems to be the optimal method for collecting information about FP clinical behaviour. Page 5 of 7 (page number not for citation purposes) Discussion This study showed that the management of childhood UTIs in the Netherlands varied substantially across patients. Only 66% of the children received antibiotics and of these 66% was prescribed first choice antibiotics. There was no follow-up in the majority of the episodes. Referral of children younger than one year was generally consistent with the guidelines, but the referral rates for boys, girls 1–4 years old with a second UTI, and girls 5–12 years old with more than one recurrent UTI should have been much higher, if we consider the guidelines. It is difficult to compare our results on FP management with other studies because our data are prospectively col- lected, had a follow-up period of three years, and focussed on primary care and individual young children; this in contrast to other studies. A study by Kwok et al. [15] already gave some insight in the management of chil- dren's UTIs in Dutch family practice. But compared to our study, this study concentrated on a much wider age range, although the children most vulnerable to renal scarring are the younger ones. The study also had a follow-up period of only one year and did not pay attention to fol- low-up after the antiobiotic treatment. One British study found that 37% of children with proven UTI were sent for renal tract imaging [16], and another Dutch study We found that the proportion of children receiving amox- icillin decreased proportionally to the number of epi- sodes. Perhaps FPs prescribe amoxicillin routinely because this medication is much older than the combined form with clavulanic acid, and has less side effects than amoxicillin/clavulanic acid. If amoxicillin alone does not work, they prescribe the combination. Prescribing ceftib- uten, ofloxacin, or nitrofurantoin suggests that not all FPs are aware that, according to the guidelines, childhood UTI should be treated as complicated UTI. This is confirmed by the fact that, for 98% of all children, the FP had regis- tered the ICPC for cystitis instead of the code for compli- cated UTI (pyelonephritis). Conclusion In order to prevent negative health outcomes, treatment of childhood urinary tract infections in Dutch family prac- tice should be improved with respect to prescription, fol- low-up, and referral. Quality improvement should address the low incidence of urinary tract infections in children in family practice. 2. Pylkkänen J, Vilska J, Koskimies O: The value of level diagnosis of childhood urinary tract infection in predicting renal injury. Acta Paediatr Scand 1981, 70:879-883. 3. Vernon SJ, Coulthard MG, Lambert HJ, Keir MJ, Matthews JNS: New renal scarring in children who at age 3 and 4 had had normal scans with dimercaptosuccinic acid: Follow up study. Brit Med J 1997, 315:905-908. J , 4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln K: Epidemiology of symptomatic urinary tract infection in childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. J 4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln K: Epidemiology of symptomatic urinary tract infection in childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. J 4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln K: Epidemiology of symptomatic urinary tract infection in childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. 5. Coulthard MG, Lambert HJ, Keir MJ: Occurence of renal scars in children after their first referral for urinary tract infection. Brit Med J 1997, 315:918-919. Authors' contributions Improvement of professional performance might sub- stantially improve clinical outcomes. This is demon- strated in Sweden, where a more aggressive approach led to no new cases of uraemia caused by non-obstructive pyelonephritis during the years 1986–1995 [20]. But, development and distribution of guidelines do not neces- sarily lead to better patient care [21]. Future research could focus on developing interventions to improve pre- scription, follow-up, and referrals, but should also con- sider motives for not following the guidelines. Because not many childhood UTIs appear in family practice in the Netherlands – our study saw an average of five children per practice in one year – interventions should not be too time consuming for the FPs. MH had primary responsibility for protocol development, analyses, and writing the manuscript. MW, JCCB, RJW, and JCvdW participated in the development of the proto- col and contributed to the writing of the manuscript. RG supervised the design and execution of the study and con- tributed to the writing of the manuscript. All authors have read and approved the final manuscript. Competing interests Competing interests The author(s) declare that they have no competing inter- ests. References 1. Jacobson SH, Eklöf O, Eriksson CG, Lins LE, Tidgren B, Winberg J: Development of hypertension and ureaemia after pyelone- phritis in childhood: 27 year follow up. Brit Med J 1989, 299:703-706. 1. Jacobson SH, Eklöf O, Eriksson CG, Lins LE, Tidgren B, Winberg J: Development of hypertension and ureaemia after pyelone- phritis in childhood: 27 year follow up. Brit Med J 1989, 299:703-706. 2. Pylkkänen J, Vilska J, Koskimies O: The value of level diagnosis of childhood urinary tract infection in predicting renal injury. Acta Paediatr Scand 1981, 70:879-883. 3. Vernon SJ, Coulthard MG, Lambert HJ, Keir MJ, Matthews JNS: New renal scarring in children who at age 3 and 4 had had normal scans with dimercaptosuccinic acid: Follow up study. Brit Med J 1997, 315:905-908. 4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln K: Epidemiology of symptomatic urinary tract infection in childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. 5. Coulthard MG, Lambert HJ, Keir MJ: Occurence of renal scars in children after their first referral for urinary tract infection. Brit Med J 1997, 315:918-919. Acknowledgements The Dutch Kidney Foundation (Nederlandse Nierstichting) provided a f hi d ( j OV02) W h k J M ld d H k d The Dutch Kidney Foundation (Nederlandse Nierstichting) provided a grant for this study (project OV02). We thank Jan Mulder and Henk van den Hoogen for their help with the data analyses. Discussion No significant differences of age or gender might imply that GPs are unaware of the Page 5 of 7 (page number not for citation purposes) Page 5 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2296/8/9 BMC Family Practice 2007, 8:9 Table 3: Follow-up and referrals in children with urinary tract infections Group 1 (183 children) Group 2 (52 children) Group 3 (43 children) Total (278 children) Pecentage (number) of episodes with more than one contact during the episode Overall episodes with more than one contact (indicator 3) 28 (52/183) 34 (35/104) 36 (46/129) 32 (133/416) First episode 28 (52/183) 35 (18/52) 33 (14/43) 30 (84/278) Second episode - 33 (17/52) 37 (16/43) 35 (33/95) Third episode - - 37 (16/43) 37 (16/43) Percentage (number) of children with a referral Overall 8 (14/183) 19 (10/52) 33 (14/43) 14 (38/278) First episode 8 (14/183) 12 (6/52) 9 (4/43) 9 (24/278) Second episode - 8 (4/52) 16 (7/43) 12 (11/95) Third episode - - 7 (3/43) 7 (3/43) Percentage (number) of children with a referral, special groups of children Children <1 year of age (indicator 4) 67 (2/3) 100 (1/1) 0 (0/0) 75 (3/4) Boys overall (indicator 5) 18 (7/40) 25 (1/4) 0 (0/3) 17 (8/47) Girls overall 5 (7/143) 19 (9/48) 38 (15/40) 13 (31/231) Girls 1–4 years, second episode (indicator 6) - 9 (2/22) 33 (7/21) 21 (9/43) Girls 5–9 years, third episode (indicator 7) - - 7 (2/27) 7 (2/27) Table 3: Follow-up and referrals in children with urinary tract infections Table 3: Follow-up and referrals in children with urinary tract infections = International Classification of Primary Care; LINH = Netherlands Information Network of General Practice; UTI = urinary tract infection = International Classification of Primary Care; LINH = Netherlands Information Network of General Practice; UTI = urinary tract infection reported 4% of the children being referred [14]. Two other British studies found much higher rates of referral [17,18]. However, these last two studies used postal question- naires to measure FP behaviour, whereas our study and the first two studies used medical records. Reporting behaviour retrospectively may lead to overestimation of guideline adherence because of social desirability bias [19]. reported 4% of the children being referred [14]. Two other British studies found much higher rates of referral [17,18]. Discussion However, these last two studies used postal question- naires to measure FP behaviour, whereas our study and the first two studies used medical records. Reporting behaviour retrospectively may lead to overestimation of guideline adherence because of social desirability bias [19]. Page 6 of 7 (page number not for citation purposes) Abbreviations , pp 5. Coulthard MG, Lambert HJ, Keir MJ: Occurence of renal scars in children after their first referral for urinary tract infection. Brit Med J 1997, 315:918-919. DCGP = Dutch College of General Practitioners; EMR = electronic medical record; FP = family practitioner; ICPC Page 6 of 7 (page number not for citation purposes) Page 6 of 7 (page number not for citation purposes) BMC Family Practice 2007, 8:9 http://www.biomedcentral.com/1471-2296/8/9 http://www.biomedcentral.com/1471-2296/8/9 http://www.biomedcentral.com/1471-2296/8/9 6. American Academy of Pediatrics: Practice Parameter: The diag- nosis, treatment, and evaluation of the initial urinary tract infection in febrile infants and young children. Pediatrics 1999, 103:843-852. 7. Royal College of Physicians: Guidelines for the management of acute urinary tract infection in childhood. Report of a Work- ing Group of the research unit, Royal College of Physicians. J R Coll Physicians London 1991, 25:36-42. J y 8. Jodal U, Lindberg U: Guidelines for management of children with urinary tract infection and vesico-ureteric reflux. Rec- ommendations from a Swedish state-of-the-art conference. Swedish Medical Research Council . Acta Paediatr Suppl 1999, 88(431):87-9. 9. van Haaren KAM, Visser HS, van Vliet S, Timmermans AE, Yadava R, Geerlings SE, ter Riet G, van Pinxteren B: NHG-Standaard Urineweginfecties - Tweede herziening [Guideline of the Dutch College of General Practitioners on urinary tract infections: second revision]. Huisarts Wet 2005, 48:341-352. 10. van der Linden MW, van Suijlekom-Smit LWA, Schellevis FG, van der Wouden JC: Tweede nationale studie naar ziekten en verrichtingen in de huisartspraktijk. Het kind in de huisartspraktijk [Second Dutch national sur- vey of general practice. The child in general practice] Rotterdam/Utrecht, Erasmus MC, afdeling Huisartsgeneeskunde/NIVEL; 2005. Erasmus MC, afdeling Huisartsgeneeskunde/NIVEL; 2005 11. Tacken MAJB, Braspenning JCC, de Bakker DH, Jabaaij L, van den Hoogen HJM, Verheij R, Groenewegen PP, Grol RPTM: The Dutch National Information Network of General Practice (LINH) and quality-of-care data. In Quality of preventive performance in gen- eral practice: The use of routinely collected data [thesis] Edited by: Tacken MAJB. Nijmegen, Radboud University Nijmegen; 2005:23-44. 12. Lamberts H, Wood M: ICPC: International classification of primary care Oxford, Oxford University Press; 1987. 13. Timmermans AE, Baselier PJAM, Winkens RAG, Arets H, Wiersma T: NHG-Standaard Urineweginfecties: Eerste herziening [Guideline of the Dutch College of General Practitioners on urinary tract infections: first revi- sion] Utrecht, Nederlands Huisartsen Genootschap (NHG); 1999. 14. van de Lisdonk EH, Verstraeten J: Kinderen met urineweginfec- ties: Verwijzen of niet? Abbreviations Grol R: Beliefs and evidence in changing clinical practice. Brit Med J 1997, 315:418-421. Abbreviations Een retrospectief onderzoek in de vier CMR-praktijken [Children with urinary tract infections: To refer or not? A retrospective study in four CMR-prac- tices]. Huisarts Wet 2000, 43:343-346. 15. Kwok WY, de Kwaadsteniet MCE, Harmsen M, van Suijlekom-Smit LWA, Schellevis FG, van der Wouden JC: Incidence rates and management of urinary tract infections among children in Dutch general practice: Results from a nation-wide registra- tion study. BMC Pediatr 2006, 6:10. y 16. South Bedfordshire Practitioners' Group: How well do general practitioners manage urinary problems in children? Br J Gen Pract 1990, 40:146-149. 17. Vernon S, Foo CK, Coulthard MG: How general practitioners manage children with urinary tract infection: An audit in the former Northern Region. Br J Gen Pract 1997, 47:297-300. g J 18. Dighe AM, Grace JF: General practice management of child- hood urinary tract infection. J R Coll Physicians London 1984, 34:324-327. 19. Paulus DL: Measurement and control of response bias. In Meas- ures of personality and social psychological attitudes Edited by: Robinson JP, Shaver PR and Wrightsman LS. San Diego (CA), Academic Press; 1991:17-59. 20. Esbjörner E, Berg U, Hansson S: Epidemiology of chronic renal failure in children: A report from Sweden 1986-1994. Pediatr Nephrol 1997, 11:438-442. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 7 of 7 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge p 21. Grol R: Beliefs and evidence in changing clinical practice. Brit Med J 1997, 315:418-421. p 21. Pre-publication history The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2296/8/9/prepub http://www.biomedcentral.com/1471-2296/8/9/prepub
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Structural analyses of wooden chairs by finite element method (FEM) and assessment of the cyclic loading performance in comparison with allowable design loads
Maderas. Ciencia y tecnología
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8,600
1Bingöl University. Department of Material and Material Process Techniques. Bingöl, Turkey. 2Muğla Sıtkı Koçman University. Faculty of Engineering. Department of Civil Engineering. Muğla, Turkey. 3Muğla Sıtkı Koçman University. Faculty of Technology. Department of Woodworking Industrial Engineering. Muğla, Turkey. ♠Corresponding author:alikasal@mu.edu.tr Received: 26.04.2020 Accepted: 19.11.2020 ABSTRACT Tests were carried out to investigate the cyclic loading performance of chairs constructed of Scotch pine (Pinus sylvestris) and Oriental beech (Fagus orientalis). Totally, 30 real size chairs were constructed and assembled with polyvinyl acetate adhesive. Chairs were tested under 3 cyclic loading directions; namely, front to back, back to front, and backrest according to American Library Association (ALA) specifications, and obtained loading performances of chairs were crosschecked with allowable design load levels. Additionally, specimens were analyzed as structures by using the finite element method in order to obtain axial, shear, and bending stresses acting on each member under loadings and compare these values with design stresses. As a result, it was concluded that finite element method analyses achieve reasonable estimates for strength performances and failure behaviors of chairs. Depending on allowable design loads by American Library Association; chairs constructed of beech could meet the medium service for front to back and back to front loading, while heavy service for backrest loading. In the case of pine chairs; they could meet the medium service for front to back and back to front loading, while light service for backrest loading. Keywords: Acceptable design loads, chair strength, cyclic loading, finite element analysis, furniture engi­ neering design, structural analysis, wooden. https://orcid.org/0000-0002-4632-0072 https://orcid.org/0000-0002-4632-0072 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 ISSN impresa ISSN online STRUCTURAL ANALYSES OF WOODEN CHAIRS BY FINITE ELEMENT METHOD (FEM) AND ASSESSMENT OF THE CYCLIC LOADING PERFORMANCE IN COMPARISON WITH ALLOWABLE DESIGN LOADS Erkan Ceylan1 https://orcid.org/0000-0002-0497-7789 Ersan Güray2 https://orcid.org/0000-0001-5349-6511 Ali Kasal3,♠ https://orcid org/0000 0002 4632 0072 Erkan Ceylan1 https://orcid.org/0000-0002-0497-7789 Ersan Güray2 Erkan Ceylan1 https://orcid.org/0000-0002-0497-7789 Ersan Güray2 https://orcid.org/0000-0001-5349-6511 Ali Kasal3,♠ h // id /0000 0002 4632 0072 Ersan Güray2 https://orcid.org/0000-0001-5349-6511 Ali Kasal3,♠ https://orcid.org/0000-0001-5349-6511 material economics, among others. (Eckelman 2003). It is well possible to utilize structural analyses and solid modelling software to accomplish the design of furniture. In addition, the finite element method (FEM) provides a furniture designer with enormous possibilities for analysis and design. In this context, furniture members, joints, and the whole system could be modelled parametrically via FEM. It is required to make simplifications while modelling the structure, for instance, the geometry can be taken as rough as possible and the connections are modelled by semi-rigid springs. If the numerical results do not well satisfy the experimental output, then some detailing modifications are done on the model to provide a better approximation. Strength calculations of the designed elements and connection points require realistic modeling and loading of the system. Then, required constraints are provided to optimize the member sizes by interpretation of these internal forces (Kasal et al. 2006). Recent studies show that the FEM has become widespread in the structural analysis of furniture systems. In (Tankut et al. 2014), a bibliographical review of the FEM, used in the analysis of wooden furniture products is listed. In another work, it is shown that the design of a chair can be very different if some simple calculations are performed. In this study, additionally, it was underlined that there had been a need for the wood analysis not in “building size” but “furniture size” (Gustafsson 1995). Some analytical models were developed for the furniture frame analysis using FEM by Kasal and Pullela (1995). The load-deformation curves were gathered by experiments and the stiffness is determined by incorporating the resulting curves with the loading. In the end, it was stated that FEM models had been helpful to simulate the furniture under loading (Kasal and Pullela 1995). A computer program was developed for the strength analysis of wooden frames. In this study, the side frame of a chair was analyzed and it was shown that accurate and practical strength analysis of the wooden members could be performed by this program (Smardzewski 1998). He developed a mathematical model for bent-mortise joints, in his other work, and tried to discover influencing factors on the strength of glued-tenon-joints. Analyses were performed with a computer assisted program and according to results, the flexural strength of the glued-mortise-tenon-joints was depending on mostly the shear strength of the adhesive and the compression strength of the wood material (Smardzewski 2016). In the study carried by Kasalet al. material economics, among others. (Eckelman 2003). The design of furniture almost rests on traditional experiences in handcraft manufacturing. As far as known, no manufacturer or designer applies static analyses to determine the internal forces inside the wooden members of a furniture body. However, some researchers have shown interest in this field (Gustafsson 1997). The section size and geometries of members that integrate the furniture frames should be designed rationally and they should carry the acceptable loads probably imposed upon them in service. In the methodology of furniture engineering; once forces and moments these acting to the ends of each member have been analyzed, the internal stresses in these members can be calculated and by comparing their magnitudes with the allowable design stresses for the material used, it can be seen whether or not each of the members has been safely designed. This process provides a methodical way of designing a piece of furniture to meet any specified service condition. In many types of furniture, members may be deliberately oversized so that no attempt is made to determine the optimum or most efficient size for each member. The principal task of the designer in such cases is to verify that each member can safely carry the forces imposed upon it without exceeding the allowable stress design values. When members are used solely for structural purposes such as in upholstered furniture, however, they often may be engineered exactly to meet specified strength requirements. The objective here is to produce an optimum overall design in which just enough material is used to produce a piece of furniture that is capable of carrying specified design loads with a given factor of safety. Such designs, being the most economical, provide savings for both the manufacturer and consumer together (Eckelman 2003). Today, technology has rapidly developed. Just like in other industrial areas, technological developments have been effective in the production of furniture from design to manufacturing. With the help of recent technological improvements and innovations, furniture could be produced at high-speed and robustness; furthermore, the constructional details are able to be analyzed in details. The precise analysis of furniture frames is a complicated process in terms of computation, since there exist a large number of internal forces to be determined for an effective solution. It is very important to use technological facilities in the strength design of furniture (Kasal et al. 2006). INTRODUCTION The design of the new furniture should satisfy three main criteria such that it should have an aesthetic looking, functional, durable and also feasible. The first one requires esthetic design; that is, the artistic +development of the furniture structure, the design of the second is necessary for the use/functionality of the furniture such that the furniture structure is planned to have the capability of its intended functionality, and the last can be obtained by an engineering design; that is, the furniture can safely resist the loads imposed upon it in its service life and manufactured with minimum cost. Until recently, the engineering design received little attention. However, it is becoming increasingly important because of various reasons such as; consumer demands for more reliable furniture, government pressures for warranties of furniture, increasing needs for Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío material economics, among others. (Eckelman 2003). (2016a), mortise and tenon joints in various sizes were analyzed by FEM and some strength properties like the bending capacity, stiffness of the joint were reported. Joints became stronger and stiffer by increasing the tenon sizes. The maximum stress was concentrated on the edge and corners of the glued surface and it was declared that the strength properties of the joints have a shape-adhesive nature (Kasal et al. 2016b). The research was aimed at designing flexible chairs consisting of lamellae using FEM. In this research, the ultimate strength 2 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 capacity of the flexible chairs was tested both experimentally and numerically. In the case, of a weight until to 110 kg chairs met the requirements of actual standards but failed for heavy loadings (Langova et al. 2019). capacity of the flexible chairs was tested both experimentally and numerically. In the case, of a weight until to 110 kg chairs met the requirements of actual standards but failed for heavy loadings (Langova et al. 2019). In the literature, there are many studies related to the ultimate and allowable strength of solid wood and wooden materials. And, safety factors were recommended for calculating the allowable design loads (Eckelman 2003, Forest Products Laboratory - USDA 2010). There are some studies related to the allowable design loads for whole furniture systems. Among these studies, the most important references are GSA (FNAE 80-214) and ALA. In these references, allowable cyclic design loads for upholstered sofas and chairs were given by classifying the light (domestic), medium, and heavy duty service (Eckelman 1982, Eckelman 1995a, Eckelman 1995b, Eckelman 1999, Eckelman and Erdil 2001). The strengths of beech chairs were investigated under cyclic and static loading. The cyclic front to back load performance of chairs was compared with the acceptable design loads such that, it was recommended that the cyclic strength could be assumed to be 56 % of the strength of static loading case (Kuskun et al. 2018). Kiliç et al. (2018) investigated the effects of tenon size, in the case of the front to back loading of Scotch pine chairs, and the strength of chairs wich were evaluated according to the acceptable design loads. Chairs with joints connected by 40 mm in width and 50 mm in length tenons were capable of meeting light service which corresponds to the domestic usage. material economics, among others. (Eckelman 2003). However, a minimum of 50 mm x50 mm size was required for the width and length of each tenon, respectively, to meet the requirement of the medium service (Kiliç et al. 2018). In the other study, the relationship between the loading capacity of beech chairs and strength of side frame joints was investigated where the chair was subjected to the front to back single push. In addition to this, a methodology was developed for predicting the strength of chairs from the strength of joints (Kasal et al. 2016a). As seen in the literature, generally, the performances of chairs were evaluated in the case of the front to back loading. There are very limited studies that evaluated the cyclic strength of the wooden chairs in all loading directions. The purpose of this study was to measure the cyclic loading performance of wooden chair frames by comparing the allowable design load levels in three loading directions given in ALA specifications. Furthermore, chair frames were structurally analyzed by using FEM and collected data were compared to the actual strength test results. As a result, it was intended to promise information about the cyclic loading performance of wooden chairs and to show the usage of structural analyses via FEM in furniture engineering design. Materials properties Chair members were constructed from beech (Fagus orientalis L.) and pine (Pinus sylvestris L.) woods. They are procured from local commercial suppliers and are used quite often in the furniture industry. The average density values of these species are 0,63 g/cm3 and 0,48 g/cm3, respectively. Other physical and mechanical properties are taken as shown in ASTM D143-94 (2000) and ASTM D4442-92 (2003) standards. Whole chair frames were constructed by utilizing grooved beech dowels. They were 10 mm in diameter and 35 mm in length. 65% solid contained PVAc (polyvinyl acetate) glue is used to assemble the chair members. This type of glue is highly preferred since it is easily spread and dried, cold applied, odorless and fire resistant. Necessary properties of it are provided by the company (Polisan, Turkey) as density of 1,1 g/cm3, viscosity of 0,16 - 0,2Pa/s, ph = 5,00, ash rate of 3%. Construction of the chairs and individual joint specimens A total of 30 (2 wood species, 3 loading direction, 5 replication) chairs were prepared in 1/1 scale. 15 chairs were constructed of beech, and the remaining 15 were constructed of pine. The general configuration of the chairs is shown in Figure 1. 3 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Figure 1: (a) Dimensions in mm (b) the real picture of the chairs. Figure 1: (a) Dimensions in mm (b) the real picture of the chairs. Figure 1: (a) Dimensions in mm (b) the real picture of the chairs. First of all, surfaces of wooden beams were smoothed at the planing machine and pieces with a thickness of 21 mm were prepared. Then, they were cut to have a 60 mm width for all members except for stretchers. The stretcher was 21 mm thick and 30 mm wide. Assembling starts with the construction of side frames. Mortise and tenon joint techniques were utilized for constructing the side frames of the chair. The length and the width of tenons were set to 40 mm at side rails, they were 30 mm for the stretcher since it was only 30 mm in width. Corresponding mortise holes were drilled on back and front legs with a 7 mm thickness (1/3 of the member thickness). Mortises were drilled and tenon joints were cut with appropriate laboratory machines. A tight fit was obtained where the average mortise–tenon clearance was 0,076 mm ± 0,025 mm. In the assembly, walls of the mortises and tenons were first adequately coated with adhesive. The adhesive was spread over approximately with 150 gr/cm2 ± 10 gr/cm2. The tenons were then inserted into the mortises, and the assembly was squeezed by bar clamps. Then a complete chair was built by these preassembled pieces utilizing dowel joints. The depth of the embedment of dowels in the edge and the face were 20 mm, 15 mm, respectively. The distance between the two dowels was chosen to be 30 mm. The dowels and corresponding holes at the top and back rails and the lower front and back rails were coated with adhesive. Bar clamps were again used to squeeze the side frames to force the dowels for a tight fit. Cyclic performance testing of chairs and moment-rotation characteristics of joints The “cyclic stepped increasing loading” is a recommended method to satisfy the requirements of the performance test system. To do this, the furniture is subjected to a specified initial load at a prescribed cyclic rate for a specified number of cycles. When cycles are accomplished, the subjected load is raised incrementally. Cycling loading is repeated after each increment. The procedure is repeated until the desired load level has been reached, or failure occurs on the furniture frame, or horizontal deflection exceeds 50 mm on the side rail and back leg connection (Eckelman 1988, Eckelman 1999). In the scope of the study, a total of 30 chair frames were constructed and tested. Chairs were tested regarding the principles of the American Library Association (ALA) specification by applying the cyclic front to back, back to front, and backrest loads to which the chairs were likely to be exposed in service (Eckelman 1999). The cyclic loading tests were performed on the furniture performance testing equipment (Mates 2011, Ankara, Turkey) at the mechanical testing laboratory in the Wood Science and Industrial Engineering Department of Mugla Sitki Kocman University. The cyclic loading test procedure was that 1) a chair was subjected to a given load for 25000 cycles at a rate of 20 cycles per minute, 2) when 25000 cycles were completed at this load level, the load was increased a specified amount and testing continued for next 25000 cycles, and 3) this procedure was repeated until the tested chair suffers disabling damage or the desired acceptance level were reached. When performing the cyclic stepped increasing loading procedure, “initial load,” “load increment,” “loading rate,” and “load cycles at each load level” practiced in the study are given in Table 1. 25000 cycles at each load level took almost 21 hours (about 1 day) in cyclic loading for chairs. In the tests, the ultimate failure loads (in Newton) and completed total cycles were recorded. Table 1: Loading program of cyclic loading tests for chair frames (Eckelman 1999). ble 1: Loading program of cyclic loading tests for chair frames (Eckelman 1999). In the front to back and back to front loading tests; horizontal loads in specified directions were applied from 420 mm away from the supports. Construction of the chairs and individual joint specimens Not only whole chairs were constructed but also representative T-type and L-type specimens were prepared for obtaining the moment rotation characteristics (stiffness coefficients) of each joint in order to introduce them as semi-rigid connections in the structural analyses of chairs. Each joint type was tested 20 times since there had been 2 wood species and tests were replicated 10 times. Therefore, 20 identical T-type joints for the connections between the “back leg to side rail” and 20 identical T-type joints for the connections between the “back/front leg to stretchers” were prepared. Separately, 20 identical L-type specimens for modelling the connection between the “front leg to side rails” were arranged. T-type and L-type joint specimens and load directions are shown in Figure 2a, Figure 2b, and Figure 2c. Figure 2: T-type joint (a, b) and L-type joint (c) specimens (measurements in mm). Figure 2: T-type joint (a, b) and L-type joint (c) specimens (measurements in mm). 4 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 As a precaution for a unique moisture content (MC), before examining, all chairs and joint specimens were kept in an environmentally controlled conditioning room to cure at least one month for reaching an average 12 % moisture content. As a precaution for a unique moisture content (MC), before examining, all chairs and joint specimens were kept in an environmentally controlled conditioning room to cure at least one month for reaching an average 12 % moisture content. Cyclic performance testing of chairs and moment-rotation characteristics of joints In these tests, reaction brackets were fixed behind each of the back legs for front to back loading while they were placed in front of each of the front legs for back to front loading in order to prevent the chair from slipping. Steel chains then passed over the seat and attached to the air cylinders that were used to apply the horizontal loads to the chair. The other end of the steel chains dropping over the front edge of the seat for front to back loading, while the back edge of the seat for back to front loading, was attached to the floor vertically. Chains provided the reaction forces preventing the chair from overturning. (Figure 3a and Figure 3b). In the case of the backrest loading test, a reaction bracket was placed just behind the back legs with the same height of side rails, and another bracket was placed in front of the front legs. Here, just similar to cases of back to front or front to back loadings, reaction brackets prevented the chair from tipping over backward when front to back loads applied to the backrest. A short steel chain was tied around the backrest and fixed to the air cylinder (Figure 3c). 5 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Figure 3: Tests; (a) front to back loading, (b) back to front loading, (c) backrest loading. Figure 3: Tests; (a) front to back loading, (b) back to front loading, (c) backrest loading. The allowable cyclic loading performance levels for light (domestic), medium and heavy usages are listed in Table 2 as given in the ALA specifications (Eckelman 1999). Tests were performed and the resulting performance values are to be declared and compared with these specified design loads in the next step. The allowable cyclic loading performance levels for light (domestic), medium and heavy usages are listed in Table 2 as given in the ALA specifications (Eckelman 1999). Tests were performed and the resulting performance values are to be declared and compared with these specified design loads in the next step. Table 2: Allowable design loads according to the loading direction (Eckelman 1999). Table 2: Allowable design loads according to the loading direction (Eckelman 1999). The moment rotation characteristics of joints in the side frame of the chair were determined to treat them as semi-rigid connections in the structural analyses. Cyclic performance testing of chairs and moment-rotation characteristics of joints The moment-rotation characteristics of the joint were determined by the relationship between the distortion angle between neighboring orthogonal members and the applied moment. It could be considered as the rotational, linear behaving, stiffness coefficient and evaluated by Equation 1: M K ϕ = (1) M K ϕ = (1) M K ϕ = (1) where K is the stiffness coefficient (Nm/rad), M is the moment (Nm) and ø designates the distortion angle at the joint (rad). T-type and L-type joint specimens were examined under bending loads to simulate the condition in which they are exposed to these loads as in the side frame of a full chair (Figure 2). Static loading tests were performed on the 50 kN capacity universal-testing machine (Mares, İstanbul, Turkey) with the 6 mm/min loading rate. The rail member of each specimen at a point 300 mm from the front edge of the leg was subjected to a concentrated load, such that, the moment arm was 300 mm according to the literature (Kasal et al. 2016a, Kiliç et al. 2018). Dial gage was clamped to the edge of joint rails and the measurements were recorded at regular intervals as specimens were loaded. 6 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Structural analysis of chairs Table 4: Physical and mechanical properties of pine and beech woods used in the study. Wood Species MOE* (MPa) Tension Strength (MPa) Compression Strength (MPa) Shear Strength (MPa) G* (MPa) MOR* (MPa) Density (kg/m3) MC* (%) Pine 11000 95 55 6 4230 90 480 10,8 Beech 12500 125 75 11 4807 120 630 11,2 *MOE: modulus of elasticity; G: shear modulus; MOR: modulus of rupture, MC: moisture content. Table 4: Physical and mechanical properties of pine and beech woods used in the study *MOE: modulus of elasticity; G: shear modulus; MOR: modulus of rupture, MC: moisture content. Then, sectional properties, i.e., the cross-sectional area, the moment of inertia values about local (Y) and (Z) axes, form factor and, torsional constant values were calculated and implemented into the workspace (Table 5). Then, sectional properties, i.e., the cross-sectional area, the moment of inertia values about local (Y) and (Z) axes, form factor and, torsional constant values were calculated and implemented into the workspace (Table 5). Table 5: Sectional properties of the chair members. Table 5: Sectional properties of the chair members. After all physical and mechanical definitions for the problem, the supports should have been assigned. For the front to back loading; front legs connection to the floor were treated with roller supports, whereas the back legs were supposed to have pin connections with the floor. In other words, it was considered that back legs were constrained to translations in (X), (Y), and (Z) directions, while they were free to rotate in any direction; but the front legs, were constrained to the translation only in (Y) direction. For the back to front loading; unlike the front to back loading, back legs of the chair were supported as roller, whereas the front legs were supported as pinned. And finally, when it was loaded at the backrest; the back legs of the chair were supported as a roller, whereas the front legs were supported as pinned, in addition, the back leg to side rail joints were supported as the roller. Other than the support joints, all internal joints (nodes) were defined with six degrees of freedom, in other words, all internal joints can carry the axial forces, shear forces, and bending moments in any direction. The numerical model was subjected to static analysis by loading properly. Structural analysis of chairs A chair structure is a frame structure where the members are assumed to be linear beams. Here, in this work, every member is a prismatic member having a width, length, and depth. The model was created with joints and the interconnected beam elements between them (Figure 4). Each joint was labelled from N1 to N20 and members were numerated from M1 to M24. This FEM modelling was performed on RISA-3D (2000) and all internal forces were determined. Figure 4: Joints/nodes (a) and members (b) of the modelled chair. b (b) f th d ll d h i Figure 4: Joints/nodes (a) and members (b) of the modelled chair. Rotational linear springs were modelled at the T-type and L-type side frame connections to treat them as semi-rigid connections. Since the software was able to assign semi-rigid connections by simply assuming springs at these joints, simply, predefined K (rad/mm), stiffness coefficients are entered into the interface of the program (Table 3). The stiffness coefficients were determined based on the bending tests of representative T-type and L-type joints, as was previously explained. Table 3: Stiffness coefficients, K, obtained for each joint type according to wood species. Table 3: Stiffness coefficients, K, obtained for each joint type according to wood species. Next, some required physical and mechanical properties (Table 4) of beech and pine obtained from the tests were entered into the software to calculate the axial, shear forces and, moments at the end of members. Poisson’s ratio (µ) was taken as 0,3 for both species (Kasal et al. 2006, Kasal et al. 2016b, Kiliç et al. 2018). 7 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Table 4: Physical and mechanical properties of pine and beech woods used in the study. Wood Species MOE* (MPa) Tension Strength (MPa) Compression Strength (MPa) Shear Strength (MPa) G* (MPa) MOR* (MPa) Density (kg/m3) MC (%) Pine 11000 95 55 6 4230 90 480 10 8 Table 4: Physical and mechanical properties of pine and beech woods used in the study. Wood Species MOE* (MPa) Tension Strength (MPa) Compression Strength (MPa) Shear Strength (MPa) G* (MPa) MOR* (MPa) Density (kg/m3) MC* (%) Pine 11000 95 55 6 4230 90 480 10,8 Beech 12500 125 75 11 4807 120 630 11,2 *MOE: modulus of elasticity; G: shear modulus; MOR: modulus of rupture, MC: moisture content. Structural analysis of chairs The ultimate loads that were obtained from the actual tests were cyclic performance, but it was performed the FEM analyses with static loads. Therefore, it was considered the relation between the cyclic and static strength. According to the literature; cyclic strength is approximately half of the static strength, so in the structural analyses, it was applied twice the loads that were obtained from the actual tests (Kasal et al. 2016a, Kuskun et al. 2018, Likos et al. 2013). Ultimate loads were symmetrically applied at corresponding nodes that were on the two side frames (Figure 5). Axial, shear forces, moments, and corresponding stresses acting on each member was obtained. Furthermore, axial force, shear force, and, moment diagrams and deflected shape of the chair was also provided as the output of the software. 8 8 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Structural analyses of wooden..: Ceylan et al. Figure 5: Loading and supports of the numerical model, loaded in direction of (a) front to back, (b) back to front, and (c) backrest. Figure 5: Loading and supports of the numerical model, loaded in direction of (a) front to back, (b) back to front, and (c) backrest. The axial forces (tension or compression), shear forces, and moments were obtained from the structural analyses for all members of the chair under each loading direction. Corresponding axial, shear and bending stresses were also available and ready to be compared with the allowable design stresses. To find the allowable design stresses, values given in Table 4 were multiplied by the appropriate reduction (safety) factors (Eckelman 2003). Table 6 also shows the allowable design stresses for the materials used in the construction of the chairs. Table 6: Allowable design stresses for the wood materials used in the construction of cha *Reduction factors are taken according to Eckelman (Eckelman 2003). Table 6: Allowable design stresses for the wood materials used in the construction of chair*. *Reduction factors are taken according to Eckelman (Eckelman 2003). *Reduction factors are taken according to Eckelman (Eckelman 2003). As a result; deformation characteristics observed in actual tests were tried to verify by structural analysis. In other words, the consistency of the joints where deformations occur in actual tests was checked with the results of structural analysis. Structural analysis results of chairs The chairs failed completely without tilting in average 10 days for front to back loading, 9 days for back to front loading, and 5 days for backrest loading. Interconnecting joints softened suddenly, but they kept holding loads after a while. In the chairs tested under the front to back or back to front loading; joint failures occurred because of the glue line fractures, i.e. tenons withdrew truly from the front and back leg members. Softening failures proceeded at the edge of the tenon and then propagated towards to another edge as the cycles and load levels increased. As the adhesive bonding failed, the tenons were lead to be loaded in bending and shear 9 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío stresses. In the case of frames tested under backrest loading; failures occurred owing to fracture of the tenons at their point of entry into the walls of the back and front legs or fracture of the back leg member at the top of the back leg to side rail joints. The exaggerated deflected shapes of a chair under the three lading directions obtained from the FEM analyses and actual tests are given in Figure 6 and Figure 7, respectively. stresses. In the case of frames tested under backrest loading; failures occurred owing to fracture of the tenons at their point of entry into the walls of the back and front legs or fracture of the back leg member at the top of the back leg to side rail joints. The exaggerated deflected shapes of a chair under the three lading directions obtained from the FEM analyses and actual tests are given in Figure 6 and Figure 7, respectively. Figure 6: Deflected shape of chair obtained from the structural analyses under, (a) front to back loading, (b) back to front loading, and (c) backrest loading. Figure 6: Deflected shape of chair obtained from the structural analyses under, (a) front to back loading, (b) back to front loading, and (c) backrest loading. Figure 7: Observed deformation characteristics of chairs under, (a) front to back loading, (b) back to front loading, and (c) backrest loading. Figure 7: Observed deformation characteristics of chairs under, (a) front to back loading, (b) back to front loading, and (c) backrest loading. Structural analysis results of chairs As seen in Figure 6 and Figure 7, deflected shape of the chairs obtained from the structural analyses under each loading direction have given reasonable estimates in terms of the overall deformation characteristics and possible failure points of the chairs. In the strength analyses of the chairs, the axial forces and corresponding axial stresses under the front to back, back to front, and backrest loadings were considered initially. Axial force diagrams are given in Figure 8 with the most critical tension and compression members. Figure 8: Axial force diagrams obtained from the structural analyses results for (a) front to back loading, (b) back to front loading, and (c) backrest loading. Figure 8: Axial force diagrams obtained from the structural analyses results for (a) front to back loading, (b) back to front loading, and (c) backrest loading. 10 10 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Regarding loading, the bottom part of the back leg member is exposed to a high compression due to the front to back loads while the bottom part of the front leg member is pulled up from the test set-up platform, and exposed to tensioning because of the pin supports. On the contrary, for the back to front loading; the bottom part of the front leg member is subjected to high compression while the bottom part of the back leg member is sustaining tension. In both loading directions, stretcher members are exposed to a considerable amount of compression. In the case of the backrest loading; the side rail member is exposed to a tension force while the stretcher member is exposed to a compression force. The members pointed out in Figure 8, were compared to the allowable axial design stress values determined for each wood material used. It was found that the maximum tension stresses (T) occurred in the bottom part of the front leg member (M14, M20) under the front to back loading, in the bottom part of the back leg member (M13, M19) under back to front loading, and in the side rail member (M3, M7) under the backrest loading. In the case of maximum compression stresses (C), has occurred in the stretcher member (M2, M6) for all three loading directions. These members are the most critical members for carrying the axial forces under the loadings. Structural analysis results of chairs The comparison results for the consequential ultimate axial stresses and allowable axial design stresses were given in Table 7. Table 7: Comparison of the maximum axial stresses values and axial design stresses (MPa). Table 7: Comparison of the maximum axial stresses values and axial design stresses (MPa). As seen in Table 7, chairs made of both wood species were found to be strong enough to carry the axial forces safely for all loading cases. The shear forces in direction (Y) and corresponding shear stresses under the front to back, back to front, and backrest loadings were provided as the second phase. Shear force diagrams were presented in Figure 9 with the most critical shear members for all loading directions. Table 7: Comparison of the maximum axial stresses values and axial design stresses (MPa). As seen in Table 7, chairs made of both wood species were found to be strong enough to carry the axial forces safely for all loading cases. The shear forces in direction (Y) and corresponding shear stresses under the front to back, back to front, and backrest loadings were provided as the second phase. Shear force diagrams were presented in Figure 9 with the most critical shear members for all loading directions. Figure 9: Shear force diagrams obtained from the structural analyses results for (a) front to back, (b) back to front, and (c) backrest loading. Figure 9: Shear force diagrams obtained from the structural analyses results for (a) front to back, (b) back to front, and (c) backrest loading. The maximum values in members, shown in Figure 9, were compared to the allowable shear design stresses. Maximum shear stresses (S) occurred in the bottom part of back leg and front leg members (M13, M19 and M14, M20) under the front to the back and back to front loading, respectively. In the case of backrest 11 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío loading, maximum shear stresses occurred in the middle part (between the side rail and stretcher) of the back leg member (M15, M16 and M21, M22). These critical members’ results for shear stresses which act upon the back leg-bottom members and allowable shear design stresses were given in Table 8. loading, maximum shear stresses occurred in the middle part (between the side rail and stretcher) of the back leg member (M15, M16 and M21, M22). Structural analysis results of chairs 12 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Structural analyses of wooden..: Ceylan et al. Table 9: Comparison of the maximum bending stresses and bending design stresses. Table 9: Comparison of the maximum bending stresses and bending design stresses. According to comparison results, the maximum bending stresses occurred in the back leg member where it was joined the side rail member among the three loading directions. Therefore, the most critical place of the chair is the back leg to side rail joints. Determined bending stress values that act upon these joints have gone beyond the allowable bending design stresses for both wood species. The overall performance of the chairs has been seriously affected by the bending capacity of these joints and the section sizes of the related members. In the engineering design of a chair, if these results are taken into consideration, the strength performance of the chair can be upgraded. Here, in this study, all possible forces and consequential stresses are investigated for both wood species. When the literature is examined, it is stated that the bending moment is the most critical internal force through them. The axial strength of even a very small sized wood member is generally much greater than needed, and shear strength is rarely of concern (Eckelman 2003). In this study, it has been understood that either beech or pine used chair members, and sectional sizes of each member of the chair had enough capacity to carry the axial (tension or compression) forces, safely, whereas the members of the back leg where it was joined the side rail or stretcher member has been imposed to over bending and shear forces. This fact is well compromised with the actual performance test results in terms of failure locations. In other words, actual test failures occurred in these joints where the structural analyses showed that the highest stresses occurred. In the backrest loading tests; firstly, softening failures occurred in the mentioned joints, and catastrophically, other joints were also untightened. In some chairs, the back leg member of the chair or tenons of the back leg to side rail joints were broken. These results helped to confirm that the FEM model of the chair was consistent with the experiment chair. Structural analysis results of chairs These critical members’ results for shear stresses which act upon the back leg-bottom members and allowable shear design stresses were given in Table 8. Table 8: Comparison of the maximum shear stresses and shear design stresses. Table 8: Comparison of the maximum shear stresses and shear design stresses. According to Table 8, it was found that, chairs, nevertheless they are made of pine or beech, had enough capability of carrying design loads in the case of both fronts to back or back to front loadings. However, the shear stresses that occurred in the middle part of the back leg under backrest loading were higher than the allowable shear design stresses for both chairs constructed of pine and beech. This result was consistent with observations in actual backrest loading tests. In the actual tests, failures occurred at these members and/or related (back leg to side rail or back leg to the stretcher) joints. In the last phase, the moments about the (Z) axis and consequential bending stresses were obtained from the results of structural analyses. Moment diagrams about the (Z) axis obtained from the structural analysis results were presented in Figure 10 with the most critical members and their ends. Figure 10: Moment diagrams obtained from the structural analyses results for (a) front to back, (b) back to front, and (c) backrest loading. Figure 10: Moment diagrams obtained from the structural analyses results for (a) front to bac (b) back to front, and (c) backrest loading. The maximum bending stresses (B) occurred in the (I) end of the bottom part of the back leg member (M13-I, M19-I) where it was joined the stretcher member (M2, M6) under the front to back loading. Therefore, the most critical point of the chair is the joint where the back leg connected to the stretcher. On the contrary, the maximum bending stresses occurred in the (I) end of the bottom part of the front leg member (M14-I, M20-I) where it was joined the stretcher member (M2, M6) under back to front loading. So, for this loading direction, the most critical point of the chair is the joint where the front leg connected to the stretcher. In the case of backrest loading, the back leg member was subjected to a considerable amount of bending stresses at the point (M18-J) where it was joined the side rail member (M3). The results are summarized in Table 9. The chairs constructed of beech had mean cyclic loading performance of 1868 N, 1801 N, and 3469 N whereas the chairs constructed of pine had mean cyclic loading performance of 1601 N, 1579 N, 1690 N for front to back, back to front, and backrest loadings, respectively. Thus, the chairs constructed of beech were 14 Assessment of the cyclic loading performance of chairs with allowable design loads The mean ultimate cyclic strength performance values of tested chairs under each loading direction with their coefficients of variation are summarized in Table 10. Table 10: Cyclic strength performance values and total completed cycles of the chairs Table 10: Cyclic strength performance values and total completed cycles of the chairs. The chairs constructed of beech had mean cyclic loading performance of 1868 N, 1801 N, and 3469 N whereas the chairs constructed of pine had mean cyclic loading performance of 1601 N, 1579 N, 1690 N for front to back, back to front, and backrest loadings, respectively. Thus, the chairs constructed of beech were 14 13 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Universidad del Bío-Bío %, 12 %, and 51 % stronger than the chairs constructed of pine for front to back, back to front, and backrest loading, respectively. Test results indicated that the ultimate cyclic loading performance of the chairs was significantly affected by the wood species. Differences in ultimate cyclic loading performance of the chairs could be explained by differences in physical and mechanical properties, especially joint strengths, shear and bending strength of the wood material used constructing the chairs. %, 12 %, and 51 % stronger than the chairs constructed of pine for front to back, back to front, and backrest loading, respectively. Test results indicated that the ultimate cyclic loading performance of the chairs was significantly affected by the wood species. Differences in ultimate cyclic loading performance of the chairs could be explained by differences in physical and mechanical properties, especially joint strengths, shear and bending strength of the wood material used constructing the chairs. According to Table 10, it has been seen that the front to back and back to front loading performance values of the chairs were close to each other for both wood species. The backrest loading performance values of the pine chairs were also close to the other loading directions. However, backrest loading performance values of the beech chairs were significantly higher than the front to back and back to front loading performances. The cyclic loading performances of chairs under the front to back, back to front, and backrest loadings were compared to the allowable light, medium, and heavy design load levels that were given in the ALA specifications (Eckelman 1999). The allowable light design load levels represent the domestic use in practice. Assessment of the cyclic loading performance of chairs with allowable design loads The assessment results of chairs are given in Table 11 for each loading direction. Table 11: Comparison of loading performance values of chairs with allowable design loads. Table 11: Comparison of loading performance values of chairs with allowable design loads. According to results, chairs constructed of pine could meet the allowable medium service load requirements for front to back and back to front loading directions, whereas they could only resist allowable light service (domestic usage) load requirements for backrest loading direction. In the case of chairs constructed of beech; they could satisfy the allowable medium service load requirements for front to back and back to front loading directions, and could meet the heavy service load requirement for backrest loading. These results were graphically shown in Figure 11. Figure 11: Assessment of performances of the chairs under the cyclic (a) front to back, (b) back to front, and (c) backrest loading. Figure 11: Assessment of performances of the chairs under the cyclic (a) front to back, (b) back to front, and (c) backrest loading. CONCLUSIONS This study was aimed to acquire quantitative information related to the cyclic front to back, back to front, and backrest loading performances of chairs, constructed of Scots pine and Oriental beech. In addition to that, it is intended to compare the cyclic loading performance of chairs with the allowable design loads that were 14 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 specified in ALA specifications. Furthermore, chairs were structurally analyzed by using the finite element method (FEM). specified in ALA specifications. Furthermore, chairs were structurally analyzed by using the finite element method (FEM). Generally, it was concluded that the overall design and construction of the pine chairs evaluated in this study would be satisfactory for light service, while beech chairs would be satisfactory for medium service. Thus, it can be said that the chairs constructed of pine and beech are extremely suitable for domestic usage. In fact, it is possible to reduce the cross-sectional dimensions of these chairs in order to obtain economic and ergonomic benefits if they are used for domestic usage. As expected, chairs constructed of beech yielded higher strength than those of pine. Although beech yielded higher strength, pine could be commonly utilized in the furniture frames in an engineering design approach because of its economic advantages. Chairs constructed of both species were given close cyclic loading performance under the front to back and back to front directions. The backrest loading performance of pine chairs was also close to other loading directions. However, the backrest loading performance of beech chairs was significantly higher than the front to back and back to front loading directions. At the end of the structural analysis results, the most critical loading was backrest loading. Under this loading, members and especially back leg to the side rail and back leg to stretcher joints of the chair were subjected to considerable amounts of shear and bending stresses, and general failures occurred at these joints. Eventually, it can be concluded that the overall strength of the chairs by considering initially the bending stress capacity of the members, secondly, section sizes and, finally the strength of the joints. Results indicated that structural analysis by Finite Element Analysis (FEA) methods provide reasonable estimates of the overall strength of these structures consistent with actual performance testing results. ACKNOWLEDGMENTS This paper is a part of MSc. thesis of the first author. The study was supported by Scientific and Technological Research Council of Turkey (TUBITAK) with the project number; 216O013, and Muğla Sıtkı Koçman University Scientific Research Project Office with the project number; 17/112. Preliminary data of this paper were verbally presented at the V. International Furniture Congress which was held in Eskişehir, Turkey. Then, data sets were expanded before converting into a scientific manuscript. CONCLUSIONS In addition, the results also showed that performance testing is an important part of furniture engineering methodology and provides quantitative feedback to designers. Performance testing enables exploration and investigation of unforeseeable failures and ensures fruitful conception to actual usage and failure conditions. In conclusion, this study confirmed that product engineering methodology, integrating structural analyses with performance testing is applicable to the design of chairs. 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